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Automated microfluidic screening and patterned illumination for

1. AQ2334 lite 1 ce314 123 IjIs123 pmec 4 ChR2 punc 122 RFP ZX899 lite 1 ce314 ljIs123 pmec 4 ChR2 punc 122 RFP zxEx621 pglr 1 MAC mCherry pelt 2 GFP ZX900 lite 1 ce314 2xXEx622 peglr 1 ChR2 H134R mCherry pelt 2 GFP SK4005 zdIs5 pmec 4 GFP lin 15 ZX819 lite 1 ce314 zxIs12 pF49H12 4 ChR2 mCherry pF49H12 4 GFP ZX1014 lite 1 ce3 14 zxEx633 pegl 46 ChR2 YFP Pelt 2 mcherry 5 7 2 Optical illumination behavioral recording and analysis All animals tested were F1 progeny of PO adults picked onto ATR plates 3 5 days prior to experiments Young adult animals were picked onto blank NGM plates 25 minutes prior to the experiments Strains with non integrated transgenes were picked based on a fluorescent co injection marker Each animal was only used for a single experiment and then discarded Single animal plates were inverted and placed on a custom made petri dish holder on a motorized X Y stage Prior Video recording and illumination was achieved using the previously discussed illumination system Chapter 3 and custom software Chapter 4 Analysis extracting the velocities and anatomical locations AP axis from the video analysis software discussed in Chapter 4 5 8 Conclusions and Discussion Optogenetics has received significant attention due to the potential for fast repeatable stimulation of genetically defined neurons We have shown here that it is possible to track a freely moving anima
2. 1 1 4 Transgenic animals C elegans nomenclature and genetic maintenance Creating transgenic C elegans is a relatively simple process and is routinely performed 56 57 Using transgenic methods genetic function and cell specificity is studied using mutant rescues protein function is analyzed using over expression investigate DNA RNA regulatory elements and investigate protein cellular localization 57 Transgenic animals are created by injecting exogenous DNA into an animal wild type or mutant The microinjection of the DNA usually using a plasmid with the desired promoter and gene of interest leads to an extrachromosomal array containing many copies of the introduced DNA The extrachromosomal array is transmissible to offspring with variable transmissibility Integrated lines are created using irradiation creating DNA breaks and integration of the extrachromosomal arrays during DNA repair 57 The extrachromosomal arrays are designated with a two letter prefix designating the lab in which they were created e g zx is the Gottschalk lab then the letters Ex followed by a number If the extrachromosomal array is later integrated through irradiation techniques then the Ex designation becomes Js The complete description of the animal can be found in its genotype designation which specifies all known differences between its genotype and wild type N2 Each difference is listed and would have a unique designation The strain name of an anim
3. UQG Optics U K Part a mm CDR 5051 38 2 568 50 Custom 50 2 CDB 5051 26 7 Green 65 b c d mm mm mm 27 7 34 6 24 8 26 7 25 4 Blue b Figure 3 4 Disassembly and insertion of custom optics into the 3 LCD projector 214 a Removal of the projection zoom lens system b Removal of the screws connecting the top of the projector case to the main body c Disconnecting the top control panel to remove projector case cover d Removal of the LAN board e Disconnecting wires and screws connecting main board f Disconnecting LCD panel cables g Removal of the dynamic iris h Removal of the cover of the main optical RGB path i Cover showing the polarizing filters j RGB optical paths k Optical path after insertion of optical filters colored boxes show location for red green and blue filter insertion 1 Removal of the diverging projection lens from the zoom lens system 3 2 3 Modification of microscope optics for infinity corrected systems The epifluorescent optical train of a microscope cannot properly relay the projector image to the sample plane as its lenses are not of the proper style or focal length and thus must be removed to make room for the custom optics In this protocol we describe the modifications for both the infinity corrected microscope and for the 160 mm fixed tube length microscopes In an infinity corrected microscope the objective lens and tube lens combine to form a two le
4. Figure 4 2 A grid of 20 points is sequentially projected and imaged determining parameters for coordinate transformation 4 3 Color Illumination and Tracking The custom software described in this section is capable of automatically tracking C elegans acquiring images identifying anatomical locations and directing the projector to illuminate the animal at the desired location with a specified color and intensity The program consists of several individual modules running in individual execution loops Fig 4 3 Each loop runs at 25 Hz PF raaa 3 RYLLLLLL LLL Camera 4 XY Stage lt ouneungunnns ounsnnnnnnes Current Position Calculate worm offset Stage qseeeeetunnnne fp Threshold Image Processing Threshold Center of Mass X Y Calculation of illumination segments Projected Image qsereeetunnnng XY Stage 4 Projector Q Q oonnnnnnnnns cuunuunenuns Figure 4 3 Software modules for performing imaging stage movements and image processing Each loop operates independently thus increasing overall processing rate 83 The front panel of the program is shown in Figure 4 4 and each component of the program will be discussed in detail below Ante Bhamnation Manus Bhuminsbon Simple Bumination Control s i i pr 4 M f E mama Scheduled i i b paiana Head Level prere Teng Reuss Figure 4
5. With our system we can achieve a greater level of stimulus control by using optical excitation These experiments will help elucidate how opposing sensory signals are integrated temporally to produce a behavioral 133 response We would hypothesize that a posterior stimulus can inhibit a subsequent anterior stimulus and the level of inhibition depends on both the stimulus intensity as well as the inter stimulus interval Furthermore we would expect to find that an anterior stimulus can inhibit a posterior stimulus to a stronger degree the intensity of anterior stimulus that can inhibit a posterior stimulus will be less than the required posterior stimulus to inhibit an anterior stimulus due to the AP asymmetry of the mechanosensory circuit The connectivity within the mechanosensory circuit is well known However the contributions of gap junction and synaptic connections and if they are excitatory or inhibitory has yet to be fully elucidated To examine the role of gap junctions and synaptic transmission in signal transduction and integration in the mechanosensory circuit one would perform optogenetic experiments using mutants defective in synaptic transmission eat 4 elimination of synaptic transmission by using cell specific expression of Tetanus Toxin TeTx 244 expressed in the mechanosensory neurons ALM AVM PLM and the RIM interneuron and RNAi knockdowns of genes for both synaptic transmission and gap junction formation in AVM
6. p 368 373 Angres B Cell microarrays Expert Review of Molecular Diagnostics 2005 5 5 p 769 779 Fernandes T G et al High throughput cellular microarray platforms applications in drug discovery toxicology and stem cell research Trends in Biotechnology 2009 27 6 p 342 349 Flaim C J et al Combinatorial signaling microenvironments for studying stem cell fate Stem Cells and Development 2008 17 1 p 29 39 Jang J H and D V Schaffer Microarraying the cellular microenvironment Molecular Systems Biology 2006 2 p 2 Park E S et al Continuously perfused non cross contaminating microfluidic chamber array for studying cellular responses to orthogonal combinations of matrix and soluble signals Lab on a Chip 2010 10 5 p 571 580 Bernard A B Michel and E Delamarche Micromosaic immunoassays Analytical Chemistry 2001 73 1 p 8 12 Sato K et al Microchip based immunoassay system with branching multichannels for simultaneous determination of interferon gamma Electrophoresis 2002 23 5 p 734 739 McClain M A et al Microfluidic devices for the high throughput chemical analysis of cells Analytical Chemistry 2003 75 21 p 5646 5655 Wei C W et al Using a microfluidic device for 1 mu 1 DNA microarray hybridization in 500 s Nucleic Acids Research 2005 33 8 Sims C E and N L Allbritton Analysis of single mammalian cells on chip Lab on a Chip 2007 7 4 p 423 440
7. vii 5 Demonstration of illumination system for neural circuit dissection 5 1 Motivation 5 2 Qualitative behavior elicited by structured illumination 5 3 Spatial activation of sensory and command neurons 5 4 Spatiotemporal control of the illumination intensity 5 5 Simultaneous multi color illumination 5 6 Optogenetic dissection of a nociceptive neural circuit 5 7 Methods 5 7 1 C elegans culture 5 7 2 Optical illumination behavioral recording and analysis 5 8 Conclusions and discussion 5 8 1 Limitations and considerations 6 Thesis contributions and future work 6 1 Thesis contributions 6 2 Future directions 6 2 1 Optimize the methods for optogenetic illumination 6 2 2 Combine optogenetics and behavioral recording 6 2 3 Utilize microfluidics optogenetics and calcium imaging techniques for exploration of integration sensory information between distinct neural circuits APPENDIX A Publications and other scientific activities APPENDIX B Detailed procedure for projector modification optical system construction and software APPENDIX C Additional contributions REFERENCES viii 103 103 103 105 112 116 120 123 123 124 124 125 127 127 130 130 133 134 138 141 155 158 LIST OF TABLES Page Table 2 1 Schedule for valve control 44 Table 3 1 Dimensions and specifications of the custom filters for insertion in to the modified Hitachi CP X605 65 Table B 1 Trouble
8. 145 iii Turn up the transmitted light intensity Using a piece of paper find the position along the epifluorescent optical path where the image of the micrometer comes into sharp focus This will be at the back focal plane of the objective located 160 mm from the nosepiece opening This will be the location IP160mm in Figure 3 2c iv Place the relay lens pair such that the edge of the lens housing is 92 mm the working distance of the lens pair from the position found in the previous step This will position the back focal plane of the lens pair at the back focal plane of the objective Fig 3 2c v Place the projector such that the primary projector image is 92 mm from the front edge of the lens tube pair This will place the primary projector image at the front focal plane of the lens pair Fig 3 2c TROUBLESHOOTING Computer setup and alignment of system e TIMING 1 h 191 With the projector connected to the computer adjust the display settings to have dual display capabilities extending the desktop onto the second monitor projector not cloning the primary monitor The projector should be configured as the secondary monitor and should be set to utilize the full resolution of the projector Hitachi CP X605 1024 x 768 The desktop should also be set to use a solid black background therefore not projecting any unwanted image to the sample 201 Place a piece of fluorescent paper or slide glass on the microscope stage and br
9. 166 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 Schultheis C et al Optogenetic analysis of GABA B receptor signaling in Caenorhabditis elegans motor neurons Journal of Neurophysiology 106 2 p 817 827 Schultheis C et al Optogenetic Long Term Manipulation of Behavior and Animal Development Plos One 6 4 Stirman J N et al Real time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans Nature Methods 8 2 p 153 U78 Nagel G et al Light activation of channelrhodopsin 2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses Curr Bio 2005 15 24 p 2279 2284 Aravanis A et al An optical neural interface in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology Journal of Neural Engineering 2007 4 3 p S143 S156 Gradinaru V et al Targeting and readout strategies for fast optical neural control in vitro and in vivo Journal of Neuroscience 2007 27 52 p 14231 14238 Guo Z V A C Hart and S Ramanathan Optical interrogation of neural circuits in Caenorhabditis elegans Nat Methods 2009 6 12 p 891 U47 EI Ali J P K Sorger and K F Jensen Cells on chips Nature 2006 442 7101 p 403 411 Whitesides G M The origins and the future of microfluidics Nature 2006 442 7101
10. 167 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 Hirsch A M et al Parallel multi time point cell stimulation and lysis on chip for studying early signaling events in T cell activation Lab on a Chip 2009 9 4 p 536 544 Gray J M et al Oxygen sensation and social feeding mediated by a C elegans guanylate cyclase homologue Nature 2004 430 6997 p 317 322 Zhang Y H Lu and C I Bargmann Pathogenic bacteria induce aversive olfactory learning in Caenorhabditis elegans Nature 2005 438 7065 p 179 184 Hulme S E et al A microfabricated array of clamps for immobilizing and imaging C elegans Lab on a Chip 2007 7 11 p 1515 1523 Chung K H M M Crane and H Lu Automated on chip rapid microscopy phenotyping and sorting of C elegans Nature Methods 2008 5 7 p 637 643 Crane M M K Chung and H Lu Computer enhanced high throughput genetic screens of C elegans in a microfluidic system Lab on a Chip 2009 9 1 p 38 40 Crane M M et al Microfluidics enabled phenotyping imaging and screening of multicellular organisms Lab on a Chip 2010 10 12 p 1509 1517 Gilleland C L et al Microfluidic immobilization of physiologically active Caenorhabditis elegans Nature Protocols 5 12 p 1888 1902 Samara C et al Large scale in vivo femtosecond laser neurosurgery screen reveals
11. 515 527 Hart A C and M Y Chao Chapter 1 From Odors to Behaviors in Caenorhabditis elegans The Neurobiology of Olfaction 2010 Purves D Neuroscience 4th ed 2008 Sunderland Mass Sinauer xvii 857 G 16 IC 7 I 29 p Liu Q G Hollopeter and E Jorgensen Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction Proceedings of the National Academy of Sciences of the United States of America 2009 p 10823 10828 Lockery S R and M B Goodman The quest for action potentials in C elegans neurons hits a plateau Nat Neurosci 2009 12 4 p 377 378 Richmond J Synaptic function in WormBook T C e R Community Editor WormBook Majewska A and R Yuste Topology of Gap Junction Networks in C elegans Journal of Theoretical Biology 2001 212 2 p 155 167 Altun Z F et al High Resolution Map of Caenorhabditis elegans Gap Junction Proteins Developmental Dynamics 2009 238 8 p 1936 1950 161 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 Fay D Genetic mapping and manipulation Chapter 1 Introduction and basics in WormBook T C e R Community Editor WormBook Priess J R and J N Thomson Cellular interactions in early C elegans embryos Cell 1987 48 2 p 241 250 Avery L and R Horvitz Pharyngeal pumping continues after laser killing of the pharyngeal nervous system of C elegan
12. each stage might have different directionality e g positive values of position might be negative in direction The way velocity is calculated might have to be changed depending on the particular system The threshold parameters can be adjusted though using the same parameters used in the Tracking and Color Illumination is advised Finally the user can choose the number of equal segments to divide the worm into An equal distance between points is found and these points and segments define the angles discussed below 4 6 2 Extracted Parameters A number of parameters are analyzed for each frame in the video The specifics for each parameter are discussed below 1 Velocity The instantaneous speed is calculated as distance traveled by the animal between successive frames multiplied by the frame rate yielding speed measured in m sec The total displacement is determined by adding the displacement of the stage from frame to frame plus the displacement of the center of mass of the animal Fig 4 15a b within the field of view from frame to frame after conversion from pixel to um The program also analyzes the direction of travel if the movement is toward the head as assigned in the Head Encode program then the speed is assigned a positive value and if toward the tail then speed is negative 98 2 Length This measures the length Fig 4 15c of the animal in the same manner 3 discussed in Section 4 3 3 1 converte
13. one can see if the overall bending angles of the animal change over time For example when an animal reverses one can see the average bending angle increase as the animal deepens its posture 99 5 Head Tail distance This is a measure of the straight line distance between the tip of the head and tail green line Fig 4 15g For example this distance decreases when an animal reverses or as it begins an omega turn 6 Amplitude The amplitude measured is the maximum right angle distance from a line connecting the head and the tail to the spline of the animal red line Fig 4 15h a Head z n pue P A gt A ia HT aA ie i A Vt Q N F ss Sai Figure 4 15 Extracted parameters from the video analysis a Bright field image of the animal b Binary image based on a c Thinned image of b From this backbone the length of the animal is determined d Segmented animal is labeled by S 1 total points starting with So at the head e Illustration of the measurement of a two point angle f Illustration of the measurement of a three point angle g Head to Tail length is the straight line distance from one end of the spline to the other h Amplitude of the animal is determined by the maximum distance from the spline of the animal to the line connecting the head and the tail 100 These parameters are the main outputs of the program A large amount of information can also be found from the IMAQ Par
14. select as many video as one wishes to have bulk analysis performed Upon selection of all videos for analysis click OK to begin analysis Upon completion of the analysis all extracted parameters are saved to a file named name of video data txt TH THRotaed Segments Segments Rotated pin 2pt Angles so m 2670 No Illumination eobsevso Enito m O i sa 6 b gt gt ba w H w i to 1 m w 150 160 170 o 150 a0 zo 29 20 Mo zo ao z0 xo xo o so sm 3 so 350 xo o s xo ao an aw ao so o do Frame 2Point apere Es W58nd2 5K 32 bit RGB image 0072 15 4 Figure 4 14 Front panel of the Complete Video Analysis program There are only a few options in the program conversion factor image binning threshold parameters and number of divisions of the animal 97 Parameters that the user can adjust are the conversion factor um per pixel for the camera and binning of the video For our system 3 3 um pixel is the full camera resolution conversion factor and the video is binned 2x2 yielding a final conversion of 6 6 um pixel These values will differ depending on specific microscope system and camera Also because the program uses both the location of the animal within the field of view and the position of the stage the conversion factor is important For our system one stage step equals one micron so no additional conversion was used for the stage coordinates Finally
15. 30 value is set such that the top border of the window is hidden and will not display The size of all images appearing within this display window is equal to the resolution of the projector 4 2 Projector Alignment After construction and rough alignment of the projector system described in Chapter 3 the system must undergo a refined alignment Furthermore a coordinate transformation must be made to convert the acquired images represented in camera coordinates XcYc defining the object of interest within the field of view of the camera and the projector coordinates XpYp defining the intended illumination pattern The parameters for coordinate transformation are saved for subsequent use in other programs 4 2 1 Initial Axial Z and In plane XY Alignment Upon starting the program a cross pattern is projected through the illumination system In order to visualize this pattern and properly align the system we must use a reflective fluorescent target The target is placed at the sample plane and should first be brought into sharp focus by focusing on dust or an imperfection scratch on the target surface Fluorescent slides or a blank NGM plate work well for this purpose This should be done first ignoring the projected light pattern After focusing on the target the projector s XYZ position should be altered such that the cross projected pattern is both centered and sharply focused Fig 4 1 The camera should be rotated such that the
16. 5 6 T 8 9 10 Time s b t 1s t 2s t 3s a 29 9 11 3 27 5 13 1 25 7 9 1 40 o o eee t 9t pa es Velocity um s t 7 t 8s t 9s t 10s 13 7 27 4 26 8 12 4 26 6 5 4 24 4 14 9 21 9 ee0 Figure 5 6 Individual animal responses to anterior stimulus 117 a Individual velocity plots of pmec 4 ChR2 animals upon anterior stimulation Blue bar indicates when the anterior portion of the animal was illuminated with blue light b Scatter plots of velocity at individual time points from the above figure Below the indication of the time point is the average velocity and in parenthesis is the standard deviation We performed similar experiments on pglr ChR2 animals which express ChR2 in the command interneurons as well as in other neurons 232 Illuminating the first quarter of the body with blue light excites the interneurons in the head including AVA 110 AVD and AVB Fig 5 7a Although this stimulation includes interneurons for both backward and forward movements the predominant effect is the backward command The velocity profile Fig 5 7b shows a robust reversal upon stimulation using this light pattern Similarly when the last quarter of the pglr ChR2 animals was illuminated and PVC excited there was a small but appreciable acceleration Although we cannot exclude the effects of photostimulation of the other g r 1 expressing cells the experiment shows specific illuminatio
17. C D O Beck and C M Chiba Caenorhabditis elegans a new model system for the study of learning and memory Behavioural Brain Research 1990 37 1 p 89 92 Rankin C H and B S Broster Factors affecting habituation and recovery from habituation in the nematode Caenorhabditis elegans Behavioral Neuroscience 1992 106 2 p 239 249 Stirman J N et al Real time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans Nature Methods 2011 8 2 p 153 U78 Zemelman B et al Photochemical gating of heterologous ion channels Remote control over genetically designated populations of neurons Proceedings of the National Academy of Sciences of the United States of America 2003 100 3 p 1352 1357 Nagel G et al Channelrhodopsin 2 a directly light gated cation selective membrane channel Proceedings of the National Academy of Sciences 2003 100 24 p 13940 13945 Banghart M et al Light activated ion channels for remote control of neuronal firing Nature Neuroscience 2004 7 p 1381 1386 Boyden E et al Millisecond timescale genetically targeted optical control of neural activity Nature Neuroscience 2005 8 p 1263 1268 Nagel G et al Light activation of channelrhodopsin 2 in excitable cells of Caenorhabditis elegans triggers rapid Behavioral responses Current Biology 2005 15 24 p 2279 2284 Szobota S et al Remote control of neuronal activity with a ligh
18. Kim J K et al Functional genomic analysis of RNA interference in C elegans Science 2005 308 5725 p 1164 1167 Maeda I et al Large scale analysis of gene function in Caenorhabditis elegans by high throughput RNAi Current Biology 2001 11 3 p 171 176 Pothof J et al Identification of genes that protect the C elegans genome against mutations by genome wide RNAi Genes amp Development 2003 17 4 p 443 448 Simmer F et al Genome wide RNAi of C elegans using the hypersensitive rrf 3 strain reveals novel gene functions Plos Biology 2003 1 1 p 77 84 Sugimoto A High throughput RNAi in Caenorhabditis elegans genome wide screens and functional genomics Differentiation 2004 72 2 3 p 81 91 Vastenhouw N L et al A genome wide screen identifies 27 genes involved in transposon silencing in C elegans Current Biology 2003 13 15 p 1311 1316 Zhang S L et al Genome wide RNAi screen of Ca2 influx identifies genes that regulate Ca2 release activated Ca2 channel activity Proceedings of the 159 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 National Academy of Sciences of the United States of America 2006 103 24 p 9357 9362 Sieburth D et al Systematic analysis of genes required for synapse structure and function Nature 2005 436 7050 p 510 517 Jorgensen E M and S E Mango The art and design
19. Nahabedian Jeffrey N Stirman Hang Lu Mutsuki Amano Guy M Benian and Kozo Kaibuchi PKN 1 a homologue of mammalian PKN is involved in the regulation of muscle contraction and force transmission in C elegans Journal of Molecular Biology 2011 407 222 231 J N Stirman Matthew M Crane Steven J Husson Sabastian Wabnig Christian Schultheis Alexander Gottschalk and Hang Lu Real time multimodal optical control of individual neurons and muscles in freely behaving Caenorhabditis elegans Nature Methods 2011 8 153 U78 Gary L Moulder Gina H Cremona Janet Duerr Jeffrey N Stirman Stephen D Fields Wendy Martin Hiroshi Qadota Guy M Benian Hang Lu and Robert J Barstead alpha Actinin Is Required for the Proper Assembly of Z Disk Focal Adhesion Like Structures and for Efficient Locomotion in Caenorhabditis elegans Journal of Molecular Biology 2010 403 516 528 J N Stirman M Brauner A Gottschalk and H Lu High throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics Journal of Neuroscience Methods 2010 191 90 93 138 M M Crane K Chung J Stirman and H Lu Microfluidics enabled phenotyping imaging and screening of multicellular organisms Lab on a Chip 2010 10 1509 1517 PRESENTATIONS presenter is underlined ORAL Jeffrey N Stirman and Hang Lu Monitoring and manipulating C elegans Behavior Workshop 18
20. SLM object plane is then transferred through a relay zoom lens and a concave diverging magnifying projection focusing lens to form the primary image and projected magnified image Fig 3 2a By removing the diverging projection lens a primary image is formed by the zoom lens a few centimeters in front of the lens This image is then relayed through a reconfigured epifluorescent optical train of an inverted microscope passing through the objective forming a demagnified image at the focal plane of the objective specimen plane Fig 3 2b c It is in this specimen plane that the object of interest e g freely moving C elegans is located and illuminated 61 SLM Spatial Light Modulator SP Sample Plane M lt 1 PZL Projector Zoom Lens RLP Relay Lens Pair PDL Projector Diverging Lens IP somm 100mm back image plane PPI Projector Primary Image OL somm Objective Lens 160 mm PI Projected Image M gt 1 FL Focal Length of the accesory tube lens ATL Accessory Tube Lens FF k p Front Focal Length of the relay lens pair OL Objective Lens Infinity Corrected BF k p Back Focal Length of the relay lens pair Figure 3 2 Optical configuration of the system and components 214 a Optical configuration of the projector in the original unmodified state b Optical configuration of the constructed illumination system for an infinity corrected microscope Optical configuration of the constructed illumi
21. a Figure 5 2 a Illustration of the positions of the six sensory neurons and a frame from Supplementary Video 3 of Reference 117 showing the 20 um bar of blue light perpendicular to the animal s longitudinal axis which was scanned at a rate of 12 5 animal body length per second 100 um s_ b Two scanning schemes along the AP axis head to tail and tail to head c Sequential frames from Supplementary Video 3 of Reference 117 showing a bar of light passing over the animal from posterior to anterior as the animal is freely crawling Initially the animal is traveling forward but when the light reaches the anterior mechanosensory neurons expressing ChR2 middle frame the animal quickly reverses direction Scale bar is 250 um Adapted from REFERENCE 117 As expected while illuminating from tail to head as long as the illumination was in the posterior half of the animal no reversals were elicited and as soon as the bar reached the anterior half animals reversed It was also evident that illuminations in the posterior initiated acceleration We quantified the body positions along the AP axis of the animal at which these behaviors were initiated Fig 5 3 as well as the anatomical positions of the touch neurons as measured by GFP fluorescence within the mechanosensory neurons pmec 4 GFP Fig 5 3 Fig 5 4 Reversals were initiated most often within the range 0 40 0 48 along the AP axis in tail to head scans consistent with the
22. anatomical data i e the positions of the ALM AVM cell bodies 229 and with our quantification of neural cell body locations Fig 5 3 5 4 In the head to tail scans animals showed a high 106 probability of reversal well before reaching the AVM or ALM cell bodies Fig 1f indicating that activation of the ChR2 in the distal processes is sufficient to elicit a response This is likely because enough ChR2 is present in neuronal processes to allow sufficient photo depolarization of the cell No behavioral response was observed for illumination in the region of the PVM neuron data not shown which is consistent with the observation that PVM plays no role in hermaphrodite mechanosensation 66 111 Tail to Head Scan Reversing n 52 50 a Head to Tail Scan Reversing n 26 PLMUR g 45 Tail to Head Scan Acceleration n 28 Measured Cell Body Locations n 54 40 AVM PVM 35 EA v ALMLIR z ae T 25 20 x on 0n onon ognon onono gnong2 eN NM TT HH OONN DOHA AH SO ne gs I I Relative AP axis position ic Figure 5 3 Histograms showing the distributions of positions along the AP axis at which point the blue light elicited a reversal response 117 Shown are the distribution of positions where accelerations elicited by the tail to head scan were observed 28 out of 52 animals showed an increase in speed two standard deviations greater than the average speed prior to illumination
23. behavior of fluids that are generally confined to channels or chambers on the micron scale and occupy small volumes nL pL fL 171 172 On this scale some properties of the fluid start to dominate the behavior such as fluid resistance viscous forces surface tension and energy dissipation As the viscous forces start to dominate the Reynold s number Re ratio of inertial to viscous forces decreases It is in this regime where microfluidics operates low Re number flow or laminar flow In laminar flow when two liquid streams come together e g two channels combine into one there is no mixing through convective forces and the dominate mechanism of fluid mixing is through diffusion across the interface Fig 1 5 The two streams will flow in parallel with no transverse perpendicular flow 23 Figure 1 5 Dye filled image of a microfluidic device There are three inputs each of a different color blue red yellow flowing at equivalent flow rates The laminar nature of the flow at low Reynolds number can be seen as the different color combine in the channels without mixing Figure courtesy of Alison Paul In addition to unique properties of the fluid there are also very practical advantages of using microfluidics Because of the small volumes and ability to prevent mixing through laminar flow the concentration of reagent in microfluidic devices can be very well defined 172 Furthermore the small volumes lead to little reagen
24. by the needed amount and finally copied into a black image of size equal to the resolution of the projector 1024x768 4 3 4 Video Recording Within the main control panel there is an option to record a video Selecting the Record button Fig 4 4 will begin recording and Stop will terminate recording Upon stopping the video acquisition the video is compressed and saved to the previously determined location The user will then be prompted for the name and location of the next video to be acquired The video records both the live image I and the selected illumination image d as side by side images Additionally the following parameters are encoded within the avi file for each and every frame current stage position Xstage Y stage and all the previously discussed parameters of illumination control 94 4 4 Scheduled Illumination By utilizing this program more complicated illumination patterns and timing schedules can be set and saved The saved illumination schedule can be called from the Color Illumination and Tracking main program discussed in section 4 3 Upon starting the program the user is prompted for the number of levels of illumination as well as the number of segments this sets the discrete number of different illumination patterns Then the user can set the specifics of the duration at each level the locations for segmentation the color and intensity for each segment When calling the save
25. cell lineages of the nematode Caenorhabditis elegans Developmental Biology 1977 56 1 p 110 156 White J G Chapter 4 The Anatomy in The Nematode Caenorhabditis Elegans ed W B Wood 1988 Cold Spring Harbor Laboratory Sulston J E et al The embryonic cell lineage of the nematode Caenorhabditis elegans Developmental Biology 1983 100 1 p 64 119 Glucksmann A Cell deaths in normal vertebrate ontogeny Biological Reviews 1951 26 1 p 59 86 Lockshin R A and C M Williams Programmed cell death Endocrine potentiation of the breakdown of the intersegmental muscles of silkmoths Journal of Insect Physiology 1964 10 4 p 643 649 Saunders J W Death in Embryonic Systems Science 1966 154 3749 p 604 612 Avery L and H R Horvitz A cell that dies during wild type C elegans development can function as a neuron in a ced 3 mutant Cell 1987 51 6 p 1071 1078 Fire A et al Potent and specific genetic interference by double stranded RNA in Caenorhabditis elegans Nature 1998 391 6669 p 806 811 Hannon G J RNA interference Nature 2002 418 6894 p 244 251 Vanderkrol A R et al Inhibition of flower pigmentation by antisense chs genes promoter and minimal sequence requirements for the antisense effect Plant Molecular Biology 1990 14 4 p 457 466 15 16 17 18 Napoli C C Lemieux and R Jorgensen Introduction of a chimeric chalcone synthase gene into petunia
26. due to the additional changes in refractive index caused by the PDMS Decreasing the experimental noise would be the most straightforward technique to decrease the required number of animals and should be explored in future work An additional problem encountered was possible low efficacy of the RNAi as well as very sick animals due to the mutation nre leading to the RNAi sensitivity These problems result in false negatives as well as small sized animals with low fertility and hence a low number of animals Furthermore many of the RNAi control populations failed to phenocopy the mutant response which could be due a few reasons One possibility is that the neural RNAi sensitive animal is still not efficiently taking up the RNAi and the transcripts are not sufficiently being knocked down This could be evaluated by expressing a GFP construct in the neurons of interest and observing the percent knock down with the RNAi for GFP A second reason possibility is that the RNAi fundamentally should not phenocopy the mutant response Many of the mutants tested in this study and in previous studies 140 were point mutation mutants loss of function or decrease of function rather than complete knockout mutants If effective the RNAi would knockout the protein to some extent rather than alter its function as the point mutation might decrease of function and therefore it is possible that the two might not exhibit similar responses Finally another pr
27. expressing ChR2 in PVD and FLP whole body illumination would activate both neurons and thus would give little information regarding the relative role for the individual neurons To test the functional role of PVD ChR2 was expressed in this neuron under the control of the F49H12 4 promoter ZX819 This promoter also expresses ChR2 in two 120 additional neurons AQR in the head and an unidentified neuron in the tail To specifically illuminate PVD and exclude the effects of photoactivation of the two other neurons the illumination tracking system and software Chapter 3 and 4 was utilized First the third quarter of the animal was illuminated with blue light This segment contains the PVD cell body AP axis position 0 65 and under photoactivation leads to rapid acceleration Fig 5 15a No perceptible change was observed for illumination of the head first quarter photoactivating AQR Fig 5 15b or illumination of the tail fourth quarter photoactivating the unknown tail neuron Fig 5 15c In order to test the sensitivity of the dendridic arbor to photostimulation we next illuminated the regions along the AP axis of 0 4 0 6 Fig 5 15d 0 3 0 5 Fig 5 15e and 0 2 0 4 Fig 5 15f Comparing the maximal velocity during illumination a decrease was observed as the illumination was shifted further from the cell body Fig 5 15g though no statistical significance was seen between 0 4 0 6 and 0 3 0 5 a b c Velo
28. filter m GREEN i 80 k Blue pre filter 80 RED Pa m Green pre filter Red pre filter 60 40 Relative Intensity Relative Intensity 20 400 450 500 550 600 650 700 Wavelength nm Pixel Value 8 bit Figure 3 5 Spectral and intensity characterization of the illumination 117 a Measured spectra before and after addition of filters internally in the projector limiting the spectral range of each RGB color b Relative intensity as a function of pixel value see text for maximal intensities The contrast ratio for each color was determined by the ratio between full on pixels set to 255 for that color Fig 3 5b to full off zero pixel value Fig 3 5b and for the modified Hitachi CP X605 projector are Red 896 1 Green 463 1 and Blue 605 1 as measured using a power meter PM100D Thorlabs These values are less than the manufacturer s stated contrast ratio of 1000 1 due to the modifications performed on the projector The contrast ratio is an important feature of the system as it determines the background light intensity and should be as low as possible to avoid causing any undesired stimulation Therefore we suggest choosing a projector with a high initial i e manufacturer s stated value contrast ratio gt 500 1 and carefully measuring these values before and after modifications are performed The main source of significant decrease greater than a 2 fold decrease in contrast ratio
29. gradually slowing At t 180 seconds the blue light was turned on to excite the GABAergic neurons releasing the neurotransmitter GABA into the neuromuscular junction This led to a hyper polarization of the muscle cells as seen in the increase in the worms length relaxation of the muscles which then decreased rapidly because of the continued presence of nicotine This additional 40 ability to perfuse drugs adds to the toolbox for these types of studies Relative Body Length Figure 2 6 Nicotine 30 mM induced contraction and ChR2 induced relaxation n 49 142 Blue bar indicates time when blue light illumination is present 2 5 Automated Robotic Liquid Handling and Integration The previously described system demonstrated relatively high throughput and the ability to easily handle and assess multiple animals from a single population In order to handle several populations in a row as would be needed in a large scale screen such as an RNAi as discussed in Chapter 1 drug library or bacterial feeding screen the device external control and computer automation needs to be further improved 2 5 1 Higher throughput device A relatively simple means to increase the number of animals simultaneously is to increase the number and density of imaging channels The number of parallel imaging channels we can image in one field of view is limited by the resolution of the imaging system at the requisite magnification The enhanced devi
30. most transformative technological discoveries in molecular biology cellular biology and physiology the green fluorescent protein GFP and other fluorescent proteins have found widespread applications as transcriptional translation reporters cell identification markers sub cellular protein localization reporter and visualization of cellular anatomy GFP can be genetically encoded negating the requirement for injection or other methods of introducing the fluorescent marker and can be expressed and visualized at all cell cycles and organism stages Green fluorescent protein is a 238 amino acid helical barrel structure protein It was purified from the jellyfish Aequorea victoria by Osamu Shimomura in the early 1960s 42 43 The wild type GFP has major and minor excitation peaks at 395 nm and 475 nm respectively It exhibits fluorescent emission peak at 509 nm Roger Tsein made a major advance in GFP through a single point mutation S65T improving the photostability fluorescent intensity and shifting the major excitation peak to 488 nm 44 making the spectral characteristics compatible with commonly used FITC fluorescent filters Dr Tsien also dedicated a significant amount of work to further improving GFP and modifying it to fluoresce at other wavelengths 44 45 He also developed mRFP 46 and modified it to include other colors of the visible spectrum 47 The fluorescent protein palette now covers all wavelengths of the visible spect
31. neural basis of behavior the processing of neural circuits and the integration of sensory signals 137 APENDIX A PUBLICATIONS AND OTHER SCIENTIFIC ACTIVITIES JOURNAL PUBLICATIONS Matthew M Crane Peri Kurchan Jeffrey N Stirman Kang Shen and Hang Lu Automated and self directed forward genetic screening of C elegans using computer vision and microfluidics In preparation Steven J Husson Jana F Liewald Jeffrey N Stirman Hang Lu and Alexander Gottschalk Microbial light activatable proton pumps as circuit breakers to dissect neuronal networks in C elegans In review Steven J Husson Wagner Steuer Costa Jeffrey N Stirman Joseph D Watson W Clay Spencer Millet Treinin David M Miller Hang Lu and Alexander Gottschalk Optogenetic analysis of a nociceptor neuron and network reveals modulatory ion channels acting downstream of nociceptive sensors In review John F Nahabedian Hiroshi Qadota Jeffrey N Stirman Hang Lu and Guy Benian A New Quantitative Assay of C elegans Locomotion Identification of Phenotypes for Mutants in Genes Encoding Muscle Focal Adhesion Components Methods Accepted Jeffrey N Stirman Matthew M Crane Steven J Husson Alexander Gottschalk and Hang Lu A Multispectral Optical Illumination System with Precise Spatiotemporal Control for the Manipulation of Optogenetic Reagents Nature Protocols In press Hiroshi Qadota Takayuki Miyauchi John F
32. neurons within and between levels are both chemical and electrical gap junctions Figure adapted from Reference 72 10 1 1 5 1 Synaptic Transmission Transmission of information from neurons to neurons and neurons to muscles is a necessary function in the nervous system Signal transmission occurs primarily though gap junctions and synaptic transmission Synaptic transmission occurs across the synaptic cleft from the pre synaptic neurons to the post synaptic target whether it is neuron or muscle The process of this transmission occurs through many steps neurotransmitter synthesis vesicle loading vesicle trafficking to axon terminal vesicle fusion to membrane release of neurotransmitter recycling of vesicle diffusion of neurotransmitter across synaptic cleft binding of neurotransmitter to post synaptic receptor enzymatic alteration of neurotransmitter reuptake of neurotransmitter and recycling of neurotransmitter 73 Defects in any of these steps can lead to diminished or elevated Synaptic transmission and synaptic dysfunction is associated with neurological and psychiatric disorders Binding of synaptic vesicles to the cellular membrane and release of neurotransmitter is initiated by the influx of calcium through voltage gated Ca channels In C elegans neurons are not spiking no action potential and are graded 74 75 As such synaptic release is also graded and is roughly proportional to the membrane potential 74 as the m
33. of nicotine Sigma Aldrich was introduced through the other inlet of the Y a b c Air Water 58 Glycerol i a P 4 n k P S Figure 2 2 Index matched solution greatly decreases contrast between flow and control layers Top row is when the valve is open no pressure and the bottom is with the valve closed positive pressure a Valve filled with air b Valve filled with water c Valve filled with a 58 glycerol solution 35 2 3 Image Acquisition and Analysis All experiments were performed on a Leica DM IRB inverted microscope with a 4x objective and a Hamamatsu Orca camera Custom software was written in LabVIEW 9 0 with Vision to control worm injection valve actuation and video acquisition Blue light illumination 0 3 mW mm was delivered via the epi fluorescent port from a Leica EL6000 metal halide fluorescent light source filtered through a GFP excitation filter 450 490 nm and electronically shuttered The microscope was properly aligned for Kohler illumination insuring uniform blue illumination across the entire field of view On chip valves and electronic shutter were controlled by the computer controlled custom control box Chapter 2 6 After loading and isolating the worms a movie was started 8 frames sec at 1280x1024 with no blue light illumination to obtain a baseline reading of the worms lengths After 2 seconds the shutter was opened to illuminate the worms for another 5 seconds after which time t
34. positive pressure control layer 29 These devices employ a number of techniques for immobilization such as cooling 187 204 mechanical 186 192 193 CO2 205 and a sol gel Pluronic 206 Typically these devices contain one or two channels because of the limited field of view These devices have demonstrated a vast increase in the speed and efficiency of screening sorting and manipulating C elegans The ease of fabrication biocompatibility and feature size lends microfluidics to high throughput genetic behavioral and physiological studies using C elegans 1 4 Thesis Outline This thesis seeks to advance the currently available technologies for research in C elegans neuroscience Over the past several years both microfluidics and optogenetics have made great contributions to research by conferring to researchers the ability to precisely manipulate the chemical and physical environment rapidly position and sort animals and allow for non invasive manipulation of neural nodes Additionally automated image processing and lab automation combined with microfluidics and optogentic technologies allows for greater speed of data collection precise and repeatable manipulations and unbiased data quantitation In the first aim Chapter 2 we combine microfluidics and optogenetics to demonstrate a vast increase in processing and data acquisition for the analysis of synaptic transmission Although demonstrated for this particular study t
35. results in reversible co suppression of homologous genes in trans Plant Cell 1990 2 4 p 279 289 Ruiz M T O Voinnet and D C Baulcombe Initiation and maintenance of virus induced gene silencing Plant Cell 1998 10 6 p 937 946 Angell S M and D C Baulcombe Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA Embo Journal 1997 16 12 p 3675 3684 Dougherty W G et al RNA mediated virus resistance in transgenic plants exploitation of a cellular pathway possibly involved in RNA degradation Molecular Plant Microbe Interactions 1994 7 5 p 544 552 19 Kumagai M H et al Cytoplasmic inhibition of carotenoid biosynthesis with virus derived RNA Proceedings of the National Academy of Sciences of the United States of America 1995 92 5 p 1679 1683 158 20 21 22 23 24 25 26 2T 28 29 30 31 32 33 34 35 36 37 38 39 Romano N and G Macino Quelling transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences Molecular Microbiology 1992 6 22 p 3343 3353 Fire A et al Production of antisense RNA leads to effective and specific inhibition of gene expression in C elegans muscle Development 1991 113 2 p 503 514 Dernburg A F et al Transgene mediated cosuppression in the C elegans germ line Genes amp Development 2000 14
36. sought to increase and enhance the available tools for C elegans neuroscience research As both microfluidics and optogenetics increases in popularity and widespread application by neurobiologists the complexity and sophistication of the questions that can be asked increases In order to keep pace with the questions biologists posed efficiently aid in answering these questions and push the frontier we must develop corresponding technologies The current limitations of some optogenetic experiments OptIoN are that they are low throughput and place a heavy burden on the researcher The manual methods employed do not lend themselves well to scaling up as would be needed in a large scale RNAi or drug screen In other optogenetic experiments the experiment is limited in interpretation and flexibility by the current illumination systems Those illumination systems rely on traditional epi fluorescent systems designed for broad field illumination or more sophisticated precise systems are prohibitively expensive and complex in their construction This thesis addresses the major limitations of current technologies and methods In Chapter 2 we demonstrated for the first time the combination of microfluidics and optogenetics Microfluidics has previously been demonstrated to increase the processing speed and efficiency when imaging small model organisms like C elegans The previous work using microfluidic devices for aiding high throughput screens was ei
37. specific interest in relating genes to behavior but believed there was no simple way to directly map the two He believed the problem could be dissected into two components the genetic determination and specification of the nervous system and how the functional nervous system leads to behavior 1 A nervous system is an interconnected network of cells and in the examination of the nervous system it would be ideal to visualize the connections between the cell and cellular anatomy and development At the time of initiating research into C elegans the main method for visualization of cell cell interaction is the electron microscope Therefore Dr Brenner s desired qualities in an organism for his research were small size existence of a nervous system yet few cells involved and amenable to genetic manipulations In is search for such an organism he came across C elegans 1 C elegans meets Dr Brenner s aforementioned requirements and has many other favorable advantages C elegans is a soil dwelling nematode roughly one millimeter long 1 It is generally found in temperate climates and can be found all over the world There are many wild type animals the most widely used wild type animals are N2 originally isolated in Bristol England and CB4856 originally isolated in Hawaii Over 99 8 of the animals found are self fertilizing hermaphrodites with the other 0 2 are males The developmental time of the animal is short
38. spot size using a 25x objective Similar measurements to above were made using the 25x objective This shows a resolution limit to be about 5um at 25x The measured spatial resolution of the system is typical for the selected objective and projector Should another projector be used the main feature of the projector that could alter this value is the size of the LCD panels 0 79 for the Hitachi CP X605 If the resolution of the system is much lower than expected the most likely source of error is the axial focus of the projector The projector must be focused at the sample plane Steps 21 23 Appendix B to ensure a high spatial resolution This is increasingly critical as the magnification and numerical aperture of the objective increases 3 3 3 3 Static Spatial Ilumination Accuracy The static spatial accuracy is the ability of the illumination system to target a pre defined specified point when the sample is stationary To determine this accuracy we randomly selected 1000 points in the camera coordinate system These were then converted to projector coordinates after careful alignment and calibration and projected onto a mirror and imaged The location of these spots is then recorded and compared to the original intended locations and a deviation was calculated The average deviation of the 1000 points was found to be less than 1 pixel at full 640x480 resolution or less than 3 um 74 3 3 4 Temporal Illumination Resolution and Acc
39. step of the cycle including valve position X closed O open location of the Gilson Liquid Handler and the approximate time for each step is shown in Table 2 1 Steps 6 8 are repeated until sufficient number of animals are processed 35 Between each animal population the tubing and device are washed and rinsed with ethanol and water 43 Table 2 1 Schedule for valve control The pinch valves are external valves located between the device and the vacuum port and control the fluid flow The capture valve and loading valve are on chip valves as seen in Figure 2 6a The aspiration pin on the liquid handler is translated XYZ into a well of the multi well plate containing the animals or to a wash stations EtOH H20 Process Entrance Exit Capture Loading Liquid Approximate Pinch Pinch Valve Valve Handler Time s Valve Valve Location 1 Wash 1 O x O O EtOH 30 2 Wash 2 X O O O EtOH 30 3 Rinse 1 O X O O H20 30 4 Rinse 2 X O O O H20 30 5 Pre load O O O O MW 60 Plate 6 Load X O O X MW 15 Plate 7 Capture X X X X MW 80 Image Plate 8 Unload X O X O MW 5 Plate 2 5 3 Measuring worm concentration In order to efficiency of load animals into the microfluidic device the concentration of animals to be loaded is critical A low concentration of animals takes a great deal of time to load animals into the device if the concentration is too high the clogging rate and the frequency of multiple animals in a single channel are greatly incr
40. support for microfluidics exists The commercially available microfluidic devices and instrumentation are largely proprietary and designed specifically for the final product and thus are not flexible in their usage for custom devices Over the past several years working closely with Matthew Crane Lu lab Georgia Institute of Technology we have transformed the external control system from a loose collection of regulators gauges valves and tubing into a self contained control box Fig 2 11 The control box is computer controlled through a USB connection and can be addressed in LabVIEW Matlab or other programs The simplified and integrated control box has significantly eased the testing and usage of microfluidic devices in our lab and more importantly has allowed the ease of transfer of technology to non engineering labs utilizing the lab s 48 microfluidic devices The discussed control box has been distributed to collaborators labs in Germany Australia England and several others throughout the United States Pressure Gauges 0666 0 60 0 15 0 15 Pressure Regulators q 7 Figure 2 11 Master microfluidic control box a Front panel of the control box Two sets of 0 60 PSI and two sets of 0 15 PSI regulators and gauges control the outlet air pressure On the front are the air outlets b On the back are additional ports for electronic control of external components such as pinch valves Also on the back are the
41. system capable of illuminating targets that are spatially distinct and with control over the intensity and spectrum of the illumination Furthermore the illumination pattern should be dynamically alterable such that moving targets could be actively tracked and illuminated To make the designed technology accessible to many labs the system should be relatively inexpensive and simple to construct and thus not requiring experts in optical design and construction to assemble 78 This chapter outlines a protocol to modify a commercially available 3 LCD projector By modifying an existing technology the price of the system is kept low The 3 LCD projector is modified such that custom inserted internal filters narrow the spectral bandwidth for each of the three colors best matching some of the available optogentic reagents By optically modifying the projector and coupling it to an inverted microscope we demonstrate the ability to create an image demagnify it and relay it to the sample plane where the object of interest is located The completed system can be dynamically controlled at 25 Hz and has full capability to alter the location intensity and color of the illumination pattern The described system is relatively simple to assemble and the protocol presented in this chapter and Appendix B does not require an optical expert and can be completed in a few days Equally important the system is 10x 100x cheaper than other assembled or commercial sy
42. the barrel structure of the GFP is open and no fluorescence can occur Upon binding to Ca the conformational change closes the barrel of GFP and fluorescence can occur As mentioned previously calcium influx though voltage gated Ca channels is in direct consequence to membrane depolarization and precedes neurotransmitter release and hence monitoring the intracellular Ca concentration is a means of monitoring neural function As with the electrophysiological methods animals can be subjected to a stimulus while monitoring Ca transients or measure these transients as specific behaviors of the animal occur Both these methods electrophysiology and Ca imaging provide a more direct means of correlating neural function to a behavior or sensory function as well as providing information about the temporal and sequential response of neurons within a circuit Combining these methods with others such as optogenetics and microfluidics discussed below provides greater flexibility and detail when measuring the neural basis of behavior and the perception of sensory information 14 1 1 5 4 C elegans mechanosensation In C elegans there are six neurons that have been identified to be part of the gentle touch response These touch receptor neurons TRN were identified both through behavioral genetic techniques as well as optical ablations 66 108 109 The six neurons ALML R AVM PVM and PLML R are named based on their anatomical location
43. to a the segmentation and illumination loop to undergo further processing ultimately calculating the relative anatomical positions of the worm and creating the desired illumination Fig 4 7c f Figure 4 7 Custom software for processing the acquired images ultimately creating illumination pattern for the real time illumination of freely behaving C elegans 214 a Acquired bright field live image I of C elegans b Binary image after applied thresholding 1 The binary image is thinned to single pixel backbone CaN representing the AP axis of the animal and segmented according to user selectable parameters I The locations for segmenting are based along the relative path length of the backbone where the head is O and the tail is 1 d Resulting segmentation of the binary image I e Selected illumination color pattern a generated based on user selectable options including segment number color RGB intensity 0 255 for each color as well as illumination duration f Resulting multi color illumination pattern projected onto the moving C elegans Image is falsely colored based on the intended illumination pattern Scale bar is 250 um 4 3 3 1 Thinning The binary image 1 is first processed to a single pixel backbone red line Fig 4 7c This is achieved by the thinning algorithm in Matlab This algorithm is accessed by 87 running a Matlab script within the LabVIEW program The specifics of the binar
44. to motor neurons that drive locomotion 1 2 Optogenetics Understanding the cellular and genetic basis of neural function and behavior of an organism is a central problem in neuroscience Recently developed optogenetic methods have contributed enormously to our experimental toolbox 118 125 Optogenetics is a method to control the excitability of cells using light on the millisecond timescale Initially opsins from Drosophila photoreceptor cells in combination with G proteins were shown to photosensitize cells when expressed 126 Light gated opsins derived from bacteria and fungi were subsequently shown to be superior as functional light gated ion channels or pumps 124 125 127 128 When expressed in cells sometimes requiring the co factor retinal illumination with the appropriate wavelength of light the opsin undergoes a conformational change thus allowing the passage of cations or anions into the cell hence depolarizing or hyperpolarizing the cell Currently a number of opsins are being used for optogenetic studies such as the non specific cation channel Channelrhodopsin 2 ChR2 from Chlamydomonas reinhardtii which depolarizes excitable cells Halorhodopsin NpHR an inward directed Cl pump from Natronomonas pharaonis the outward directed proton pumps MAC bacteriorhodopsin from Leptoshaeria maculans and Arch archaerhodopsin 3 from the archaeon Halorubrum sodomense all of which hyperpolarize cells 124 125 128 1 2 1 Channelr
45. wall muscle cells Upon blue light excitation GABA released from these neurons into the NMJ synaptic cleft binds to GABA receptors on the muscle cell leading to a hyperpolarization and thus relaxation of the muscle This can be seen as a lengthening of the animal Fig 2 5a b zx s6 is a worm strain carrying ChR2 in the cholinergic neurons which act in an excitatory 208 fashion at the NMJ When zx s6 worms are exposed to blue light a shortening of the animal is observed Fig 2 due to the release of acetylcholine which induces muscle depolarization and thus contraction Animals mutant in the unc 49 gene encoding the GABA receptor carrying the zx s6 transgene showed an additional decrease in body length Fig 2 under blue light illumination This is because the cholinergic motor neurons connect to GABAergic neurons such that GABA release is co activated by ACh release which reduces the activating effect of ACh unless the UNC 49 GABAaR is absent The data in Figure 2 5a b agree extremely well with those previously obtained by standard manual methods 140 This demonstrates that the microfluidic devices coupled with the automation and image processing tools give comparable experimental results but are faster and more easily standardized 39 o pppn i BAN g 5 1 00 Eeoa a re iiag gt 0 96 E m 0 94 4 ii E 0 924 4 2xIs3 Haig RH S 9 904 4 zxis3 ATR zxls6 ATR fina o zxls6 HRAHH HHHH Relative Body Length ag
46. 0 A number of genome wide RNAi screens have been performed and used to discover genes involved in synaptic transmission longevity and aging regulation of small molecules fat storage genome protection development and many sensory functions 24 31 40 These screens are extremely time consuming and relatively low throughput Those that have demonstrated high throughput and generally low content end point assays drug resistance or can be done imaging many animals at low magnification 41 As screens become increasingly dependent on identification of subtle behavioral differences or necessitate high magnification imaging new methods for performing these RNAi screens must be developed 1 1 3 Tissue cell and protein visualization in C elegans Upon first examining C elegans under the microscope one can immediately see a striking feature of this animal it is optically transparent This allows for easy visualizations of the internal structures organs and cells This fact certainly aided in tracking the cell lineages observing cell divisions and annotating cellular patterning of tissue and organs Osamu Shimomura Martin Chalfie and Roger Y Tsien received the 2008 Nobel Prize in Chemistry for for the discovery and development of the green fluorescent protein GFP nobelprize org Though the research on GFP did not necessitate the use of worms its power was certainly emphasized through the use in C elegans Perhaps one of the
47. 0 0 0 Fig 4 11 This pane displays the illumination determined by the control in Figure 4 9 In the example shown here the control has the 2 and 4 segment turned on and the blue value has been set to 200 and all other color set to 0 To create the illumination pattern according to the previously discussed user input the binary image of the segmented animal I is first divided into S number of segments individual binary images each containing only one segment 1 5 Each of these images is then multiplied by the corresponding color value for that segment RGBs and by the binary value of the segment Bs on 1 off 0 as defined in the Illumination Control panel or in Scheduled Illumination The final selected illumination image is then the sum of all the individual segment images 93 S Pes gt x RGBs x Bs 1 Examples of final illumination image I can be seen in Figure 4 7e and Figure 4 11 This image is calculated every iteration of the Segmentation and Illumination loop at a set rate of 25 Hz Finally when the user selects the Illuminate button this image is transmitted to the projector and thus to the specimen This will continue until the pre set illumination time has elapsed or until the user deselects this button To accurately transmit the image I to the projector the previously found section 4 1 2 X and Y scaling and offset factors are utilized The I is scaled and offset
48. 0 7 10 p 848 U117 Liewald J F et al Optogenetic analysis of synaptic function Nature Methods 2008 5 10 p 895 902 Mahoney T et al Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans Proceedings of the National Academy of Sciences of the United States of America 2008 p 16350 16355 Stirman J N et al High throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics Journal of Neuroscience Methods 2010 191 1 p 90 93 Leifer A M et al Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans Nature Methods 2011 8 2 p 147 U71 Schultheis C et al Optogenetic Long Term Manipulation of Behavior and Animal Development Plos One 2011 6 4 Fiala A et al Light induced activation of neurons in Drosophila using channelrhodopsin 2 Journal of Neurogenetics 2006 20 3 4 p 115 116 Schroll C et al Light induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae Current Biology 2006 p 1741 1747 Suh G S B et al Light activation of an innate olfactory avoidance response in Drosophila Current Biology 2007 17 10 p 905 908 165 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 Zhang W W P Ge and Z R Wang A toolbox for light co
49. 0x240 1X 8 bitimage0 0 0 Figure 4 5 Live image acquisition and binary image creation The binary image is used to determine animal position offset within the field of view as well as used in subsequent image processing steps After creation of the binary image the image is filtered such that only the largest object in the field of view is kept all others particles are removed A few binary image processing steps are performed on the image to enable the most accurate determination of the center of mass and for subsequent processing steps 1 the image is first eroded x1 2 holes are filled in and 3 the image is dilated x1 These steps are part of the binary image processing pallet within LabVIEW and eliminate small spurs or bumps on the outside edge of the binary image fill small holes and return the binary image to its original size From the binary image I of the animal the center of mass of the animal 85 is calculated using the built in binary image analysis routines These values Xcom and Ycom are passed to the motorized stage subroutine 4 3 2 Motorized Stage Control For our system we used a Prior motorized stage Control of the motorized stage was achieved through the serial port and RS232 commands were sent through the VISA read write subVIs in LabVIEW The command language used can be found on the Prior website www prior com Upon each new execution of the motorized stage loop the stage is first queried f
50. 13 p 1578 1583 PalBhadra M U Bhadra and J A Birchler Cosuppression in Drosophila Gene silencing of Alcohol dehydrogenase by white Adh transgenes is Polycomb dependent Cell 1997 90 3 p 479 490 Kamath R S and J Ahringer Genorne wide RNAi screening in Caenorhabditis elegans Methods 2003 30 4 p 313 321 Boutros M et al Genome wide RNAi analysis of growth and viability in Drosophila cells Science 2004 303 5659 p 832 835 Silva J M et al RNA interference microarrays High throughput loss of function genetics in mammalian cells Proceedings of the National Academy of Sciences of the United States of America 2004 101 17 p 6548 6552 Ahringer ed J Reverse genetics in WormBook T C e R Community Editor WormBook Timmons L and A Fire Specific interference by ingested dsRNA Nature 1998 395 6705 p 854 854 Timmons L D L Court and A Fire Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans Gene 2001 263 1 2 p 103 112 Tabara H A Grishok and C C Mello RNAi in C elegans Soaking in the genome sequence Science 1998 282 5388 p 430 431 Ashrafi K et al Genome wide RNAi analysis of Caenorhabditis elegans fat regulatory genes Nature 2003 421 6920 p 268 272 Hamilton B et al A systematic RNAi screen for longevity genes in C elegans Genes amp Development 2005 19 13 p 1544 1555
51. 4 Front panel of the LabVIEW custom program for tracking illumination and video acquisition The program controls all functions described in this section 4 3 4 3 1 Image Acquisition For our system a Guppy AVT firewire camera was chosen to be used This camera utilizes a 1 3 format CMOS chip and can acquire images at 640x480 at a maximum rate of 60 Hz We found that using a firewire camera rather than a USB camera frees up resources on the main motherboard and due to the increased data transfer rates it can more accurately maintain the set rate 25 Hz The live images I are acquired at a resolution of 640x480 and are resized to 320x240 this done to increase processing time in future program modules At the objective magnification 4x and C mount camera coupler 0 4x the measured calibration was 6 6 um pixel For each acquired image a binary image was created I by performing a simple threshold process to the live image In most cases we kept the parameters of the binary image creation to be 1 for pixel 84 values between 0 100 and equal to O for pixel values of 101 255 Fig 4 5 The bright field illumination intensity is adjusted such that the binary image is an accurate representation of the animal 320x240 2X 8 bit image 177 231 229 ie J CameraAttributes AutoExposure Mode Attribute Value Ignored Set Image Options 2x2 Image Size _ X Resolution 320 Y Resolution z0 vi 32
52. 5 mm x 2 29 mm yielded the average intensity across the entire projected image To correct for the illumination intensity within the central region we multiplied this average intensity value by the ratio of the average value of normalized intensity across the entire projector image 0 882 to the average value of normalized intensity across the central region of the projector image 0 977 This yielded a correction factor of 1 11 The values reported in the text are the centrally corrected intensity and any references to intensity have an uncertainty of about 3 Spectral measurements were made using a USB 1 spectrometer Thorlabs 70 a 768 Relative Intensity 704 E 0 94 1 00 640 c E 0 88 0 94 O 576 E 0 82 0 88 a W E 0 76 0 82 4 E 0 70 0 76 y E 0 64 0 70 320 p oO 256 o 192 128 64 0 Oo st 0 N wo Oo q co N wo Oo t 0 N wo O st wo N a wo N co t a Tt Oo wo m a wo N a a m m Tt n w wo N o fon Q Projector X axis position b c Relative Intensity Relative Intensity 0 128 256 384 512 640 768 896 1024 Projector X axis position Projector Y axis position Figure 3 6 Measuring the uniformity of illumination across the entire projector image 117 a Heat map showing the variation of intensity across the 1024x768 pixel projector image Black dotted line represents the field of view of the camera The red dotted circle represents the central region 1mm diameter where the
53. ALM PVM and RIM using cell specific RNAi sensitive strains sid 1 213 6 2 3 Utilize microfluidics optogenetics and calcium imaging techniques for exploration of integration sensory information between distinct neural circuits In C elegans there are a variety of sensory modalities Though we can attempt to isolate and understand individual neural networks responsible for each sensory modality it would be of great interest to examine the relationship and inter sensory processing between these senses Organisms are subjected to a wide range of stimuli and must choose an appropriate response In this section the goal is to investigate the integration of distinct sensory modalities by combining optogenetics and microfluidics and observe 134 both behavioral response as well as use calcium imaging as an indicator of neural activity C elegans are known to migrate to temperatures on which they were cultivated 245 The neural circuit for thermosensation 246 is distinct from the mechanosensory circuit though there are some overlapping interneurons and motor neurons In this section the behavioral response would be investigated when animals are placed on a temperature gradient and allowed to freely move while at the same time optogenetically exciting the escape response via the touch neurons Fig 6 2 As the animals enter certain regions of temperature one would stimulate anterior touch cells and observe the behavioral response
54. AP axis position Once this element is found obtaining i the correspond point x y is retrieved as well as the i 3 and i 3 points From these three points a least square line is determined A line is then drawn extending through the point 91 x yl and at a right angle to the previously determined line In this manner a line extending through the desired segmentation point and normal to the AP axis is found The length of this line is determined by user input Width Fig 4 8 and should be set to a value slightly larger than the width of the animal around 15 20 pixels This is repeated for all values in the input panel equal to the number of segments minus one S 1 To decrease processing time the total number of segments should be kept as low as needed for the particular experiment In other words if you wish to illuminate a segment between 0 5 and 0 55 it would be best to choose three segments with divisions of 0 0 5 0 55 and only utilize the middle segment rather than choose 20 equally divided segments and utilize the 11 segment From the defined line segments a binary image is created consisting only of these segmentation lines a A binary image of the segmented animal a is then found by the following image logic operation Fig 4 7d y g JSA JB N JSL The final image I now contains S individual segments and are labeled from 1 to S where the 1 segment contains the head and the s segment contains th
55. AUTOMATED MICROFLUIDIC SCREENING AND PATTERNED ILLUMINATION FOR INVESTIGATIONS IN CAENORHABDITIS ELEGANS NEUROSCIENCE A Dissertation Presented to The Academic Faculty by Jeffrey N Stirman In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Bioengineering program in the School of Chemical amp Biomolecular Engineering Georgia Institute of Technology May 2012 AUTOMATED MICROFLUIDIC SCREENING AND PATTERNED ILLUMINATION FOR INVESTIGATIONS IN CAENORHABDITIS ELEGANS NEUROSCIENCE Approved by Dr Hang Lu Advisor School of Chemical amp Biomolecular Engineering Georgia Institute of Technology Dr Athanassios Sambanis School of Chemical amp Biomolecular Engineering Georgia Institute of Technology Dr Jennifer Curtis School of Physics Georgia Institute of Technology Dr Philip Santagelo School of Biomedical Engineering Georgia Institute of Technology Dr Alexander Gottschalk Institute of Biochemistry and Frankfurt Institute for Molecular Life Sciences FMLS Johann Wolfgang Goethe University Frankfurt Frankfurt Germany Date Approved 5 December 2011 ACKNOWLEDGEMENTS I would like to foremost thank my advisor Dr Hang Lu who graciously accepted me into her lab She had provided me with invaluable guidance support and when needed freedom to explore I have truly enjoyed my experience as her graduate student and am grateful to her I express my gratitude to m
56. Check that all lenses are located as described in Figure 1 Alter the LabVIEW code to communicate with the specific 24 C ix Program gives error upon start was written for Altered camera from what program was written for Altered motorized stage from what program was written for 154 camera used See also supporting documentation of Ref 12 Alter the LabVIEW code to communicate with the specific camera used See also supporting documentation of Ref 12 Alter the LabVIEW code to communicate with the specific stage used See also supporting documentation of Ref 12 APPENDIX C ADDITIONAL CONTRIBUTIONS In addition to the research presented in the preceding chapters there was some additional work of interest Dr Guy Benian s Emory University Department of Pathology research focuses on muscle physiology and utilizes the nematode C elegans for his investigations Over the years he has discovered a number of mutants defective in some aspect of their musculature Though these mutants have altered phenotypes when looking muscle structure electron microscopy and immunohistochemistry no easily observable behavioral phenotype was observed Along with Gina Cremona Lu lab Georgia Institute of Technology and Dr Hang Lu we developed a quantitative assay that could distinguish many mutants from wild type In this assay animals are prodded on their head with a platinum wire this excites the harsh touch escape response a
57. Fig 1 7c Simultaneously they imaged calcium transients in downstream neurons The microfluidics not only provided a means of defining the stimulus it also provided a means to immobilize the animal restrictive immobilization well enough to acquire high magnification fluorescent images 27 iy _ o _ iz uaBAxO Q O 4 2 D zi D 2 O fo S Radius uol es UBDU0D JOPO High Left Right 2 Time Worm Loading Area worm nose PDMS 7 Bacteria Targets Figure 1 7 Microfluidics for precise control of the external environment 189 a Attractive and aversive bacteria are assessed within a microfluidic decision arena b A precise O2 gradient is established in a microfluidic device allowing for evaluation of preferred O2 concentration c Laminar flow in a microfluidic channel can precisely stimulate sensory neurons in C elegans Microfluidics when combined with automated image processing has been shown to greatly increase the speed and efficiency of handling imaging analysis and sorting of cells and small organisms like C elegans 142 186 188 200 204 Controlling the size of microfluidic features and the fluid flow properties allows the control distribution and manipulation of C elegans within the microfluidic device Hulme et al have previously demonstrated a microfluidic device for isolation and imaging of multiple individual worms 186 This device utilizes microfl
58. GUI easy to operate Furthermore several user guides were made to explain the usage of both the microfluidics and the illumination system The microfluidic master control box Chapter 2 7 that I designed and constructed was a direct consequence of the need to have a user friendly end product that could be given to our collaborators all over the world This represents a significant improvement on the initial loose collection of unreliable valves wires tubing and often failing regulators technologies so unreliable that transfer of early systems would be nearly impossible Although the development of the discussed technologies is very satisfying I have found the transfer of the technologies to others who find them valuable to be equally satisfying and perhaps a more telling descriptor of the impact and contribution of this thesis research 157 p 10 11 12 13 14 REFERENCES Brenner S Genetics of Caenorhabditis elegans Genetics 1974 77 1 p 71 94 Brenner S Autobiography nobelprize org accessed 9 25 2011 Cassada R C and R L Russell The dauerlarva a post embryonic developmental variant of the nematode Caenorhabditis elegans Developmental Biology 1975 46 2 p 326 342 Byerly L R C Cassada and R L Russell The life cycle of the nematode Caenorhabditis elegans I Wild type growth and reproduction Developmental Biology 1976 51 1 p 23 33 Sulston J E and H R Horvitz Post embryonic
59. Hang Lu High Throughput Study of Synaptic Transmission Enabled by Optogenetics and Microfluidics Neuroscience Topic Worm Meeting 2010 Jeffrey N Stirman and Hang Lu Microfluidic Platforms for Neuroscience and Systems Biology TRAM Method Seminar Series Biocenter Campus Riedberg Max von Laue Str 9 Frankfurt a M 2010 Invited presentation Kwanghun Chung Jeffrey Stirman Matthew Crane and Hang Lu Large Scale In Vivo Genetic Screens and Laser Microsurgery Enabled by Automated Microsystems Association for Laboratory Automation 2009 139 POSTER Steven J Husson Jana F Liewald Jeffrey N Stirman Hang Lu Alexander Gottschalk Microbial proton pumps as hyperpolarizers complement the optogenetics toolbox in Caenorhabditis elegans 18 International C elegans Meeting 2011 Matthew M Crane Peri T Kurshan George J Wang Jeffrey N Stirman Kang Shen Hang Lu Computer automated forward genetic screening using sub cellular fluorescent reporters 18 International C elegans Meeting 2011 Sebastian Wabnig Jasper Akerboom Jeffrey N Stirman Hang Lu Loren Looger Alexander Gottschalk Addition of the genetically encoded red shifted Ca sensor RCaMP to the C elegans optogenetic toolbox 18 International C elegans Meeting 2011 Karen Erbguth Matthias Prigge Franziska Schneider Jeffrey N Stirman Hang Lu Peter Hegemann Alexander Gottschalk Red shifted optical excitatio
60. ICS MACHINE VISION AND LAB AUTOMATION FOR HIGH THROUGHPUT OPTOGENETIC SCREENING Much of the work presented in this chapter was originally published 142 Stirman et al Microfluidic system for high throughput studies of synaptic functions using ChR2 Journal of Neuroscience Methods 2010 191 1 90 93 Additionally reference to the original publication of the associated figure can be found in the figure caption 2 1 Motivation and Overview Over the past several years optogenetic techniques have become widely used to help elucidate a variety of neuroscience problems The unique optical control of neurons within a variety of organisms provided by optogenetics allows researchers to probe neural circuits and investigate neuronal function in a highly specific and controlable fashion Recently optogenetic techniques have been introduced to investigate synaptic transmission in the nematode Caenorhabditis elegans 140 For synaptic tranmission studies although quantitative this technique is manual and low throughput 140 As it is this technique is difficult being applied to large scale genetic screens In this chapter we enhance this new tool by combining it with microfluidics technology machine vision and lab automation This allows us to increase the assay throughput by a couple of orders of magnitude as compared to standard approach currently We also demonstrate the ability to infuse drugs to worms during optogenetic experiments us
61. International C elegans Meeting 2011 Invited presentation Steven J Husson Wagner Steuer Costa Jeffrey N Stirman Joseph D Watson W Clay Spencer Millet Treinin David M Miller II Hang Lu Alexander Gottschalk Optogenetics dissection of the nociceptive PVD network RNAi of PVD specific genes reveals TRP channels as signal amplifiers 18 International C elegans Meeting 2011 Matthew M Crane Jeffrey N Stirman and Hang Lu Autonomous synaptogenesis screening via SVM generated quantitative phenotypical space Cold Spring Harbor Lab Automated Imaging and High Throughout Phenotyping 2010 Husson S J Steuer Costa W Stirman J N Watson J D Spencer W C Miller D M Treinin M Lu H Gottschalk A Optogenetics assisted functional analysis of a PVD mediated nociceptive neuronal network in Caenorhabditis elegans European Worm Neurobiology Meeting 2010 Husson S J Steuer Costa W Stirman J N Watson J D Spencer W C Miller D M Treinin M Lu H Gottschalk A Optogenetics assisted functional analysis of a harsh touch nociceptive neuronal network in Caenorhabditis elegans Structure and Function of Neuronal Circuits EMBO EMBL symposium 2010 Jeffrey N Stirman Matthew M Crane Alexander Gottschalk and Hang Lu Spatial and temporal optical activation of neurons in freely behaving worms Neuroscience Topic Worm Meeting 2010 Jeffrey N Stirman Alexander Gottschalk and
62. M et al Green fluorescent protein as a marker for gene expression Science 1994 263 5148 p 802 805 Boulin T J F Etchberger and O Hobert Reporter gene fusions in WormBook T C e R Community Editor WormBook Fire A S W Harrison and D Dixon A modular set of lacZ fusion vectors for studying gene expression in Caenorhabditis elegans Gene 1990 93 2 p 189 198 Gregory J P Green fluorescent protein a bright idea for the study of bacterial protein localization FEMS Microbiology Letters 2001 204 1 p 9 18 Mello C C et al Efficient gene transfer in C elegans extrachromosomal maintenance and integration of transforming sequences Embo Journal 1991 10 12 p 3959 3970 Evans ed T C Transformation and microinjection in WormBook T C e R Community Editor WormBook Horvitz H R et al A uniform genetic nomenclature for the nematode Caenorhabditis elegans Molecular and General Genetics MGG 1979 175 2 p 129 133 160 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 11 78 Hodgkin J Nomenclature http wiki wormbase org index php UserGuide Nomenclature accessed 10 12 2011 Consortium C e S Genome sequence of the nematode C elegans A platform for investigating biology Science 1998 282 5396 p 2012 2018 Ward S et al Electron microscopical reconstruction of the anterior sensory anat
63. Nat Neurosci 2008 11 8 p 865 867 Gray J M J J Hill and C I Bargmann A circuit for navigation in Caenorhabditis elegans Proc Natl Acad Sci USA 2005 102 p 3184 3191 Nagel G et al Channelrhodopsin 2 a directly light gated cation selective membrane channel Proc Natl Acad Sci USA 2003 100 24 p 13940 13945 170 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 Husson S J et al Optogenetic analysis of a nociceptor neuron and network reveals modulatory ion channels acting downstream of nociceptive sensors in review Way J C and M Chalfie The mec 3 gene of Caenorhabditis elegans requires its own product for maintained expression and is expressed in 3 neuronal cell types Genes amp Development 1989 3 12A p 1823 1833 Chatzigeorgiou M and W R Schafer Lateral Facilitation between Primary Mechanosensory Neurons Controls Nose Touch Perception in C elegans Neuron 2011 70 2 p 299 309 Kaplan J and H Horvitz A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans Proceedings of the National Academy of Sciences of the United States of America 1993 p 2227 2231 Wicks S R and C H Rankin Integration of Mechanosensory Stimuli in Caenorhabditis elegans Journal of Neuroscience 1995 15 3 p 2434 2444 Rankin C H Interactions between 2 antagonistic reflexes in the nematode Caenorhabditis elegans Jo
64. Plos Genetics 2008 4 3 Wang S et al All optical interface for parallel remote and spatiotemporal control of neuronal activity Nano Letters 2007 7 12 p 3859 3863 Grossman N et al Multi site optical excitation using ChR2 and micro LED array Journal of Neural Engineering 2010 7 1 169 219 220 221 222 223 224 225 226 221 228 229 230 231 232 233 234 235 236 237 Delica S and C M Blanca Wide field depth sectioning fluorescence microscopy using projector generated patterned illumination Applied Optics 2007 46 29 p 7237 7243 Itoga K et al Cell micropatterning using photopolymerization with a liquid crystal device commercial projector Biomaterials 2004 25 11 p 2047 2053 Aravanis A et al An optical neural interface in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology Journal of Neural Engineering 2007 p S143 S156 Campagnola L H Wang and M J Zyka Fiber coupled light emitting diode for localized photo stimulation of neurons expressing channelrhodopsin 2 Journal of Neuroscience Methods 2008 169 1 p 27 33 Schoenenberger P et al Optimizing the spatial resolution of Channelrhodopsin 2 activation Brain Cell Biology 2008 36 1 4 p 119 127 MathWorks R201 1b Documentation Image Processing Toolbox http www mathworks com help toolbox images accessed 19 Novembe
65. R 30 20 10 0 2 0 29mW mm 1 17mWmm 4 67 mW mm Intensity of Illumination 520 msec pulse 2 1 0 29 mW mm 40 for each 1 41 17 mW mm 2 of animals n a 2 4 67 mW mm Figure 5 9 Quantification of behavioral responses elicited by different anterior illumination intensities Adapted from REFERENCE 117 a Patterns used for illumination location and their intensity b Distribution of the four responses observed at the three intensity levels n 40 for each of the three illumination levels Next we asked whether it is possible to simultaneously stimulate neurons in spatially distinct locations and with sophisticated light intensity patterns We were interested in the animals responses to simultaneous stimuli in anterior and posterior regions i e past which intensity threshold a reversal is produced and how this changes when a competing signal is present something that would be very difficult to address using the traditional manual approach We compared pmec 4 ChR2 animals that were stimulated only in the head in an increasing step function of illumination intensity to a maximal intensity of 1 17 mW mm Fig 5 10a to animals stimulated with an identical pattern in the head but that additionally were being stimulated in the tail at a constant intensity 1 17 mW mm Fig 5 10b When the threshold of intensities when an animal initiates a reversal for a population of animals were compi
66. RNAi Furthermore the main purpose of washing the animals is to remove bacteria from the suspension Bacteria cause significant clogging in microfluidic devices and can cause problems for the image processing steps Animals cultured in liquid culture 51 would still need to be washed and diluted appropriately and would therefore save little time The most significant amount of time involved in the process of data collection is acquiring a sufficient number of animals for statistical significance To explore the possibility that fewer animals are needed we performed a power analysis test Based on the data collected experimentally Fig 2 5 we found a standard deviation of 2 08 before the illumination and 2 94 after the illumination was initiated The standard deviation during the illumination period is the most important factor in the power analysis and therefore we use 2 94 as the sigma value for both populations In addition to this we would need to know the absolute difference in the means of the animal populations In the investigations using the mutant animals Fig 2 5 a difference of 6 was observed between zx s6 and unc 49 zxIs6 This however is an extreme case and much smaller differences are expected If we choose an expected or desired difference of 2 5 true difference of means and perform a power analysis test two tailed t test a 0 05 we see that for a predictive power of 0 8 80 an n value of 23 n 46 total spli
67. USB input main air inlet power supply connector and power switch c Within the control box there are both pneumatic and electronic connectors the micro solenoid valves and the USB control board 2 7 1 Components Pressurized air enters the back of the control box This is split into four lines 1 A set of four solenoid valves regulated at 0 60 psi 2 a second set of four solenoid valves regulated at 0 60 PSI 3 a set of two solenoid valves monitored and regulated at 0 15 49 PSI and 4 an unvalved outlet regulated 0 15 PSI The miniature solenoid valves are Asco Series 188 valves Part 18800056 and were selected based on their ability to actuate at high pressure up to 115 PSI and their reliable performance We have previously tested Lee Company and Hargraves valves and found their performance to be unreliable and they ultimately fail after routine usage of 3 6 months The pressure regulators utilized are Polysulfone Panel Mount Pressure Regulator McMaster part numbers 43275K13 and 43275K14 and the gauges are Panel Mount Multipurpose Gauge McMaster part numbers 3846K41 and 3846K43 The valves gauges and regulators are connected through push to connect fittings and 5 32 tubing The power and control for the solenoid valves and the external ports are supplied by a USB output driver board PacDrive Ultimarc The board is controlled by DLL calls written in LabVIEW Matlab or other custom programs The completed control box
68. We expect the normal behavioral response due to mechanosensation to be altered depending on the temperature in which animals currently reside and the cultivation temperature further enhancing the escape response when on a temperature not to their liking It will also be interesting to determine if an animal can be trained to reside at temperatures other than those they were cultivated on based on negative feedback applied though optogenetic excitation of the mechanosensory escape response This would require the illumination system described in 6 3 1 3 135 15 C 25 C Fig 6 2 Investigation of sensory integration between the optogenetically elicited mechanosensation and thermosensation In this thesis Chapter 2 we demonstrated combining microfluidic technologies and optogenetics for the high throughput analysis of synaptic function at the C elegans neuromuscular junction 142 Combining microfluidics and calcium imaging has been demonstrated to study the neural and genetic basis of odor detection 99 and oxygen sensation in C elegans 247 Recent work demonstrated the use of microfluidics for precise spatial and temporal definition of chemical attractant while monitoring C elegans behavior 248 One can use a microfluidic device 99 to apply a chemical attractant such as sodium which is primarily sensed by ASE and using Ca imaging can monitor the downstream interneurons AIB ATY RIA RIM as well as those interneuron
69. about 3 5 days from fertilization to adulthood 1 and the average life span is 2 3 weeks 3 The rapid developmental time and the large brood size of a hermaphrodite about 300 per cycle means a large number of animals can be generated in a short period of time ideal for genetic biochemical and developmental analysis C elegans are easily kept and maintained in a laboratory setting typically grown on agar plates seeded with a bacterial food supply Furthermore C elegans has a number of features which though might have not been known initially have emerged as powerful aspects in C elegans research The most informative way to illuminate the biological power and to introduce some important features of C elegans is to discuss the Nobel Prizes that have been awarded to researchers utilizing this small yet powerful nematode 1 1 1 C elegans Development In 2002 the Nobel Prize in Physiology or Medicine was awarded to Sydney Brenner Robert Horvitz and John Sulston for their discoveries concerning genetic regulation of I organ development and programmed cell death nobelprize org Dr Sulston and Dr Horvitz were postdoctoral researchers in Dr Brenner s lab in the 1970 s Their work focused on the lineage tracking of cells in larval development The life cycle of C elegans consists of embryonic development and post embryonic development consisting of four larval stages and adulthood and the timing of development in temperat
70. al unc 49 e407 43 45 30 i zxls6 amp S amp F F EK OF PF S NS F X SH 1 2 Ss F L y Time s X Da Figure 2 5 Contraction and relaxation of C elegans muscles under photoactivation of motor neurons 142 a Changes in body length over a 7 second interval Body length is relative to an average of the body length 2 seconds prior to blue light illumination Blue light was turned on at t 0 b Mean relative body length measured 2 seconds after continuous blue light illumination t 2 seconds The numbers of individual animals tested are indicated at the bottom of the bars Blue bar indicates time when blue light illumination is present ATR was added to the growth media to yield functional ChR2 unless otherwise indicated Error bars are s e m An additional advantage of using the microfluidic setup is the ability to infuse drugs during experiments To demonstrate the utility of this microfluidic chip we combined the stimulation of the neurons with ChR2 along with exposure of the animal to a soluble drug Nicotine is a known acetylcholine receptor agonist present on the muscle cells at the NMJ that causes a depolarization and thus contraction of the muscle cells 209 The microfluidic device was used to deliver M9 buffer containing nicotine to zx s3 worms this strain relaxes under blue light illumination Within seconds of the nicotine stimulation the worms began to contract Fig 2 6 n 49 rapidly at first and then
71. al represents the unique two letter prefix of the lab that originally created or isolated the strain flowed by a number This two letter code is usually different from the two letter code designated for extra or integrated constructs For instance the strain KG1180 genotype is lite 1 ce314 and indicated the unique mutation in the lite 1 gene originally found by the Miller lab designated by KG Another example is AQ2334 which has the genotype lite 1 ce314 jIs123 pmec 4 ChR2 punc 122 RFP this strain describes an animal containing the lite 1 genotype that has been injected and integrated with a chromosomal array ljIs123 both AQ and Jj designate the Schafer lab This chromosomal array has the channelrhodopsin 2 ChR2 gene downstream from the mec 4 promoter promoter designated by the prefix p as well as the gene encoding RFP downstream from the unc 122 promoter Creation of the chromosomal array is accomplished through microinjection of two plasmids one containing the ChR2 construct and another containing the RFP construct into the KG1180 strain thus giving the lite 7 background After microinjection animals are selected either through behavioral phenotyping or usually through co injection markers which can either be fluorescent such as the punc 122 RFP construct or a phenotype rescue marker such as lin 15 These animals can either be maintained as extra chromosomal arrays by selecting animals with the appropriate phenotype or t
72. ally reference to the original publication s of the associated figure can be found in the figure caption 3 1 Motivation and Overview Recently there has been significant interest in optically targeting optogenetic reagents for non invasive excitation and inhibition of cultured cells 124 125 130 138 139 and neurons and muscles in small model organisms such as the nematode Caenorhabditis elegans 74 117 122 137 140 144 the fruitfly Drosophila melanogaster 145 148 the zebrafish Danio rerio 136 149 153 and mice 133 134 154 157 As discussed in detail in Chapter 1 optogenetic reagents are light gated ion channels and pumps and when expressed in excitable cells neurons and muscles illumination with the appropriate wavelength of light cause depolarization e g Channelrhodopsin 2 or ChR2 130 or hyperpolarization e g Halorhodopsin or NpHR 124 MAC and Arch 125 of the cell In cultured cells and small model organisms the ability to excite or inhibit a subset of the cells would allow for probing circuits and functions in real time However there are few single cell specific promoters in C elegans and thus optogenetic reagents are generally expressed in a larger population of cells Although there are techniques for single cell expression including Cre lox 215 and FLP recombinase 216 these can be unreliable or do not allow for sufficient expression of optogenetic reagents Furthermore 56 to investigate int
73. als zx s6 and zx s3 in a neuronal RNAi sensitive background nre 210 We screened 90 genes for both strains using the microfluidic device and liquid handler discussed above The 90 genes were chosen based on positive hits from an aldicarb resistance screen done previously 40 and were fed to C elegans following standard methods 27 From this screen about 70 of the genes screened from the zx s6 cholineregic line were found to have some synaptic transmission defects Additionally several positive controls known synaptic transmission mutants were included as well as negative controls both empty vector and no ATR An example of a length time series plot is shown below Fig 2 10 In this example animals were fed RNAi for unc 11 an AP180 clathrin adaptor homolog involved in endocytosis 211 It was previously demonstrated that a unc 11 e47 mutant demonstrated enhanced contract when assessed by OptloN 140 Using the RNAi knockdown and the microfluidics we also observe this phenotype Fig 2 10 however because it is involved in synaptic vesicle recycling it is expected that the contraction would gradually relax during the experimental time A Gottschalk personal communication This was not observed leaving some questions about the ability of the RNAi to phenocopy the genetic mutant Although many of the RNAi tested demonstrated a difference from the control empty vector the results were largely inconclusive the reasons and limit
74. analyze the video and extract the discussed parameters 4 7 Conclusions The goal of this research was to create a comprehensive set of software for control of the illumination system described in Chapter 3 The software needed to posses sufficient speed and accuracy to illuminate intended targets within a freely moving animal Furthermore the software should possess the ability to automatically locate anatomical 101 structures within the animal and illuminate those areas As demonstrated in this chapter the designed software meets the above stated goals The software acquires images of animals freely moving on an agar plate and through image processing routines determines the AP axis of the animal and hence can locate a priori known anatomical structures The software can then instruct the projector the illuminate user determinable locations of the animal with full control over the intensity color and duration of the illumination Subsequent programs are used for extraction of a number of parameters associated with C elegans locomotion and are used for quantitative phenotyping The final programs are designed for flexibility and a friendly user interface such that non programming experts find them easy to use 102 CHAPTER 5 DEMONSTRATION OF ILLUMINATION SYSTEM FOR NEURAL CIRCUIT DISSECTION Much of the work presented in this chapter was originally published 117 Stirman et al Real time multimodal optical control of neur
75. and the distributions of the anatomical positions of the touch neurons in pmec 4 GFP animals Figure 5 4 107 This experiment suggests that the resolution of the system can be used for precise interrogation of the neuronal network at the single cell level if used in combination with appropriate promoters that drive ChR2 expression in single cells e g cre lox system 230 and provided that the cells expressing ChR2 have cell bodies or processes farther apart than the spatial resolution of the system PLML R oor r E A r a F ALML R aine ane PLMU R Ai r Fag Figure 5 4 Representative fluorescent images of pmec 4 GFP animals 117 Images were processed in the same manner as in all experiments and backbone of the animal determined The gentle touch sensory neurons were identified and quantified as a location along the AP axis defined by the backbone This data was used in Figure 5 3 In the C elegans touch circuit command interneurons integrate signals from sensory neurons and ultimately produce locomotor behaviors 66 114 228 231 Fig 108 1 2 To quantify these behaviors we excited the head or tail touch neurons and the head or tail interneurons using ChR2 and the structured illumination system and measured the animals velocity First pmec 4 ChR2 animals Fig 5 5a were stimulated either in the second 25 or the last 25 of the body We illuminated a quarter of the body length beca
76. arget specific areas supporting methods must be developed to increase the processing power of optogenetic screens and software for control of developed hardware must be made flexible and approachable for all users Due to its relative neural simplicity and the wealth of resources available C elegans is a popular model organism for neuroscience research The use of optogenetics in C elegans research has seen a vast increase over the past several years and has been utilized to study synaptic function neural basis of behavior transfer characteristics of synaptic connections mating behavior among several of areas of neurobiology Thus optogenetics is a powerful and rapidly emerging technique for investigations of the nervous system in C elegans These studies will only increase in sensitivity complexity and throughput as corresponding advances in the hardware and software are developed This thesis seeks to enhance the optogenetic toolbox through the design construction and evaluation of a number of hardware and software modules for research in C elegans neuroscience In the first aim we combine optogenetics microfluidics and automated image processing to create a system capable of high throughput analysis of synaptic function Furthermore the system was further enhanced by combining it with a commonly used liquid handling system for increased processing power In the second XIV aim we develop a multi modal illumination system for
77. as well as the observation of 15 protofilament microtubules 110 The anterior neurons ALML R and AVM comprise the anterior sensory field and upon stimulation by touching the anterior portion of the animal the animal responds with a rapid reversal 109 The posterior sensory field is made from the PLML R neurons and upon mechanical stimulation of the posterior the animal responds with rapid forward acceleration 109 The PVM neuron is not implicated in the mechanosensory response 66 111 The complete mechanosensory wiring diagram Fig 1 2 has been reconstructed through genetic and optical methods as well as the reconstructed wiring diagram of White 62 Both gap junctions and chemical synapses connect the sensory level to the internuerons as well as from the interneurons to the motor neurons The interneurons responsible for forward movement receive connection from both anterior and posterior sensory fields as do the interneurons responsible for reversals This would indicate that the interneurons receive antagonistic signals compare and integrate the signals ultimately leading to the appropriate behavioral response Mechanosensory behavioral assays are traditionally performed by touching the animal with an eyelash attached to a pipette tip or toothpick 109 The location of the sensory stimulus is determined by the researcher as they manually track the animal The duration and intensity of the stimulus is also determined by the ability of t
78. ashion To achieve efficient operation a number of external pinch valves Cole Palmer were incorporated for automated flushing and washing of the microfluidic device between processing of populations of animals A schematic of the integrated system is shown in Figure 2 8 42 Q Pinch Valve PV Microfluidic Chip Q s VA En PV mance Gilson 215 Liquid Handler P LERLE wus Figure 2 8 Schematic of the automated robotic system integrating a Gilson 215 liquid handler system microfluidic device and external control components We chose to use negative pressure applied at the outlet to draw animals into the device rather than positive pressure This simplifies integration with the liquid handler as no pressure sealing with the multi well plate has to be achieved Negative pressure was applied with a vacuum pump Bio rad HydoTech and controlled with a vacuum regulator McMaster A second vacuum source was located at the entrance to the microfluidic device connected with a 20 gauge Y connector Smallparts This second source is able to draw animals and wash fluid into the main entrance tubing without having to go through the device This decreases flow resistance and speeds the time for animal loading Additionally after a sufficient number of animals are processed the remaining animals that are in the entrance tubing are drawn into the entrance vacuum port bypassing the device and decreasing clogging rates The schedule for each
79. ations of the system and analysis are discussed in Chapter 2 7 We found an average processing time of 15 minutes per population easily allowing 24 populations to be processed in a day The washing routine utilized 46 demonstrated sufficient clearing of animals as well as air bubbles Although many of the RNAi tested demonstrated a difference from the control empty vector the results were largely inconclusive The limitations of the system and analysis are discussed in Chapter 2 7 1 02 Empty Vector Control ad unc 11 RNAi d 0 92 Normalized body length g S R 8 ee a S a OE a a O S O O G 5 0 2 5 0 0 2 5 5 0 7 5 10 0 12 5 15 0 17 5 20 0 22 5 25 0 27 5 30 0 32 5 35 0 37 5 40 0 42 5 45 0 47 5 50 0 52 5 55 0 57 5 60 Time seconds Figure 2 10 Example of a length time series plot from the preliminary RNAi screen Blue illumination was turned on at t 0s and lasted until t 60s Enhanced contraction of animals is observed from knock down of the unc 1 gene encoding a clatherin adaptor protein AP180 which functions in clatherin mediated endocytocis 211 Error bars represent s e m 2 6 C elegans culture All worm strains used in this study were grown at 22 C in the dark on standard nematode growth medium NGM plates with OP50 bacteria All trans retinal ATR is a required cofactor for channelrhodopsin and must be supplimented to C elegans in order to have active channelrhodopsin Those experiments usin
80. bles connecting 141 5I 6l 7 Sl the top control panel to the main circuit board two cables for the Hitachi CP X605 Disconnect these cables from the main unit Fig 3 4c The case cover can now be completely removed and set aside TROUBLESHOOTING The topmost metal casing is the LAN board Disconnect the large set of blue wires connecting the LAN board to the main circuit board Locate the four screws holding the LAN board down and unscrew There is also a black grounding wire connected to the left side of the LAN board that should be disconnected The LAN board can now be carefully removed and set aside Fig 3 4d A CRITICAL STEP We suggest that a photograph of the projector and the location of the wires is obtained before disconnecting to assist with accurate reassembly later You will now be able to see all the wires connecting to the main board as well as the three LCD panel connections Fig 3 4e Disconnect all wires taking note where the wires were connected Unlatch the LCD panel cable connector and slide out the LCD panel cable from the main board Fig 3 4f There are three screws on the right side of the main board which need to be removed as well as an additional one on the back left of the metal bracket connected to the main board A CRITICAL STEP We suggest that a photograph of the projector and the location of the wires is obtained before disconnecting in order to assist with accurate reassembly later Remove t
81. ce has 16 parallel imaging channels Fig 2 7 connected to a single inlet and a single outlet The field of view of the imaging system is 3 2 mm by 2 4 mm when imaged with a 4x objective red box Fig 2 7a Fig 2 7b The inlet of the device consists of a diverging channel 1 2 4 8 16 this 41 design serves to distribute the animals among the imaging channels The large chamber outlets allow for rapid animal unloading and minimize clogging The operation of the device is similar to previously discussed 2 a Figure 2 7 Device for increased throughput a Dye filled device showing flow layer green and control layer red Loading in this device is left to right in the direction of the arrow b Zoomed image of red region from a showing loaded C elegans Scale bars are 500 um 2 5 2 Integration of microfluidic device and liquid handler In order to increase the throughput as well as decrease burden on the end user we have integrated an automated liquid handler and the microfluidic system The general idea is that a number of washed and diluted populations 6 24 of animals can be loaded into a multi well plate The animal populations can be individually addressed in the multi well plate by the Gilson 215 liquid handler The animals are drawn into the microfluidic device through negative pressure at the outlet and can be sequentially processed with very little dead time between and performed in an automated f
82. cell expression is often difficult therefore whole body illumination generally does not permit the cell specificity required to interrogate circuits at the single neuron level To truly understand a specific circuit one would ideally probe multiple distinct nodes cells with temporally separate signals There have been a few techniques that demonstrate restricted illumination capable of targeting individual neurons or sub areas of cells Two techniques involve the use of two photon microscopy coupled with temporal focusing 138 or generalized phase contrast combined with temporal focusing 139 It has been also been shown that in constrained worms ChR2 can be used to stimulate specific spatially separate neurons while Ca transients are recorded from neurons connected to them using a commercial digital micromirror device DMD 170 22 These techniques allow the interrogation of neural circuits in a cell specific manner and greatly advance the state of the art for optogenetic illumination However these techniques for precise and localized illumination are quite expensive and relatively difficult to implement for laboratories without significant experience in optical design Furthermore many behavioral neuroscience problems would additionally benefit from the ability to control and monitor a particular behavior in a freely moving animal 1 3 Microfluidics Microfluidics is a very general term that refers to the manipulation control and
83. city mm s PVD Velocity mm s AQR Velocity mm s Tail neuron 0 4 0 4 ea 0 4 14s light zxis12 1s light 7xI812 0 3 0 3 No ATR ATR n 18 No ATR ATR n 15 0 24 n 19 i n 15 0 1 15 15 0 1 Time s 0 1 Time s 0 4 d PVD cell body e PVD cell body f PVD cell body p reaction Velocity mm s reaction Velocity mm s reaction Velocity mm s a 4s light 0 3 J 1s light ge 0 3 J 1s light soji so 02 al 2 40 0 1 4018 o1 P S 20 20 20 0 0 0 0 5 10 15 0 5 10 15 0 5 10 15 Time s Time s Time s D E F Figure 5 15 Illumination of PVD expressing ChR2 238 a Photoactivation of PVD b Photoactivation of the head neuron AQR c Photoactivation of the unknown tail neuron a c Red line indicated mean and gray shading indicates the s e m Number of animals tested is indicated in the figure d f Photostilulation of the dendridic branches of PVD Fractions of responding animals are indicated by the bar graphs d Illumination of region 0 4 0 6 n 28 e Illumination of region 0 3 0 5 n 22 f Illumination of region 0 2 0 4 n 30 g Maximal speed observed for the animals tested in d f Statistical significance was determined using a t test p lt 0 05 121 These results indicate that PVD is primarily responsible for initiating escape behavior through rapid acceleration forward When prodded by a platinum wire the primary response is rapid reversal 239 this suggests t
84. cross pattern is perfectly horizontal and vertical using the guidelines This can also be slightly offset to maximize the region where the projector image intensity is the flattest Camera values 81 can be adjusted to see the image clearly Successfully completing these steps will ensure that the object specimen plane and projection image plane are coincident Figure 4 1 Cross pattern used for projector alignment The camera is rotated such that it is aligned both horizontally and vertically and the projector is XYZ adjusted so the pattern is centrally located and in sharp focus 4 2 2 Coordinate Transformation After alignment click Continue on the program A grid of 20 solid circles is projected known center positions Xp Yp sequentially through the constructed optical system left side Fig 4 2 These images are seen on the target surface and imaged with the camera The locations of the projected circles are determined Xc Yc by measuring the center position of the threshold image right side Fig 4 2 After completion of this program a file is saved containing the translational offset and scaling factor used in the main program Upon successful completion a dialog box will pop up stating proper completion or error occurred If an error occurred check alignment and perform again This program is generally run throughout the day to ensure proper alignment is maintained throughout the experiments 82
85. d file within the main program the program sequentially creates the selected illumination image I from the saved parameters and sends these images to the projector for the set amount of time ts tt ad a ta ia aaa is gt HHH a ht Ca cece cine cine TC LCi cece eine cece cies Pole cc cies cle Bee eee a a wia aa a a TICC cee o o TC T A Is HEHE aT e TCT TIe Ta A ts gt A ef gt Figure 4 12 Control panel for setting complex custom illumination patterns In this example there are 4 segments and fours levels or time periods The first time period lasts 2 sec during which nothing is illuminated The second period lasts 3 sec and the second spatial segment is illuminated in blue The third time period lasts 3 sec and the first spatial segment is illuminated in green Finally the last time period is 2 sec long and again nothing is illuminated 95 4 5 Head Encode The purpose of this program is to encode the location of the head of the animal into the video This location information is used in the subsequent video analysis Several automated methods can be used to perform head tail discrimination but all have non zero failure rates The videos processed in this study Chapter 5 are relatively short lt 45 seconds and thus manually annotating the position of the head is an acceptable method Upon starting the program a dialog box will then prompt the user for a video file Selec
86. d from pixels to um based on the conversion factor Angles To measure the angles or curvature of the animal the animal is first descretized into a user defined number of segments S Each segment is of equal length measured along the contour of the animal s AP axis Segmentation of the animal is done as described in Sections 4 3 3 1 and 4 3 3 2 Points along the AP axis of the animal at each segmentation point are determined plus the points at the head and the tail yielding S 1 points describing the animal Fig 4 15d a Two point angles This measures the angle between two successive points along the animal relative to 90 degrees The angle is then normalized 117 such that the expectation value of all angles along the animal equal to zero Fig 4 15e The normalization of these angles subtracting the average of all measured angles from the angle serves to adjust the coordinate system such that it is in the frame of reference of the animal and thus the absolute orientation of the animal is ignored There are a total of S number of segments two point angles These angles can be used in subsequent eigenshape analysis as was performed in Reference 225 Three point angles This measures the angle between three successive points along the animal relative Fig 4 15f There are a total of S 1 three point angles 4 Average angles This is an average of the absolute values of the previous angles From the average angles
87. e excitation epifluorescent shutter as the projector can switch from full on pixel value 255 to full off pixel value 0 at a maximum rate of 60 Hz refresh rate of the projector and therefore we envision that this could also replace a shuttering system Lastly because the light intensity is defined by the value of the pixel from 0 to 255 8 bit the projector can also modulate the intensity of illumination and thus potentially replace neutral density filters 3 1 2 Comparison with other methods Details of other optogenetic illumination are addressed in Chapter 1 Here they are briefly reviewed and compared to the illumination system presented in this chapter Many of the existing techniques for optogenetic illumination are performed by positioning optical fibers in the vicinity of the target 149 169 221 222 statically focused laser illumination 223 or static shadowing of illumination regions 136 These methods are frequently imprecise or are performed in static samples limiting their applicability Current state of the art illumination systems involve the use of two photon microscopy 138 139 LED arrays 218 DLP digital light processing mirrors 137 143 217 or commercially available LCD projectors 117 148 to spatially restrict light and have the ability to dynamically alter illumination pattern These techniques allow for a high degree of light localization to target individual or groups of neurons or muscles and can fo
88. e tail thus keeping the order of the segments along the AP axis 4 3 3 3 Illumination Control Within the illumination control panel Fig 4 9 the controls for colors RGB for each defined segment RBGs are found The user can input a value 0 to 255 for each color and for each segment and thereby determine the relative intensity Fig 3 6 for that segment Also within the illumination control panel are buttons to turn illumination of the particular segment on or off Bs on 1 off 0 A preview of the desired illumination pattern is seen in the main panel Fig 4 4 Fig 4 11 under the Selected Illumination pane The switch between Simple and Scheduled Fig 4 9 determines the illumination scheme Simple uses the parameters set in the previously discussed 92 Illumination Control For Simple there are also options for Timed illumination and Untimed where the duration of illumination can be set Scheduled illumination option utilizes a previously saved illumination schedules for more complicated illumination see section 4 3 The Level indicator shows the current parameter line of the illumination schedule Finally to transmit the current illumination pattern for either Simple or Schedules the Illuminate button is pressed Illumination will only occur if the Thinning and Segmentation is currently ON 320x240 1X 32 bit RGB image 0 0
89. e the filters have been successfully placed and secured Fig 3 4k the projector can be reassembled by reversing steps 4 to 9 A CRITICAL STEP For the projector to function correctly all cables must be reattached in the original position otherwise an error will occur when powering on the projector Refer to the photographs acquired in the previous steps for accurate reassembly 11 To remove the projection lens from the lens assembly remove the screws attached to the zoom ring four screws for the Hitachi CP X605 A CRITICAL STEP For those projectors where the lens assembly cannot be removed the projection lens can simply be removed by completely unscrewing counterclockwise 12 Slide the zoom ring back as far as possible and rotate to see the small inner screws Fig 3 41 These are stops for the projection focus lens preventing it from being fully 143 unscrewed Loosen these screws until the diverging projection lens can be fully rotated counterclockwise and off the zoom lens assembly 13 Reattach the zoom ring The zoom lens should now be reinserted into the projector by lining up the notches and rotating clockwise until a click is heard A CRITICAL STEP The projection lens portion of the zoom lens assembly must be removed for optimal performance However the diverging projection lens can be reinserted to use the projector in its original function magnify and project an image Adjustment of the projector settings e TIMING 0 25
90. e two sets of experiments Adapted from REFERENCE 117 suoJn N JOJON suoIn N JOON asuodsey Jesolneyog asuodsey jesolIAeyog 117 As processes of the glr 1 MAC expressing cells pass the region of mec 4 ChR2 expressing neurons behaviors evoked in mec 4 neurons may be slightly dampened Supplementary Note 2 The behavior of the animals was tracked over time while they were manipulated following the two photostimulation schemes depicted in Fig 5a Velocity averages from multiple animals are shown in Fig 5b When the anterior sensory neurons ALM AVM were stimulated by blue light for 4 seconds at 1 17 mW mm intensity Scheme 1 Fig 5a c the animals produced a robust reversal behavior When ALM AVM neurons were illuminated the same way while the head interneurons were inhibited by green light 2 seconds after the blue light was on Scheme 2 Fig 5a c the animals first produced the expected reversals but upon silencing of the interneurons the reversals were inhibited and the velocity became positive Fig 5b Figure 5 13 Sequential frames from Supplementary Video 8 of Reference 117 demonstrating the multi spectral dynamic capacity of the illumination system The animal is illuminated with blue light in the region of the anterior mechanosensory neuron which express ChR2 thus eliciting a reversal The animal is subsequently illuminated with green light in the region of the command interneurons which express the hyper
91. eased For all experimentation described quantification of the concentration of animals and dilution to optimal levels for introduction into the microfluidic device is required Animals are grown on agar plates containing the bacterial food source Animals are rinsed from the agar plate with M9 buffer Animal concentration is measured using a designed a worm densitometer This simple instrument measures the scattering of incident light red light by the worms and quantifies it either by a photodiode We have tested this instrument at relevant worm concentration Shown below is a plot of scattered light 44 intensity versus time for various worm concentrations Fig 2 9a As the worms settle into the scattering area area of illumination the intensity of scattered light increases We chose to measure worm concentration at t 60s time after washing animals from the plate into a microcentrifuge tube At this time we found most of the L4 and adult animals had settled while the unwanted L1 L3 animals and eggs remain in the supernatant Additionally shown is a plot of scattered light intensity concentration at t 60s showing a linear relationship between concentration and scattered light intensity Fig 2 9b We found experimentally that the optimum worm concentration for our device is 900 worms mL Therefore after washing the worms off the plate and measuring the relative number the optimum density of worms was achieved by removing the s
92. ed in Figure 3 8a From this we see that as the smallest spots are approached the measured width of the spot 10 intensity levels off This places a lower limit of illumination resolution at about 14 um using a 4x objective This however is thought to be the worst case scenario because this measurement involves two contrast transfer functions one from the projector to the object plane and one from the object plane to the camera actual illumination onto the object plane only involves the first contrast transfer function Lower limits of illumination resolution can be achieved by increasing the objective power thereby increasing the amount of demagnification but similar shape curves would be expected just shifting to lower limits As a demonstration we have also characterized the resolution of a 25x objective as shown in Figure 3 8b With this objective the spatial resolution can be as high as 5 um 73 O o N o wo a N a wo o 150 Measured N a N a N ua o ua o Measured Spot Size um width at 10 max intensity a a Measured Spot Size um width at 10 max intensity X 3 o Ee N o 0 25 50 75 100 125 150 175 200 0 5 10 15 20 25 30 Projected Spot Size um Projected Spot Size um Figure 3 8 Measuring the limits of spatial resolution 117 a Measured spot size using a 4x objective This shows a resolution limit to be about 14 um at 4x b Measured
93. egans has been powerful in elucidating the mechanisms control and determination of cell division lineage tracking and apoptosis Primarily for these two studies the significance and enormity of the finding the three researchers Brenner Horvitz and Sulston were awarded the Nobel Prize 1 1 2 RNAi In 2006 the Nobel Prize committee awarded the Nobel Prize in Physiology or Medicine to Andrew Z Fire and Craig C Mello for their discovery of RNA interference gene silencing by double stranded RNA nobelprize org RNA interference RNAi is a cellular process in which the level of protein expression is regulated through the interference of RNA translation by either small interfering RNA siRNA or microRNA mRNA 12 13 The process of gene silencing had previously been recognized in plants 14 19 and animals 20 23 Fire et al found efficient gene knock down could be achieved by injection of double stranded DNA dsDNA 12 It soon then became apparent that all these studies were controlled by similar mechanisms Although RNAi is an inherent mechanism of gene regulation researchers have harnessed this mechanism for experimental control of gene expression 13 Large scale screens utilizing RNAi have been performed in the multicellular organisms C elegans 24 and D melanogaster 25 and cells 26 Administration of RNAi in C elegans is relatively simple 27 the animal can be fed 28 29 injected 12 or soaked in RNAi 3
94. egration of distinct neural signals expression in multiple cells is required To fully utilize the potential of the optogenetic reagents the toolbox must be expanded to include techniques for specific and localized optical targeting of excitable cells Additionally because currently available optogenetic reagents cover a broad range of the optical spectrum the ability to have multi spectral optical illumination is valuable In this chapter we present a procedure to modify a commercially available three panel liquid crystal display 3 LCD projector and integrate it with most inverted epifluorescent microscopes for the purpose of patterned illumination on a sample The presented protocol allows for fully reversible modification of the microscope system Once completed the illumination system is capable of multi color illumination and can be applied to both static and moving samples The illumination pattern is defined by a computer and sent to the projector as a second video output the image is then relayed from the projector to the microscope and de magnified determined by the objective and the accessory optics Images for projection can be easily defined statically through programs such as Microsoft PowerPoint or can be dynamic and more complex in design through the use of image processing techniques in Matlab or LabVIEW 117 Compared to other custom assembled systems and commercially available products the protocol discussed in this chapter al
95. em to display an object Another critical feature is the spatial accuracy or how close an intended target can be illuminated If the object is not moving then this can simply be measured by selecting a target and illuminating it and measuring the distance from intended target to actual illumination This was performed and yielded a spatial accuracy of less than 3 um If the target is moving then another critical aspect is the temporal accuracy as defined and measured above We can therefore define another spatial accuracy as Ad vxAt where Ads represents spatial accuracy and tia represents temporal accuracy Assuming a representative C elegans moving forward at 250 um sec then the spatial accuracy would be 28 um thus our illumination would be off target by as much as this amount In all experiments performed the boundaries of our illumination extended well beyond this amount relative to the intended target neuron or muscle cells It is suggested knowing this temporal and spatial accuracy the intended illumination region is chosen such that it is greater than the target area by at least the amount defined by the spatial accuracy Ad For very slow moving or non moving objects such as cultured cells the spatial accuracy would be determined by the previously reported value of less than 3 um 3 4 Conclusions The goal of this research was to design and create a multi modal optical illumination system The design goals were to create a
96. embrane is depolarized below the resting potential more neurotransmitter is released as the membrane is hyperpolarized potential more positive than resting potential neurotransmitter release is decreased or halted Although many genes and proteins involved in synaptic transmission have been identified in C elegans new genes are being discovered and new functions of known genes are being attributed continuously albeit slowly by traditional genetic methods such as the RNAi screens discussed earlier There are a variety of neurotransmitters in the C elegans nervous system These neurotransmitters can either be excitatory or inhibitory depending on the nature of the post synaptic receptor complex Examples of some common neurotransmitters are 11 Acetylcholine ACh Serotonin SHT Dopamine DA Tyramine TA Octopamine OA Glutamate Glu and Gamma aminobutyric acid GABA 76 1 1 5 2 Gap Junctions A second primary method of signal transmission in C elegans occurs through gap junctions 77 78 Gap junctions represent about 1 3 of the connection in the nervous system 6 62 and are additionally important in connecting cell populations in the early development of the animal Gap junctions are intercellular connections formed by multiple channels connecting the cytoplasm of cells The channel formed allows the direct passage of ions and small molecules and therefore provide a mechanism for rapid signal transmission As a neuro
97. ent RNAi susceptible strains were quite sickly and did not fully demonstrate phenocopying the mutant phenotype As improvements in the ability to deliver RNAi to neurons are made this should increase the applicability of the method Although greatly diminished when using the microfluidic channels coiling of the animals still represent a significant problem for image processing This can possibly be addressed by altering the geometry of the channels or putting the animals in a mutant background that suppressed this effect Finally combining robotic liquid handling systems and microfluidics is a significant step in bridging the gap between traditional and more recent high throughput screening technologies though additional work remains to be done standardizing connections and the supporting control systems 2 8 1 Limitations and considerations Our average processing time for a single population was about 15 minutes Furthermore a long time between sets of populations usually 6 24 populations each run was incurred because of the manual processing and washing of animals These manual steps represent a way in which the process can be further improved though automated washing of animals from plates is a substantial hurdle There is a possibility to culture the animals in liquid culture rather than on the solid agar though animals cultured in liquid are generally longer and thinner and the culture method has an unknown effect on the efficacy of the
98. entary Software from Reference 117 In order to begin the segmentation Fig 2d e select the Thin and Segment option The small white dot in the Backbone display should be located at the animal s head If this is incorrect press the Flip HT button 150 xvi xvii xviii xix xx xxi xxii xxiii xxiv xxv With the options set as desired begin the video recording with the Record button Begin the illumination by clicking Illuminate When completed stop the video acquisition to save the movie and enter the name for the next video when prompted Stop the program with Complete Stop To implement Head Encode on the saved video open the Head encode program and start with the play button Place the mouse cursor over the head of the animal and hit Enter button on keyboard Follow the position of the head with the cursor as video is played The encoded video will automatically be saved with the name of the original file plus HE To analyze the completed video open the Complete video analysis program Enter the calibration value for micrometers per pixel at full resolution and select the binning of the camera used For the data obtained 117 we measured 3 3 um per pixel and used a 2x2 binning thus providing a calibration of 6 6 um per pixel for our recorded videos Start program with the play button When prompted select
99. f PDMS for construction of microfluidic devices is accomplished in a process called micro molding 198 199 In this process the mixed two component precursors are poured over a mold and cured The mold called a master is a negative of the final intended channel structure and can be composed of multiple layers After curing the PDMS is peeled from the master and the master can be reused multiple times replica molding The structures in the master can be created using traditional MEMS processing techniques such as wet etching Additionally the molds can be formed using micro milling techniques However these processes are either time consuming and expensive or have low fidelity and resolution The most commonly used method for creating masters for replica molding is photolithography In this method a photoresist is spun onto a substrate usually a Silicon wafer to the desired height The photoresist can be positive or negative and the pattern is defined by a mask positive or negative The mask is a high resolution transparency whose features can be created on drawing programs such as AutoCAD Multiple layers of PDMS can also be made using multiple masters The individual layers can be aligned and bonded thus creating complex geometries multi layer soft lithograph 198 Using this process on chip valves can also be made ref On chip valving technology greatly increases the flexibility and control of a microfluidic device allowing for la
100. f the National Academy of Sciences of the United States of America 2004 101 29 p 10554 10559 Sulston J M Dew and S Brenner Dopaminergic neurons in the nematode Caenorhabditis elegans The Journal of Comparative Neurology 1975 163 2 p 215 226 Chalfie M and J Sulston Developmental genetics of the mechanosensory neurons of Caenorhabditis elegans Developmental Biology 1981 82 2 p 358 370 Chalfie M and J N Thomson Structural and functional diversity in the neuronal microtubules of Caenorhabditis elegans The Journal of Cell Biology 1982 93 1 p 15 23 Wicks S R C J Roehrig and C H Rankin A dynamic network simulation of the nematode tap withdrawal circuit Predictions concerning synaptic function using behavioral criteria Journal of Neuroscience 1996 16 12 p 4017 4031 Chalfie M and M Au Genetic control of differentiation of the Caenorhabditis elegans touch receptor neurons Science 1989 243 4894 p 1027 1033 Rankin C H Interactions between 2 antagonistic reflexes in the nematode Caenorhabditis elegans Journal of Comparative Physiology a Sensory Neural and Behavioral Physiology 1991 169 1 p 59 67 163 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 Wicks S R and C H Rankin Integration of mechanosensory stimuli in Caenorhabditis elegans J Neurosci 1995 15 3 p 2434 2444 Rankin C H
101. g other animals to enter during optogenetic excitation and behavioral recording They also provide a means by which the animals that are trapped can subsequently be released thus providing the needed reversibility 33 b Valve Open Valve Closed partially Control Layer Flow Layer Slide glass Figure 2 1 Microfluidic chip used in this investigation 142 a Microfluidic device for parallel investigation of C elegans responses to light stimulation Green channels are the flow layer where C elegans are located and the red channels are the valve control layer Arrow indicates direction of worm loading Scale bar is 500 um b Schematic representation of the partially closed valves used in this device The main imaging channels are 60 um wide slightly larger than the width of a young adult worm It is important that the channels not provide any resistance to the animals when the animals contract when activated by ChR2 The cross sections of the valves are rectangular and are therefore only partially closed allowing some fluid flow while preventing larger animals to pass through the channels Fig 2 1b The partially closed valve also known as sieve valves also allows young animals and eggs to pass through the channel providing a simple method to filter unwanted animals and debris We chose to use a single large valve which covers all channels instead of eight individual valves This greatly simplifies the device co
102. g plates containing ATR Sigma Aldrich were made by diluting a 50 mM stock ATR solution in ethanol in 300 ul OP50 to a final concentration of 100 uM and spreading on a 5 5 cm NGM plate All animals tested were F1 progeny of PO adults picked onto ATR or no ATR plates 3 5 days 47 prior to experiments For the RNAi experiments animals were grown on either an empty control vector or the vector containing the RNAi construct for the gene of interest The strains used in this chapter include zx s3 punc 47 chop 2 H134R yfp lin 15 zlIs6 punc 17 chop 2 H134R yfp lin 15 and unc 49 e407 zxIs6 These strains were created by Martin Brauner in Dr Alexander Gottschalk s lab Frankfurt Germany RNAi worm strains include RNAi sensitive strains with ChR2 in the cholinergic neurons nre I hd 20 lin I15b zxIs10 punc 17 chop 2 H134R YFP unc 119 and RNAi sensitive strains with ChR2 in the gabaergic neurons ones to nre 1 hd 20 lin 15b zxIs8 punc 47 chop 2 H134R YFP unc 119 These strains were constructed by Sebastian Wabnig in Dr Alexander Gottschalk lab The RNAi bacterial feeding strains came from the Ahringer Lab RNAi library Geneservice Ltd 2 7 Master microfluidic control box A major limitation in the implementation of microfluidics is the lack of an integrated control method for the devices Microfluidics is a relatively young field and is primarily limited to research labs and therefore little commercially available
103. h 14 Reinsert the projection lens Turn on the projector and focus on a wall or a screen TROUBLESHOOTING 15 The settings of the projector must be set to ensure optimal performance Follow the manufacturers user s manual instructions and set the following e All keystone setting should be zero offset e Brightness contrast color and tint should be set to the middle position usually default 0 on the Hitachi CP X605 e The active iris should be turned off 16 Adjust the vertical and horizontal lens shift setting to a neutral zero offset position by following manufacturers user s manual instructions 17 Remove the projection lens by unscrewing counterclockwise Assembly of projector and microscope system e TIMING 3h 181 These steps describe the process for modification of an inverted microscope and integration of the projector into the system Either an infinity corrected microscope A or 160mm fixed tube length microscope B can be used for these steps A Assembly of projector and microscope system Infinity corrected 144 i ii iii iv v Remove the epifluorescent optical train from the inverted fluorescent microscope Follow the manufacturers user s manual for schematics and description A CRITICAL STEP All optical components should be handled with care Save all optical components noting locations from which they came for later reassembly if necessary Place the accessory tube lens
104. h behavioral genetic techniques Animals are mutagenzied 41 and assessed for deficiencies in one or more behavioral assays 68 These behavioral assays test for defects in sensory processes such as mechanosensation chemosensation and olfaction The deficiencies are typically behavioral responses that differ from those observed in wild type animals After determination of the specific behavioral abnormality the mutation can be mapped to a specific gene 79 The neurons expressing this defective protein can be determined through cell specific rescue or expression of GFP 53 In this way neurons implicated in a specific sensory function can be determined Another powerful technique for determining the function of neurons is optical laser ablation 7 63 66 71 80 85 This method targets specific neurons in early larval stage animals L1 with a tightly focused nanosecond UV to blue laser beam and destroys the cell Again behavioral assays are performed on populations of animals and assessed for deficiency thereby correlating behavior to neurons 66 71 81 83 Similarly genetic ablations can be performed 86 though usually with less specificity Although powerful these techniques only provide static information about the neuron and its relation to the behavior in its absence There are newer techniques that can directly assess the functions of neurons under application of a stimulus Electrophysiological recording from neurons is a direct met
105. h hyperpolarize and silence cells are both activated in the green yellow region Simultaneously exciting and inhibiting different cells in a circuit particularly in behaving animals can greatly enhance our ability to understand circuits and their functions By using an LCD projector we have three independently controllable LCD panels that can be used for three independent illuminations We used two of these channels to interrogate the mechanosensory circuit using pmec 4 ChR2 pglr 1 MAC animals In these animals MAC inactivates the g r interneurons when illuminated by green light 550 nm Because MAC can also be activated although less efficiently by blue light 125 we illuminated the second quarter of the body along the A P axis i e avoiding illumination of g r neuron cell bodies using blue light and the first quarter of the body using green light This allowed exciting the ALM AVM sensory neurons while inhibiting the g r neurons only in the anterior part of the animals inhibiting all backward command neurons but only one of two pairs of forward command neurons eS See LJ suoJn u1 u SUOJNON yono ys x LAMS Si SUOJNEN yono y SUOJINBUS U Chemical Synapse apusan Gap Junction vs DB Chemical Syn Synapse Gap Junction Figure 5 12 The neural gentle touch circuit showing the neurons that are either stimulated or silenced and the resulting behaviors at different points in th
106. hR2 is shown in Figure 1 4 it is maximally excited at 470 nm 130 Upon illumination with the appropriate wavelength of light the all trans retinal cofactor undergoes a conformational change converting to all cis retinal thus opening the pore of ChR2 to approximately 6 A After excitation and in the absence of light ChR2 relaxes to its closed all trans state in a few milliseconds 18 Relative Sensitivity 400 450 500 550 600 650 700 Wavelength nm Figure 1 4 Action spectrum of two common optogenetic reagents ChR2 is a depolarizing reagent and MAC is a hyperpolarization reagent Figure courtesy of Dr Steven Husson Gottschalk laboratory 1 2 2 Optogenetics and neuroscience In 2005 several research groups brought optogenetics to the field of neuroscience As it was previously shown that ChR2 can alter the polarization state of cells Karl Deisseroth s research group at Stanford imaginged optogenetic reagents could confer precise control over excitable cells such as neurons They demonstrated that when expresed in cultered mammilian hypocampal neurons ChR2 evoked deplarization could enduce spiking patterns precisely controlled through the blue light illumination 121 This was achieved with millisecond precision and single spikes could be evoked with a high degree of repeatability This spiking appeared electrophysiologically indistinguishable from natural spiking as did the ability of the spiking neurons to induce synaptic t
107. hat another neuron responsible for the reversal behavior gets coactivated by this mechanosensory insult Recently the neuron FLP has been implicated in the mechanical nociceptive response 240 To further investigate the nociceptive response and circuit responsible for this behavior FLP neurons as well as the integrating interneurons were then photostimulated The mechanical nociceptive circuit Fig 5 16a shows both PVD and FLP have synaptic connectivity to both the forward and reverse command interneurons As demonstrated previously Fig 5 15 the primary response upon photostimulation of PVD is acceleration forward To test the role of the interneurons ChR2 expressing animals in PVD were stimulated with both synaptic partner interneurons present N2 zxIs12 and in animals in which the PVC interneuron was degenerated deg I d zxIs12 As before when both interneurons were present photostimulation of PVD resulted in rapid forward acceleration Fig 5 16b When the command interneuron responsible for forward movement PVC was absent photostimulation of PVD resulted in rapid reversal Fig 5 16b This indicates both interneurons are active and respond to PVD stimulation however the forward command interneuron PVC dominates perhaps due to increased synaptic connections and strength to PVD To test the role of FLP in this response animals expressing ChR2 in FLP HSN and PVD ZX1014 were photostimulated Illuminating only the anterior reg
108. he combination of microfluidics and optogenetics would be valuable for any study where a large number of animals need to be optogenetically probed In the second aim Chapter 3 we develop and optical illumination system capable of defining an illumination pattern in both space and time for the purpose of specifically illuminating neurons and muscles expressing optogenetic reagents Furthermore we have the ability to dynamically alter the color and intensity of the illumination in near real time thus exploiting the ever expanding optogenetic reagent palette To control the illumination system providing the needed control to illuminate specific areas of freely moving C 30 elegans we develop a set of software in aim 3 Chapter 4 The software is responsible for acquiring images of the animal analyzes the images to determine anatomical locations of the animal and directs the illumination system to optically illuminate the intended targets There is an additional software module for analysis of the resulting behavior and quantitative phenotyping In the final aim Chapter 5 we demonstrate the power and utility of the illumination system and software by targeting specific nodes of the mechanosensory circuit We demonstrate capabilities that are not possible using traditional assays We finally summarize the thesis contribution and suggest future work following up on the research presented in this thesis Chapter 6 31 CHAPTER 2 MICROFLUID
109. he first time these behaviors could be directly interrogated and done so with some spatial specificity Additional capabilities of the system were demonstrated with the experiments investigating the probabilistic behavior of the animals upon differential stimulus intensity and the simultaneous illumination of the anterior and posterior TRNs These experiments were not previously possible using other methods and thus our method opens up the door to a new manner of investigation properties of a 129 neural circuit propagation of a signal and the integration of signals within a neural network Finally the multi color capabilities of the system allow for simultaneous activation of many optogenetic reagents This means that true multiplexing of optogenetic reagents is now possible and complex assessment of the function of neurons can be accomplished In summary this thesis combines several technologies that are rapidly advancing C elegans neuronscience microfluidics optogenetics lab automation and automated machine vision The developed technologies discussed in this chapter should allow increasingly high content complex interrogation into the nervous system of C elegans both in regard to the genetic and neural basis of behavior 6 2 Future Directions Throughout the thesis I have noted areas in which the developed technologies or application of those technologies could be improved In this section I outline some specific improvements and e
110. he researcher to control this and is highly variable and difficult to control Another commonly used technique for mechanical stimulation is through a plate tap 109 112 114 By tapping or vibrating the agar plate containing the animal a non specific mechanical stimulus is delivered to the animal This stimulus presumably activates both anterior and posterior 15 TRNs and thus the resulting behavior is an integrated response between the two sensory fields 114 The stimulus intensity and duration can be controlled though the variation in intensity could be quite high depending on the location of the animal on the plate and the mechanical characteristics of the agar Both of these methods have allowed for investigation of the role of the TRNs and other characteristics such as the habituation response 115 116 However neither method provides sufficient control for a detailed investigation of the temporal and spatial integration and processing of antagonistic responses Furthermore these methods are difficult to integrate with other methods of neural examination Optogenetics discussed below provides a non invasive method of stimulating neurons with great control and flexibility SUOJNON yono suoJn u1 U Chemical Synapse Gap Junction SUOJNEN JOJO asuodsey jesolneyag Figure 1 2 Mechanosensory circuit of C elegans 117 Sensory signals are transmitted from the sensory level to internuerons finally
111. he screws on the cover to the dynamic iris Fig 3 4g Remove the cover and then slide out the dynamic iris unit Disconnect the green grounding wire The cover to the main optical train of the projector must now be removed The Hitachi CP X605 has four screws holding down the cover Fig 3 4h to be removed as well as two plastic brackets Fig 3 4h that can be unlatched with a flathead screwdriver or spatula Remove the cover 142 A CRITICAL STEP Connected to the optical train cover removed in this step are three polarizing filters Fig 3 41 which are positioned directly in front of the LCD panel when the cover is in place Care should be taken not to damage these filters These filters have also been aligned at the factory rotationally to maximize the contrast of the projector These filters should not be rotated or altered 9 The internal optical path can now be seen the left path is for red the middle for green and the right for blue Fig 3 4j Locations of the insertion of the custom filters are indicated with boxes in Figure 3 4k Insert the pre cut optical filters dimensions for filters for the Hitachi CP X605 can be found in Table 4 1 into the appropriate locations The filters should be secured to the case with high temperature epoxy Alternatively the filters can be temporarily secured from the top side with electrical tape TROUBLESHOOTING A CRITICAL STEP All optical components should be handled with care 101 Onc
112. he shutter was closed Then the worms were unloaded and another cycle begun Decisions on when to isolate the animals and initiate the recording and illumination cycle were determined by the user The timing of video acquisition and illumination were controlled via the LabVIEW program The acquired videos were post processed using custom software written in LabVIEW Movies were analyzed frame by frame first identifying each of the eight channels Fig 2 3a b Channel identification was done by matching regions in the first video frame that matched a template image The template image identified the region of the isolation valve green box Fig 2 3a From this matched image region and the known dimension of the microfluidic device the entire channel could be matched and extracted red box Fig 2 3a d These isolated images were then processed to identify the worm filtering out the edges of the channel and threshold and finally the worm was thinned to a single pixel backbone Fig 2 3e f The length of the backbone was then calculated for all frames of the video Specifics of 36 calculating the backbone and the length of the backbone are discussed in Chapter 4 6 The relative length of the backbone was calculated initial length was taken as the average length during the 1 second before blue light illumination and plotted versus time Comparisons of mean relative body length can be made between wild type animals and those with mutations i
113. hey can be integrated as with the AQ2334 strain through irradiated and subsequent selection A more in depth discussion and nomenclature guidelines can be found in References 58 59 C elegans was also the first multicellular organism to have its genome completely sequenced which was completed in 1998 60 This has made gene identification genetic screens and genetic analysis much simpler The website www wormbase org is a comprehensive listing of identified genes functions and phenotypic information for C elegans Additionally the web resource www wormatlas org and www worm is a database of the known structural and behavioral anatomy of C elegans A large number of the known genetic knock out animals and transgenic animals are maintained at a central respository at The Caenorhabditis Genetics Center CGC and is located at the University of Minnesota Twin Cities 1 1 5 The neuroanatomy and neurophysiology of C elegans As mentioned previously the adult hermaphrodite has exactly 302 neurons Of these neurons 282 belong to one distinct large somatic nervous system and the remaining 20 belong to a small pharyngeal nervous system 5 7 61 62 Early work by Drs Sulston and Horvitz mapped the lineage off all cells including neurons 5 7 The knowledge of this lineage is important in relating structure function relationships among neurons as well as intelligent choice of precursor neurons that one can optically ablate 63 Furthermo
114. hniques to investigate sensory integration within the mechanosensory circuit of C elegans The C elegans touch circuit was discussed in Chapter 1 4 5 4 interneurons integrate signals from sensory neurons and ultimately produce behaviors 66 228 241 242 The anterior touch cells when activated lead to a reversal and the posterior cells when activated lead to a forward acceleration Presumably in the soil both anterior and posterior sets of mechanosensory neurons are excited often simultaneously and the animal must decide the appropriate response How these sensory signals are integrated remains largely unknown Investigation of the integration of sensory signals from the mechanosensory neurons through optical excitation of ChR2 within these neurons using the structured illuminated system outlined in this thesis and in 6 3 1 2 would provide a significant step forward in understanding how neural circuits operate We have demonstrated both anterior and posterior escape behavior elicited by optical activation and how complex illumination pattern can be used to interrogate anterior posterior signal integration Chapter 5 To deepen the understanding of processing by a neural circuit experiments of sequential posterior illumination pulses followed by anterior illumination pulses and vice versa at different inter stimulus intervals would be performed Similar experiments have been performed by Rankin et al by mechanical touching and tapping 243
115. hod of measuring activity of neurons Although very challenging due to the small size of neurons in C elegans and the tough cuticle of the animal electrophysiology in C elegans is well established 87 91 Animals are immobilized usually by gluing them and putative neurons involved in a particular sensation are patched The animal can then be subjected to a stimulus mechanical chemical etc and electrical recordings are made thus directly observing the response of the neuron Another technique is similar in function though less invasive 13 and less sensitive is calcium imaging There are a number of genetically encoded calcium indicators GECI that have been used in C elegans neuroscience 92 101 Cameleon indicators have two fluorescent proteins usually CFP and YFP connected by calmodulin CaM and the calmodulin binding domain of myosin light chain kinase M13 102 103 Upon the introduction of the calcium ion CaM binds free Ca and then M13 can bind to the CaM This causes a conformational change in the protein bringing the two fluorescent proteins together allowing fluorescent resonant energy transfer FRET to occur The fluorescence arising from the FRET can be imaged thus indicating relative changes in the concentration of the intracellular calcium The other main calcium indicator is based on a GFP or YFP variant 101 104 107 CaM and M13 are introduced into the middle of the fluorescent protein When unbound to calcium
116. hodopsin 2 Channelrhodopsin 1 was first discovered and characterized to be involved in the phototaxis and photophobic responses in the green algae Chlamydomonas reinhardtii 127 The isolated protein was shown to be an ion channel primarily passing protons and when expressed in Xenopus laevis oocytes could evoke channel currents that depend on illumination of light Later Channelrhodopsin 2 ChR2 also isolated from Chlamydomonas reinhardtii was demonstrated to also be an ion channel non specific to cations and when expressed could depolarize cells in a light dependent manner 129 17 130 Because it is allows the passage of ions directly it is ionotropic ChR2 is composed of seven transmembrane domains which form the pore of the ion channel The chromophore is made of all trans retinal ATR and is coupled to the protein through covalent linkage Fig 1 3 The C terminus of the protein extends into the intracellular space of the cell and to this terminus a fluorescent protein can be fused During this allows for direct visualization and confirmation of expression of ChR2 121 122 130 all trans retinal cytosol Na Figure 1 3 Structure of ChR2 Illumination of blue light allows for passage of cations into the intracellular space Fluorescent proteins are often fused to the C terminus thus allowing direct visualization of the expression of ChR2 Figure courtesy of Dr Alexander Gottschalk The action spectrum of C
117. in and green algae channelrhodopsin Proceedings of the National Academy of Sciences of the United States of America 2005 102 49 p 17816 17821 Deisseroth K et al Next generation optical technologies for illuminating genetically targeted brain circuits Journal of Neuroscience 2006 26 41 p 10380 10386 Wang H et al High speed mapping of synaptic connectivity using photostimulation in Channel rhodopsin 2 transgenic mice Proceedings of the National Academy of Sciences of the United States of America 2007 104 19 p 8143 8148 Huber D et al Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice Nature 2008 451 7174 p 61 U7 Gradinaru V et al Optical Deconstruction of Parkinsonian Neural Circuitry Science 2009 324 5925 p 354 359 Douglass A D et al Escape behavior elicited by single Channelrhodopsin 2 evoked spikes in zebrafish somatosensory neurons Current Biology 2008 18 15 p 1133 1137 Guo Z V A C Hart and S Ramanathan Optical interrogation of neural circuits in Caenorhabditis elegans Nature Methods 2009 6 12 p 891 U47 Andrasfalvy B K et al Two photon single cell optogenetic control of neuronal activity by sculpted light Proceedings of the National Academy of Sciences of the United States of America 2010 107 26 p 11981 11986 Papagiakoumou E et al Scanless two photon excitation of channelrhodopsin 2 Nature Methods 201
118. in the epifluorescent optical path near the filter cube centering it along the optical axis Remove the transmitted light optical filter With the filter cube in place with the dichroic and emission filter but no excitation filter as it has been inserted internally in the projector place the stage micrometer calibration slide on the microscope stage and bring the slide into focus Turn up the transmitted light intensity Using a piece of paper find the position along the epifluorescent optical path where the image of the micrometer comes into sharp focus This should be at the back focal plane of the accessory tube lens Place the projector such that the primary projector image coincides with the location of the focal plane of the accessory tube lens determined in step 18A iv Fig 3 2b TROUBLESHOOTING B Assembly of projector and microscope system 160 mm i ii Remove the epifluorescent optical train from the inverted fluorescent microscope Follow the manufacturers user s manual for schematics and description A CRITICAL STEP All optical components should be handled with care Save all optical components noting locations from which they came for later reassembly if necessary Remove the transmitted light optical filter With the filter cube in place with the dichroic and emission filter but no excitation filter place the stage micrometer calibration slide on the microscope stage and bring the slide into focus
119. ing it into focus through the eyepieces or camera 211 With the projector turned on bring up an image on the second monitor projector from the computer A checkerboard pattern will work well for this step Without adjusting the focus of the microscope bring the pattern into focus on the paper by adjusting the position of the projector and lens Gross adjustments can be made by observing the pattern on the paper by eye 146 TROUBLESHOOTING 221 To make fine adjustments in the projector position and focus begin by placing a highly reflective material on the microscope stage this can be a front coated silver mirror Also a blank NGM plate works well for this purpose Bring the front surface of the reflective material into focus by focusing the microscope on an imperfection or dust on the surface 231 With the projector on and projecting an image make further adjustments of the X Y and Z position of the projector and lens system to bring the projected image into sharp focus These positions should be noted and the lens and projector system can be fixed A CRITICAL STEP This is a critical step to ensure the projected image is focused on the sample of interest When the sample of interest is focused through the microscope the projector image will be demagnified and focused on the sample If the system is not moved these focusing steps need not be repeated although it is suggested doing once in a while weekly to ensure proper alig
120. ing microfluidics Finally by combining microfluidics liquid handling robotics and lab automation we demonstrate the capability of merging traditional high throughput screening technologies HTST with 32 current HTST microfluidics Together these technologies will enable high throughput genetic studies such as the investigation of genes regulating synaptic function 2 2 Microfluidic Device Design Fabrication and Operation The first goal is to develop a microfluidic system in which animals can be loaded into an imaging area illuminated with blue light and imaged and then unloaded This process must be reliably repeated many times to acquire sufficient number of ananimals for analysis To achieve high throughput in a controllable integrated system we designed and fabricated a two layer polydimethylsiloxane PDMS Dow Corning device using multi layer soft lithography 198 Details of multi layer PDMS device fabrication can be found in Chapter 1 3 1 The device is composed of eight parallel imaging channels connecting a large loading and un loading chamber The loading chamber contains multiple pillars that serve two functions support for the large chamber and an additional mechanism to distribute worms throughout the array of channels These channels can be isolated trapping the worm by actuating a set of two valves Valve 1 and Valve 2 Fig 2 1a The on chip valves serve to isolate animals within the imaging area not allowin
121. ing this multispectral illumination system and can often be found more cheaply In a 160 mm tube length microscope the specimen is placed slightly in front of the front focal plane of the objective and the intermediate image is formed 160 mm behind the nosepiece opening To reverse this process and demagnify the projector image the primary projector image PPI Fig 3 2c should be placed 160 mm from the nosepiece opening However due to mechanical restrictions this is usually not possible Therefore the primary projector image must be transmitted to the plane 160 mm from the nosepiece opening This is accomplished by using a relay lens RL Fig 3 2c consisting of a 1 1 matched relay lens pair The relay lens pair should be located such that the front focal plane FFLrip of the lens pair and 67 160 mm plane coincide and the back focal plane BFLrrp of the lens pair and primary projector image coincide Fig 3 2c 3 2 5 System Assembly The projector is mounted on a stable lab jack to provide z translational ability the accessory tube lens or relay lens and the projector must be centered along the optical axis of the epifluorescent port Fine adjustments to the location of the lenses and projector are made to ensure the demagnified projector image and the object of interest e g C elegans is coincident When connected to a computer and set up as a dual monitor display the completed system will relay the image for the second mo
122. ing yielding an overall temporal accuracy of 111 ms 76 1000 o w 5 8 Measuresed Pulse Width ms gt S 0 200 400 600 800 1000 0 50 100 150 200 250 300 Applied Pulse Width ms Relative Inten 0 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 Time s Relative Intensity 0 50 O55 0 60 065 0 70 0 75 0 80 085 090 0 95 1 00 Relative Intensity 0 50 0 51 0 52 0 53 O54 O55 0 56 O57 0 58 0 59 0 60 Time s Figure 3 9 Measurement of temporal accuracy and resolution 214 a Measurements of pulse width full width half maximum over a range of applied widths 40 ms 50 ms 75 ms 100 ms 125 ms 150 ms 250 ms 500 ms 750 ms 1000 ms with a 50 duty cycle N 100 for each b Expanded region of a c Example measured pulse train for an applied pulse width of 250 ms Blue regions indicate when the pulse was applied and the red trace indicated the measured relative intensity d Expanded view of one cycle on to off from c indicating the measured pulse width Atpwum Blue represents when the signal applied e Expanded view of the onset of the pulse from d indicating the time lag Atag as well as the rise time Atrise These two factors determine the overall temporal accuracy 77 3 3 4 3 Moving Spatial Illumination Accuracy The static spatial resolution of the system was found to be 14 um using a 4x objective and 5 um at 25x This can be thought of as the limits of an optical syst
123. ion containing FLP resulted in rapid reversal of the animal Fig 5 16c 122 b N2 zxIls12 Time s deg 1 d zxls12 1 15 Time s f PVD PVC degenerated Figure 5 16 Optogenetic dissection of the nociceptive response 238 a Simplified wiring diagram of the nociceptive neural network b Photostimulation of PVD in animals with both forward and reverse interneurons and with the forward interneuron degenerated c Response of photostimulation of the FLP neuron Together these results suggest that both FLP and PVC contribute to the nociceptive response While photostimulation of PVD results in forward acceleration and photostimulation of FLP results in reversal together this suggests that both FLP and PVC are have an active role in the harsh touch sensation and FLP is the primary anterior sensor and PVD is the primary posterior sensor 5 7 Methods 5 7 1 C elegans culture Worm strains were cultured at 22 C in the dark on standard nematode growth medium NGM plates with OPSO bacteria The required cofactor for channelrhodopsin all trans retinal ATR Sigma Aldrich was supplemented ATR was supplemented to the NGM plates by diluting a 50 mM stock ATR solution in ethanol in 300 ul OPSO to a final concentration of 100 uM and spreading on a 5 5 cm NGM plate The strains used in this paper include ZX299 lin 15 n765ts zxEx22 pmyo 3 ChR2 H134R YFP lin 15 ZX460 N2 zxIs6 punc 17 ChR2 H134R YFP lin 15
124. is capable of controlling eight on chip valves 2x4 each controlled at 0 60 PSI has two worms injection ports 0 15 PSI a single outlet 0 15 PSD that is used for flushing fluid not utilized or described in this work and four back ports for control of external components such as pinch valves and LEDs 2 8 Conclusions The work presented in this chapter demonstrated for the first time the combination of two recently advancing techniques in C elegans biological research optogentics and microfluidics By combining the rapid positioning and imaging of C elegans utilizing microfluidics with the non invasive specific activation of neurons with optogenetics we demonstrated reliable repeatable and fast assessment of synaptic function using OptIoN The machine visions programs allowed for automated control of illumination image acquisition and data analysis The combined system demonstrated at least an order of magnitude increase in the speed of data collection Furthermore the developed supporting hardware such as the worm densitometer and the master microfluidic 50 control box increase the ease of use and efficiency of the microfluidic system Finally utilizing automated liquid handling systems further increases the processing ability of the microfluidic system and decreases researcher burden Although a significant step forward in the application of OptlIoN there are still a number of barriers that must be overcome The curr
125. is sized filter Internal disconnected cables Other errors associated with projector Bulb is near the end of life Disconnected LCD panel cable Shifted or broken filter Unsecured loose LCD panel cable Accessory tube lens focal plane located within body of microscope Relay lens focal plane located within body of microscope Mis positioned lenses Altered camera from what program 153 Solution Check both the back and the bottom of the projector to ensure all necessary screws have been removed Measure the opening at the location for the filters and check dimension of the custom filters Alter the filters as necessary to fit If all cables were not correctly connected when reassembling projector then unit will not power up Take projector apart and ensure all cables are connected Check error blinking codes and consult user s manual for solution Check projector for bulb hours and consult user s manual on replacing Disassemble projector to ensure that LCD panel cables have been securely reattached Disassemble projector and check all inserted filters to check if they have shifted or possibly broken Replace if necessary Disassemble projector to ensure that LCD panel cables have been securely reattached Extend the accessory tube lens to the rear of the microscope allowing the focal plane to be located outside the body of the microscope Select a matched lens pair of greater focal length
126. ivation and overview 3 1 1 Potential applications of the method 3 1 2 Comparison with other methods 3 1 3 Overview of the procedure 3 2 Experimental design 3 2 1 Choice of 3 LCD projector 3 2 2 Modification of the projector and insertion of custom optics 32 32 33 36 38 41 41 42 44 46 47 48 49 50 51 56 56 57 58 60 63 63 63 3 2 3 Modification of microscope optics for infinity corrected systems 66 vi 3 2 4 Modification of microscope optics for 160 mm fixed tube length systems 67 3 2 5 System assembly 67 3 3 Characterization of the illumination system 68 3 3 1 Spectral and intensity characterization 68 3 3 2 Illumination distribution across the field of view 69 3 3 3 Spatial resolution and accuracy 71 3 3 4 Temporal illumination resolution and accuracy 75 3 4 Conclusions 78 4 Software for selected area illumination of freely moving C elegans and behavioral analysis 80 4 1 General computer setup 80 4 2 Projector alignment 81 4 2 1 Initial axial Z and in plane XY alignment 81 4 2 2 Coordinate transformation 82 4 3 Color illumination and tracking 83 4 3 1 Image acquisition 84 4 3 2 Motorized stage control 86 4 3 3 Image processing segmentation and illumination 87 4 3 4 Video recording 94 4 4 Scheduled illumination 95 4 5 Head encode 96 4 6 Complete video analysis 97 4 6 1 Using the program 97 4 6 2 Extracted parameters 98 4 7 Conclusions 101
127. kfurt Germany 238 As will be discussed in Chapter 6 1 both the illumination system and software were transferred to Dr Alexander Gottschalk s lab during an extended visit in October 2010 Working closely with Dr Gottschalk s lab I have continued to assist the lab with troubleshooting and maintenance of the system and software A variety of noxious stimuli mechanical chemical and thermal have the potential to damage tissue Nociceptive neurons in C elegans help to protect the animal by evoking a fast withdrawal upon neural activation Two neurons whose dendridic arbors cover a large receptive field in C elegans are FLP and PVD these neurons respond to harsh mechanical touch Traditionally the harsh touch response in C elegans is assessed by prodding the animal with a platinum wire harsh touch The response is complicated by the co activation of the other mechanically sensitive neurons such as those previously discussed ALM AVM PLM Furthermore distinguishing the roles of FLP and PVD in this response is very challenging by traditional techniques Optogenetics presents a powerful method to investigate the nociceptive response by photoactivating FLP and PVD as well as the downstream integrating interneurons without concurrent activation of other mechanically sensitive neurons However traditional whole animal illumination would activate both PVD and the other neurons expressing ChR2 under the promoter used Furthermore when
128. l and spatiotemporally excite and or inhibit specific nodes of neural networks This illumination system is capable of delivering light stimuli to genetically modified optically excitable cells with high repeatability and light intensity control It also enables the use of combinations of optogenetic tools with non overlapping activation spectra By using a three color LCD we were able to achieve true simultaneous multi color illumination allowing this spatial and spectral separation to probe neuronal networks more precisely 124 In addition to the experiments shown here multimodal real time optogenetic control will allow further studies of other sensory circuits Furthermore studies related to the integration of different sensory modalities and behaviors will be considerably advanced by the ability to track and stimulate freely moving animals Real time illumination and behavior tracking as presented here can also be combined with calcium imaging or with other methods capable of perturbing the circuit such as using microfluidic devices to deliver well defined sensory stimuli analyzing animal mutants for particular neurotransmitters or performing laser ablation of cells axons or synapses to remove single nodes or connections within the circuit Lastly one could imagine using the illumination system with other photostimulation methods such as uncaging of small molecules 5 8 1 Limitations and Considerations We demonstrated that few o
129. l responses 214 a Using the dorsal coiling effect to cause a worm to crawl in a triangle b Showing direct muscular control of a paralyzed worm Images are falsely colored to show illumination pattern Scale bar is 100 um 104 5 3 Spatial Activation of Sensory and Command Neurons To demonstrate the spatial resolution of our system when performed in freely moving animals we performed experiments analyzing the mechanosensory behavior in C elegans There are six major mechanosensory neurons in C elegans AVM and ALML R anterior and PVM and PLML R posterior 66 228 as discussed in Chapter 1 X Animals carrying pmec 4 ChR2 AQ2334 express ChR2 in these six touch neurons Fig 5 2a By traditional touch assay and laser ablation it has been established that stimulating the anterior neurons causes the animal to move backwards whereas stimulating the posterior neurons causes forward movement or acceleration 66 In our experiment we used a 20um wide bar of blue light and scanned it along a pmec 4 ChR2 animal s anterior posterior AP axis at a relative velocity of 12 5 body length per second 100 um s while monitoring the locomotor behavior of the animal Fig 5 2b c The line was scanned in both the head to tail and tail to head directions Fig 5 2b 105 a Head PT Head to Tail AP Axis P 0 sh N ai a AP Axle P 1 lt AVM PVM i c Time s Head aN Aan EA EA ei Tail i i h
130. l control and the risk of circuit compensation during development optogenetics is non invasive reversible and can be employed at any developmental stage It has been successfully used in C elegans to investigate neural circuits synaptic transmission and the cellular basis of behavior 122 140 158 166 1 2 3 1 Optogenetic investigation of neurotransmission OptloN Optogenetics has been applied to studying synaptic transmission in C elegans 140 142 Liewald et al demonstrated the ability to distinguish mutants defective in synaptic transmission from wild type using optogenetics 140 They expressed ChR2 in the cholinergic depolarizing neurotransmitter at the C elegans NMJ or gabaergic hyperpolarizing neurotransmitter at the C elegans NMJ motor neurons zx s6 and zxIs3 Upon stimulation with blue light ChR2 depolarized these neurons releasing Ach or GABA causing muscle contraction or relaxation respectively In animals containing the zx s6 or zx s3 transgene in the wild type N2 background when illuminated the animal will decrease or increase in length When a number 10 40 of animals are subjected to illumination and the length versus time plots are averaged a length time series plot is generated that is characteristic Length time plots from mutant animals can be compared to wild type and can indicate animals defective in synaptic transmission 140 The absolute amount of contraction or elongation as functions of time is info
131. le Contraction and Force Transmission in C elegans Journal of Molecular Biology 2011 407 2 p 222 231 Nahabedian J F et al Bending Amplitude A New Quantitative Assay of C elegans Locomotion Identification of Phenotypes for Mutants in Genes Encoding Muscle Focal Adhesion Components Methods Accepted 171
132. led we observed that holding constant tail illumination intensity increases the average head intensity at which animals respond Fig 5 10c This suggests the activation of the antagonistic 114 sensory field modulates the response of the other sensory field and that the two are integrated leading to the ultimate behavioral outcome of the animal Scheme 1 b Scheme 2 5 5 S x Oo amp REI Relative intensity o a Relative Intensity o oa 0 4 mi 0 4 0 2 0 2 0 0 o 123 4 5 7 8 8 a 12 18 O42 3 4 5 6 TFT amp 8 10 11 12 13 Time s Time s c I Scheme 1 Hj Scheme 2 of animals N o 0 1 0 2 0 2 0 3 0 3 0 4 0 8 0 9 NR 0 4 0 5 0 5 0 6 0 6 0 7 0 7 0 8 0 9 1 0 S 7 2 Relative illumination intensity Figure 5 10 Illumination patterns used to explore the integration of anterior posterior signals and behavior generated from the stimulation Adapted from REFERENCE 117 a Illumination locations and plot of the temporal variation of the intensity for the two patterns tested Normalized intensity of 1 corresponds to blue light of intensity 1 17mW mm b Histogram distributions of intensity at which animals initiated a reversal under two illumination patterns anterior alone and anterior and posterior simultaneously n 40 for each illumination scheme To further investigate the integration of competing signals we stimulated one set of animals with a single light
133. low a researcher to assemble the illumination system for a fraction of the cost and can be completed within a few days 3 1 1 Potential applications of the method One set of applications of this illumination system as well as similar systems is for the dissections of various neural circuits and synaptic functions in C elegans 117 137 143 as will be demonstrated in Chapter 5 In addition this technology can replace or supplement other technologies used for illumination in other model systems including D melanogaster 146 148 D rerio 136 149 150 and cells 217 218 where region specific illumination of optogenetic reagents is beneficial We also envision that this 57 method might be applied to cultured cell lines for instance for monitoring homeostasis in a network of neurons in a culture dish Furthermore because the protocol describes a method to create a system for patterned illumination the system can be used in place of existing techniques that use spatially defined illumination including enhancing resolution by reconstruction of samples using structured illumination technique 219 and patterned photo crosslinking 220 Additionally the illumination intensity is sufficient to perform standard fluorescent imaging and the multi spectral capability of the illumination system can allow for simultaneous multi color fluorescent imaging When extremely fast shuttering lt 15 ms is not needed the projector can replace th
134. ls will yield greater intensity at the sample plane Therefore a projector that maximizes the brightness minimum suggested is 2 000 ANSI lumens with the smallest panels should be chosen maximum panel size suggested is 1 inch The Hitachi CP X605 is a 4 000 ANSI lumen projector with 0 79 inch LCD panels and is used in this dissertation Also important is the contrast ratio Both DLP and LCD based systems have no true zero intensity even when the DLP or LCDs are in the off state there is a finite amount of background illumination To minimize the background illumination thus preventing unwanted excitation of the optogenetic reagents a high contrast ratio projector at least 500 1 should be selected The Hitachi CP X605 has a stated contrast ratio of 1 000 1 3 2 2 Modification of the projector and insertion of custom optics The protocol to reconfigure the 3 LCD projector Hitachi CP X605 begins by removing the diverging projection lens and inserting custom filters internally Fig 3 3 The filters are selected to best match the requirements of the illumination for fluorescent protein excitation or optogenetic reagent activation 63 600nm LP 568 50 nm 475nm SP Filter add Filter add Filter add UHB Mercury Lamp Primary Projector Image Plane Relay Zoom Focusing Diverging Lens Projection Lens to be removed Figure 3 3 Schematic of final configuration of the modified projector Internal filters are added to the 3 LCD
135. luidics for in vivo imaging of neuronal and behavioral activity in Caenorhabditis elegans Nature Methods 2007 4 9 p 727 731 Kerr R A Imaging the activity of neurons and muscles in WormBook T C e R Community Editor WormBook Tian L et al Imaging neural activity in worms flies and mice with improved GCaMP calcium indicators Nature Methods 2009 6 12 p 875 U113 Miyawaki A et al Dynamic and quantitative Ca2 measurements using improved cameleons Proceedings of the National Academy of Sciences of the United States of America 1999 96 5 p 2135 2140 Miyawaki A et al Fluorescent indicators for Ca2 based on green fluorescent proteins and calmodulin Nature 1997 388 6645 p 882 887 Baird G S D A Zacharias and R Y Tsien Circular permutation and receptor insertion within green fluorescent proteins Proceedings of the National Academy of Sciences of the United States of America 1999 96 20 p 11241 11246 Nagai T et al Circularly permuted green fluorescent proteins engineered to sense Ca2 Proceedings of the National Academy of Sciences of the United States of America 2001 98 6 p 3197 3202 Nakai J M Ohkura and K Imoto A high signal to noise Ca2 probe composed of a single green fluorescent protein Nature Biotechnology 2001 19 2 p 137 141 Nagai T et al Expanded dynamic range of fluorescent indicators for Ca2 by circularly permuted yellow fluorescent proteins Proceedings o
136. mately 25 minutes on the plate to allow the animal to recover from the mechanical disturbance of picking and adjust to the lack of food 249 Open the main program Color illumination and tracking and start with the play button TROUBLESHOOTING Select the location and name of the video to be saved when prompted With the transmitted light filter in place and the transmitted light turned on invert the plate and place it on the custom microscope stage Locate the animal and center it within the field of view and bring it into focus Adjust the bright field illumination intensity such that the binary image is an accurate representation of the animal Fig 2c With the animal in the center of the field of view select the TRACK button to begin automated tracking of the animal In the upper right of the program interface there is a block labeled Illumination Control In this block the number of segments and location of the segment divisions should be set Fig 2d e Additionally within this block set the values 0 255 of the individual red green and blue light and select turn on the segments to illuminate Finally set the timing to Timed and adjust the illumination duration or alternatively set to Untimed These setting can be adjusted with the slide bar set to Simple More complicated illumination patterns can be set with the Scheduled option see Read Me Program Overview in Supplem
137. muscles in freely moving C elegans we developed a set of software described in Chapter 4 The software was written to acquire images of freely moving animals and provide a frame of reference on the animal such that one can define the intended anatomical structures The software provides full control over the previously mention factors color intensity time location Flexibility within the software enables the user to define complex illumination pattern that can create sophisticated patterns of neural activation when used with optogenetic reagents thus allowing the interrogation of the neural basis of behavior The interface of the software was designed for ease of use and approachability to non engineering laboratories The other software presented allows for extraction of a multitude of parameters that can be used for quantitative phenotyping thus allowing quantitative comparisons between different neural patterns and activity In Chapter 5 we demonstrated that the completed illumination system and software is capable of targeting neurons and muscles in C elegans We showed for the first time the optogenetic elicitation of a behavior by specifically illuminating the anterior or posterior touch receptor neurons TRNs Furthermore we could elicit similar behavior through the activation of the interneurons AVA AVB AVD PVC responsible for forward or reverse locomotion Although the function of these neurons has been well established this was t
138. n s membrane potential is altered through the influx or efflux of ions these ions can diffuse to neurons connected through gap junctions thus altering the connected neurons membrane potential The extent to which the connected neuron s membrane potential is altered is due to the properties of the gap junction selectivity conductance In vertebrates gap junctions are formed between two hemichannels one in each cell and are composed of connexin proteins In the region of the gap junctions the intercellular space is about 4 nm In invertebrate animals gap junctions are composed of proteins called innexins invertebrate analog of connexins The innexins protein family is functionally similar but with no sequence homology to vertebrate connexins In both vertebrates and invertebrates a hemichannel is composed of six connexins or innexins The hemichannel can be homomeric if the connexins innexins are the same while those with differing connexins innexins are heteromeric Additionally the gap channel can be composed of the same hemichannel homotypic or different hemichannels heterotypic Depending on the composition of the channel it can exhibit single channel conductances from about 30 pS to 500 pS Furthermore the selectivity to small molecules is greatly altered by the components that make up the gap junction channel 12 1 1 5 3 Assessment of neural function in C elegans Traditionally the role of neurons was assessed throug
139. n genes involved in synaptic transmission Only those animals of age L4 to adults based on length were included in the analysis and animals not meeting these criteria were rejected Figure 2 3 Schematics of computer data processing 142 Worm strain is zxls6 expressing ChR2 in the cholinergic neurons a Bright field image of loaded worms prior to blue light illumination b Bright field image of loaded worms 2 seconds after blue light illumination The analysis program identifies each channel red box and separates it for further processing c d Zoom in view of the areas selected by the red boxes e f For each channel section the worm is first identified and separated from the rest of the image and then thresholded white Then a curve black is fit to the midline of the animal and its length is measured This process is done for every frame of the movie 8 fps From this length data the curves in Fig 2a are generated Scale bars are 250 um 37 2 4 Results We successfully designed and fabricated the microfluidic devices and integrated with the off chip hardware and the control image analysis software The microfluidic chip design and operation features lead to a low occurrence in which there is more than one worm in a single channel lt 5 Fig 2 4 Frequency ON ON amp O 1 2 3 4 5 6 rd 8 Number of Worms Loaded Figure 2 4 Histogram of the worm loading efficiency 142 Channels with zero o
140. n in freely moving animals in ways that is not possible to perform with previous methods The behavior is consistent with the known roles of the locomotive interneurons Previous interrogations of the interneurons relied on traditional genetic techniques or optical ablations where the absence of the behavior was correlated with the role of the neuron 66 114 This demonstrated direct elicitation of the behavior through ChR2 activation of the interneurons using the presented illumination thus allowing a direct causation of the behavior rather than a correlation a b pglr 1 ChR2 600 AVA AVB AVD PVC 400 200 200 Velocity um s 400 0 1 2 3 4 5 6 F 8 9 410 Time s Figure 5 7 Optical stimulation of forward backward command interneurons Adapted from REFERENCE 117 a Illustration of the positions of neurons expressing ChR2 in pmec 4 ChR2 and pglr 1 ChR2 transgenic worms b Average velocity plots of pelr I ChR2 animals under illumination conditions shown as a blue bar above n 24 posterior illumination n 12 anterior illumination Error bars s e m 111 5 4 Spatiotemporal Control of the Illumination Intensity Traditionally the study of C elegan s touch circuit has been largely performed using a manual assay either by touching the head or tail of a freely moving animal with an eyelash or by tapping the agar plate containing animals 66 114 One difficulty associated with this assay is cont
141. n overlapping and communicate through interneurons thus producing a hierarchy of execution The sensory responses are conferred by neural circuits that are generally composed of 3 level of organization 72 The first level is composed of sensory neurons which transduce an external stimulus into a polarization state of the neurons This is transferred to the second levels primarily composed of interneurons and can have a few layers Interneurons are neurons which relay signals from upper levels afferent neurons to lower levels efferent neurons The interneurons usually contain multiple inputs signals from multiple sensory neurons and can thus compare and integrate multiple inputs The final level is composed of motor neurons which control the musculature These levels neural classes Fig 1 1 are not completely restrictive some neurons are polymodal and can belong to more than one layer Similarly sensory neurons can belong to one or more sensory modalities Head Motor Neurons Forward Motor Neurons VB DB Command Internuerons Layer 1 Internuerons gt Layer 2 enso Internuerons Neurons x EN AVA AVB AVD AVE PVC Reverse Motor Neurons VA DA Sensory Interneuron Motor Neuron Level Level Level Figure 1 1 Organization of a neural circuit Three levels of neurons transmit signals from sensory neurons to motor neurons which ultimately lead to a behavior Connections between
142. n with a Chlamydomonas Volvox hybrid Channelrhodopsin 18 International C elegans Meeting 2011 J N Stirman Matthew M Crane Steven J Husson Christian Schultheis Alexander Gottschalk and Hang Lu Real time multimodal optical control of individual neurons and muscles in freely behaving Caenorhabditis elegans Georgia Tech Research and Innovation Conference 2011 J N Stirman Matthew M Crane Steven J Husson Christian Schultheis Alexander Gottschalk and Hang Lu Real time multimodal optical control of individual neurons and muscles in freely behaving Caenorhabditis elegans Clayton State WI2STEM conference on C elegans and other model organisms 2010 Best Poster Graduate Division Gina Cremona Jeffrey Stirman and Hang Lu Quantitative Phenotyping of C elegans in an Automated Microsystem Twelfth International Conference on Miniaturized Systems for Chemistry and Life Sciences 2008 PATENT APPLICATIONS Jeffrey Stirman Matthew Crane and Hang Lu Real time multi spectral optical illumination of model organisms or cells US Patent Office Provisional Patent GTRC 5514 filed January 14 2011 140 APPENDIX B DETAILED PROCEDURE FOR PROJECTOR MODIFICATION OPTICAL SYSTEM CONSTRUCTION AND SOFTWARE Modification of the LCD projector e TIMING 2 5h CAUTION All steps in this section should be performed with the projector unplugged and after at least 30 minutes if the projector
143. nation system for a 160 mm microscope The image projected onto the sample plane can be constructed through programs such as Microsoft PowerPoint or other graphic illustrators for simple static patterns 137 or for patterns that change in time in a predefined manner 148 These projected images would be suitable for immobilized animals or cells or objects that vary slowly in time as there is no real time feedback For freely behaving animals or for dynamic events one must use software that can provide and process real time feedback Custom programs can be written in LabVIEW MatLAB or C that can dynamically alter the illumination patterns based on user inputs or closed loop automated analysis of images e g targeting neurons and muscles in C elegans 117 143 The specifics of the program we utilized 62 to control the projector for illumination of freely moving C elegans are presented in Chapter 4 3 2 Experimental Design 3 2 1 Choice of 3 LCD Projector A few considerations must be taken into account when selecting a 3 LCD projector The main specifications of importance are the brightness the size of the LCD panels and the contrast ratio The combination of the brightness reported in lumens and the size of the LCD panels define the maximum possible intensity of the demagnified image at the sample plane Because the etendue of an optical system cannot decrease a projector with the same reported brightness yet smaller LCD pane
144. nd causes the animal to rapidly reverse By quantitatively measuring the maximal bending amplitude upon reversal animals mutated in some musculature associated proteins could be distinguished from wild type 250 Because many behavioral assays are end point assays or primarily observe forward locomotion we believe the subtleties of the mutants were previously overlooked In a reversal we believe not only are different sets of muscles engaged in a different pattern but also due to the rapid escape response the musculature is more heavily engaged For these reasons we believe the defects are illuminated with this assay The software discussed in Chapter 4 6 was used to measure the length of the animal as well as the amplitude and from this the amplitude length A L ratio was calculated Fig 6 1 Though simple this assay has proven to be very powerful in finding mutants both deficient in their ability to bend as well as hyper flexible 250 252 155 A L 0 383 A L o N a Time s Figure C 1 Quantitative measurements of bending amplitude upon reversal 252 a Bright field image of C elegans undergoing a reversal b Binary image calculated from a c Backbone spline was found from the binary image and from this the length L was calculated d The amplitude A was found by measuring the maximum straight line distance from a line connecting the head and tail to the backbone e Ratio of amplitude to length A L
145. ng systems any well defined spatial pattern spreads out over a larger area To determine the effects of the lens s contrast transfer function on spatial spread of illumination patterns we projected spots of known diameter onto a mirror and imaged them using the full 640x480 resolution of the camera Fig 3 7a shows a spot of diameter 59 6 um 20 projector pixels in diameter projected onto a mirror and imaged using a 4x objective A line scan of intensity was taken and compared against the ideal case Fig 3 7b As expected the intensity spreads out over a larger diameter than the ideal case measured to be 68 5 um at 10 relative intensity 72 a b 1 0 Measured 0 8 Relative Intensity o i gt oa N o 150 100 50 0 50 100 150 Position um Figure 3 7 Measuring the effect of lens contrast transfer function on illumination spatial spread 117 a A spot of diameter 59 6 um 20 pixels was projected onto a mirror using a 4x objective Dotted line shows location of an intensity line scan across image used for part b b Intensity line scan showing measured and perfect ideal profile A spatial spread in intensity is due to the contrast transfer function of the illumination imaging system The width of the spot at 10 intensity was measured to be 68 5 um Different spot diameters were projected measured and used to generate Figure 3 8 This was done for a number of different spot sizes and is plott
146. nitor projector through the microscope which reduces it in size projecting it onto the sample A step by step procedure for modification of the projector alteration of the microscope assembly and alignment of the system is presented in Appendix B 3 3 Characterization of the illumination system 3 3 1 Spectral and Intensity Characterization After insertion of the internal filters in the 3 LCD projector the spectrum of the three color planes Red Green Blue are spectrally restricted Fig 3 5a based on the specifications of the filters Table 3 1 as measured using a spectrometer CCS100 Thorlabs The narrow band width of the spectrum allows for sufficient separation of wavelength to excite distinct optogenetic reagents These results are expected based on the band pass values of the filters If other filters are chosen the modified spectra should reflect those filter specifications Note that the modification of the individual color spectra can only further narrow the individual color spectra it cannot extend the limits of the color spectrum as those are determined by the dichroic mirrors within the projector s optical train which are not modified in this protocol Each pixel element is defined by an 68 8 bit integer 0 255 for each color and thus defines the relative intensity at that location Fig 3 5b a b Blue post filter 100 ee 100 anm Green post filter BLUE eeo Red post
147. nment Small offsets in the axial location of the lens and projector from the ideal locations Fig 3 2b c will make only slight alterations in the amount of demagnification Example applications Methods of illumination control 24l These steps provide three different approaches for performing targeted illumination with the constructed system option A is suitable for rapid evaluation single point white illumination and human feedback option B is suitable for multi color static pattern generation or pre defined pattern generation and projection with no feedback and option B utilizes custom software 117 for real time automated illumination of samples that may vary in space and time A Simple illumination using a mouse pointer e TIMING 0 10h 147 i ii Place the sample on the microscope and bring it into focus Move the mouse cursor from the primary monitor to the secondary monitor projector A small point of light moving in the area of the sample will be observed as the mouse is translocated The mouse can be placed over the intended target area by observing through the eyepieces or camera In this way one can rapidly evaluate the constructed illumination system as well as qualitatively assess the reaction of the sample B Static or pre defined dynamic illumination using Microsoft PowerPoint e TIMING 0 25 h i ii iii iv v Create a new presentation in Microsoft PowerPoint Set the backgro
148. ns Lab on a Chip 2010 10 5 p 589 597 Hung P J et al Continuous perfusion microfluidic cell culture array for high throughput cell based assays Biotechnology and Bioengineering 2005 89 1 p 1 8 168 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 Rowat A C et al Tracking lineages of single cells in lines using a microfluidic device Proceedings of the National Academy of Sciences of the United States of America 2009 106 43 p 18149 18154 Chung K et al Microfluidic chamber arrays for whole organism behavior based chemical screening Lab on a Chip 2011 11 21 p 3689 3697 Chung K and H Lu Automated high throughput cell microsurgery on chip Lab on a Chip 2009 9 19 p 2764 2766 Chokshi T V A Ben Yakar and N Chronis CO 2 and compressive immobilization of C elegans on chip Lab on a Chip 2009 9 1 p 151 157 Krajniak J and H Lu Long term high resolution imaging and culture of C elegans in chip gel hybrid microfluidic device for developmental studies Lab on a Chip 10 14 p 1862 1868 DowCorning Refractive Index of Glycerine Water Solutions at 20 C 69 F http msdssearch dow com PublishedLiteratureDOWCOM dh_0032 0901b8038 00322b7 pdf filepath glycerine pdfs noreg 115 00667 pdf amp fromPage GetDoc accessed 19 November 2011 White J G et al The Structure of the Nervous System of the Nemat
149. ns system and when imaging the amount of magnification is determined by the ratio of the focal lengths of two lenses M TLr OLg gt 1 In order to 66 transfer the projector s primary image PPI Fig 3 2b to the sample plane SP Fig 3 2b an accessory tube lens ATL Fig 3 2b must be inserted in the optical path between the projector and the objective The magnification in this direction is again determined by the ratio of the focal lengths of two lenses M FLo FL ri which will yield M lt 1 or demagnification Tube lenses from different microscope manufacturers have different focal lengths Leica 200 mm Nikon 200 mm Olympus 180 mm and Zeiss 165 mm The accessory tube lens to be inserted should be chosen to best match the focal length of the tube lens of the microscope manufacturer in this way the power of the objective closely matches the amount of demagnification The distance between the accessory tube lens ATL and the projector primary image PPI should be equal to the focal length of the ATL FLar Fig 3 2b The distance between the accessory tube lens projector combination and the objective lens is not as critical however it is generally recommended that this distance be kept as short as possible 3 2 4 Modification of microscope optics for 160 mm fixed tube length systems Although the 160 mm fixed tube length microscopes are an older style microscope they are more than adequate for the purpose of construct
150. nse that mimics the respnose naturally observed Since these initial studies demonstrating the utility of ChR2 in nueroscience research ChR2 has gone on to be the reagent of choice for specific non invasive control over the state of excitability in neurons and muscles Optogenetics a term coined by Deisseraoth in 2006 132 has been utilized for a number of studies including mapping of synaptic connectivity in mice 133 studying learned behaviour in mice 134 dissecting neuronal circuitry in brains with Parkinsons disease 135 examining the escape response in zebrafish 136 and examinging an avoidance circuit in C elegans 137 Multiple animal systems have been used for optically targeting optogenetic reagents for non invasive excitation and inhibition including cultured cells 124 125 130 138 139 and neurons and muscles in small model organisms such as the nematode Caenorhabditis elegans 74 117 122 137 140 144 the fruitfly Drosophila melanogaster 145 148 the zebrafish Danio rerio 136 149 153 and mice 133 134 154 157 20 1 2 3 Optogenetics in C elegans research The nematode C elegans is an ideal organism for optogenetic studies as it is transparent has 302 neurons with known wiring and a host of genetic tools as discussed earlier Unlike cell ablations and genetic manipulations of neurotransmitters to probe neuronal circuitry which have specific limitations such as an inability to probe networks with tempora
151. nstruction set up and operation To aid in downstream image processing we filled the valve channels with a 58 W W glycerol solution This solution has a refractive index closely matching that of PDMS n 1 41 as estimated from Reference 207 at the location where a valve channel crosses a flow channel only a faintly contrasting line is seen a dramatic improvement from when the valve channel is filled with air or water Fig 2 2 To operate the device initially Valves 1 is closed and Valve 2 is open Worms 34 are pushed into the device by pressure driven flow and are stopped at Valve 1 The injection pressure used was between 2 psi and 4 psi As an animal enters an imaging channel the resistance of that channel increases and thus the flow through that channel decreases Subsequent animals are then diverted to unloaded channels When worms have filled the channels Valve 2 is actuated and closed and the driving fluid pressure is turned off After the completion of the illumination and imaging fluid pressure is turned back on and Valve 1 is opened flushing the animals from the imaging area Valve 1 is then closed and Valve 2 opened beginning another cycle For the infusion of soluble drugs a 20 gauge stainless steel hypodermic tubing Y connector SmallParts was used in the inlet After introducing the worms into the device through one inlet of the Y the inlet was closed with an off chip pinch valve Cole Parmer and a 30 mM solution
152. ntrol of Drosophila behaviors through Channelrhodopsin 2 mediated photoactivation of targeted neurons European Journal of Neuroscience 2007 26 9 p 2405 2416 Arrenberg A B F Del Bene and H Baier Optical control of zebrafish behavior with halorhodopsin Proceedings of the National Academy of Sciences of the United States of America 2009 106 42 p 17968 17973 Arrenberg A B et al Optogenetic Control of Cardiac Function Science 2010 330 6006 p 971 974 Schoonheim P J et al Optogenetic Localization and Genetic Perturbation of Saccade Generating Neurons in Zebrafish Journal of Neuroscience 2010 30 20 p 7111 7120 Umeda K et al Transgenic zebrafish expressing an optimized channelrhodopsin variant under regulation of Gal4 UAS systems optogenetic stimulation of Rohon Beard neurons Journal of Physiological Sciences 2010 60 p S118 S118 Zhu P X et al Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the Tet system Frontiers in Neural Circuits 2009 3 Arenkiel B R et al In vivo light induced activation of neural circuitry in transgenic mice expressing channelrhodopsin 2 Neuron 2007 54 2 p 205 218 Aravanis A M et al An optical neural interface in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology Journal of Neural Engineering 2007 4 3 p S143 S156 Ayling O G S et al Automated light based mapping of mo
153. oblem exists with using the RNAi 54 It is possible that the RNAi in fact does efficiently knockdown or knockout the gene and the gene is essential and therefore those animals did not develop or died only those animals not effectively taking up the RNAi survived and were tested leading to false negatives These factors should be taken into consideration and perhaps it is not completely appropriate to compare RNAi animals to their mutant counterpart Currently Sebastian Wabnig is assessing a new RNAi sensitive strain 213 which should have increased sensitivity while maintaining the viability and health of the animals The over expression of the sid gene in neurons has been shown to have an increased ability of neurons to effectively take up RNAi and demonstrate a more potent knock down effect 213 Furthermore these animals were observed to be healthy and appear much closer in size and offspring number to N2 when compared to other RNAi sensitive animals such as the nre strain 213 We believe this strain might be more suitable to use for the OptloN RNAi screen 55 CHAPTER 3 DESIGN CONSTRUCTION AND CHARACTERIZATION OF A MULTI MODAL OPTICAL ILLUMINATION SYSTEM Much of the work presented in this chapter was originally published 214 Stirman et al Assembly of a multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents Nature Protocols accepted Addition
154. ode Caenorhabditis Elegans Philosophical Transactions of the Royal Society of London Series B Biological Sciences 1986 314 1165 p 1 340 Richmond J E and E M Jorgensen One GABA and two acetylcholine receptors function at the C elegans neuromuscular junction Nature Neuroscience 1999 2 9 p 791 797 Schmitz C P Kinge and H Hutter Axon guidance genes identified in a large scale RNAi screen using the RNAi hypersensitive Caenorhabditis elegans strain nre 1 hd20 lin 15b hd126 Proceedings of the National Academy of Sciences of the United States of America 2007 104 3 p 834 839 Nonet M L et al UNC 11 a Caenorhabditis elegans AP 180 homologue regulates the size and protein composition of synaptic vesicles Molecular Biology of the Cell 1999 10 7 p 2343 2360 Lenth R V Java Applets for Power and Sample Size Computer software from http www stat uiowa edu rlenth Power retrieved 10 November 2011 Calixto A et al Enhanced neuronal RNAi in C elegans using SID 1 Nat Meth 2010 7 7 p 554 559 Stirman J N et al Assembly of a multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents Nature Protocols In Press Macosko E Z et al A hub and spoke circuit drives pheromone attraction and social behaviour in C elegans Nature 2009 458 7242 p 1171 U110 Davis M W et al Gene activation using FLP recombinase in C elegans
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156. omy of the nematode Caenorhabditis elegans The Journal of Comparative Neurology 1975 160 3 p 313 337 White J et al The structure of the nervous system of the nematode Caenorhabditis elegans Philos Trans R Soc Lond B Biol Sci 1986 314 1165 p 1 340 Bargmann C I and L Avery Laser killing of cells in Caenorhabditis elegans Methods in Cell Biology Vol 48 1995 48 p 225 250 Varshney L R et al Structural Properties of the Caenorhabditis elegans Neuronal Network Plos Computational Biology 2011 7 2 p 21 Durbin R M Studies on the development and organisation of the nervous system of C elegans Ph D thesis University of Cambridge United Kingdom 1987 Chalfie M et al The neural circuit for touch sensitivity in Caenorhabditis elegans Journal of Neuroscience 1985 5 4 p 956 964 Hart A C S Sims and J M Kaplan Synaptic code for sensory modalities revealed by C elegans GLR 1 glutamate receptor Nature 1995 378 6552 p 82 85 Hart ed A C Behavior in WormBook T C e R Community Editor WormBook Joshua M K Sensory signaling in Caenorhabditis elegans Current Opinion in Neurobiology 1996 6 4 p 494 499 Tobin D M and C I Bargmann Invertebrate nociception Behaviors neurons and molecules Journal of Neurobiology 2004 61 1 p 161 174 Bargmann C I E Hartwieg and H R Horvitz Odorant selective genes and neurons mediate olfaction in C elegans Cell 1993 74 3 p
157. on Figure 4 12 Control panel for setting complex custom illumination patterns Xi 61 62 64 66 69 71 73 74 77 82 83 83 84 85 86 87 89 90 91 93 95 Figure 4 13 Front panel for encoding the position of the head in the video file Figure 4 14 Front panel of the Complete Video Analysis program Figure 4 15 Extracted parameters from the video analysis Figure 5 1 Sequential frames from acquired videos showing qualitative behavioral responses Figure 5 2 Illumination line scan Figure 5 3 Histograms showing the distributions of positions along the AP axis at which point the blue light elicited a reversal response Figure 5 4 Representative fluorescent images of pmec 4 GFP animals Figure 5 5 Optical stimulation of anterior posterior mechanosensory neurons Figure 5 6 Individual animal responses to anterior stimulus Figure 5 7 Optical stimulation of forward backward command interneurons Figure 5 8 Velocity plots from pooled data from animals receiving different illumination intensities Figure 5 9 Quantification of behavioral responses elicited by different anterior illumination intensities Figure 5 10 Illumination patterns used to explore the integration of anterior posterior signals and behavior generated from the stimulation Figure 5 11 Distributions among the four response states for anterior illumination alone or simultaneous anterior posteri
158. ons and muscles in freely behaving Caenorhabditis elegans Nature Methods 8 153 158 2011 Additionally references to the original publication s of the associated figure can be found in the figure caption 5 1 Motivation The ability to optically excite or silence individual cells using optogenetics has provided a powerful tool to interrogate the nervous system Optogenetic experiments in small organisms have mostly been performed using whole field illumination but genetic methods do not always provide adequate specificity Targeted illumination can be a valuable alternative but to date it has only been shown in non moving animals without the ability to observe behavior output The real time multimodal illumination technology presented in Chapter 3 and 4 allows both tracking and recording the behavior of freely moving Caenorhabditis elegans while stimulating specific cells that express light sensitive proteins We use this system to optically manipulate nodes within the C elegans touch circuit and study the roles of sensory and command neurons and the ultimate behavioral output Together with optogenetic reagents this technology significantly enhances our ability to control alter observe and investigate how neurons muscles and circuits ultimately produce behavior in animals 5 2 Qualitative Behavior Elicited By Structured Illumination We performed two simple experiments to show spatiotemporal control over gross C elegans behavior
159. or illumination at the same intensity 1 17mW mm Figure 5 12 The neural gentle touch circuit Figure 5 13 Multi spectral dynamic capacity of the illumination system Figure 5 14 Simultaneous two color illumination Figure 5 15 Illumination of PVD expressing ChR2 Figure 5 16 Optogenetic dissection of the nociceptive response xii 96 97 100 104 106 107 108 109 110 111 113 114 115 116 117 118 119 121 123 Figure 6 1 Schematic representation of a system for the simultaneous imaging of fluorescently labeled neurons and optogenetic illumination 132 Figure 6 2 Investigation of sensory integration 136 Figure C 1 Quantitative measurements of bending amplitude upon reversal 156 xiii SUMMARY The field of neuroscience has recently seen optogenetics emerge as a highly utilized and powerful method of non invasive neural activation and inhibition As optogenetics becomes a common method to probe neural circuits and function a great amount of research has been dedicated to advancing and enhancing the optogenetic toolbox Thus far much effort has been devoted to the optogenetic reagents themselves increasing sensitivity altering ion channel pump properties and selectivity and altering the activity spectrum While important improvements of the hardware and software used in optogenetic experiments must also be improved the methods of illumination must be made more specific to t
160. or its current position Xstage Ystage Next the X and Y offsets of the center of mass values of the animal X com Ycom from the center of the field of view are calculated AX offer and AY offset These values are converted from pixel measurements to microns using the previously determined calibration factor 6 6 um pixel The stage is then instructed to move the appropriate number of steps to re center the animal In our case one step equals one micron thus the stage is instructed to move AXoffset and AY offset microns in the appropriate direction The stage control loop operates at 25 Hz and thus the animal is maintained within the center of the field of view This allows for both continuous animal tracking for extended time periods and also maintains the animal within the area of the least variance in relative illumination intensity Fig 3 6 The front panel control for stage control is shown below Fig 4 6 Tracking Hz X mult Measured period Y wo F060 SS 1 Figure 4 6 The stage only tracks the object within the field of view when the TRACK button is selected The tracking rate and conversion factors are user defined and the measured tracking loop period and current X and Y positions of the stage are indicated 86 4 3 3 Image Processing Segmentation and Illumination After the live images I are acquired Fig 4 7a and the filtered processed binary image I is created Fig 4 7b the image is sent
161. polarizing MAC thus halting the reversal Scale bar is 250 um 118 a b 500 400 wo o So qt ML li we i N o i lia ji A 4 la fan sill il ng o o o Velocity um s 100 yo 3 id at na i it Scheme 1 n 19 300 ic a ai 400 500 Figure 5 14 Simultaneous two color illumination 117 a Illustrations of the two illumination schemes b Velocity plots of pmec 4 ChR2 and pglr 1 MAC mCherry animals subjected to the illumination schemes in a Error bars s e m n 19 for scheme 1 n 12 for scheme 2 Spontaneous reversals could also be inhibited by green illumination Supplementary Video 8 of Reference 117 suggesting that this is not an artifact by the optogenetic stimulation but a direct interference with the neuronal circuit This experiment illustrates our ability to illuminate a behaving animal with spatial temporal spectral and intensity control The method yields quantitative behavior data that cannot be obtained by manual touch assays laser cell ablation or genetic manipulation of neurotransmitters 119 5 6 Optogenetic Dissection of a Nociception Neural Circuit In this section I will briefly review and discuss additional studies which largely utilize the illumination system and software Chapters 3 and 4 The experiments were performed by Dr Steven Husson in Dr Alexander Gottschalk s lab Goethe University Fran
162. projector thus narrowing the bandpass for each RGB color Adapted from 117 The spectrum of each color of the unmodified Hitachi CP X605 projector is quite broad and would thus cause significant cross activation between optogenetic reagents Similar spectra would be observed for other 3 LCD projectors Therefore to limit the spectral width of the excitation custom filters are added inside the projector the filters in this protocol are chosen to maximize optogenetic activation and minimize cross activation for the optogenetic reagents used later Chapter 5 ChR2 and MAC Fig 1 4 To fit in the projector the new filters must either be custom sized by a filter company e g Semrock or Chroma or cut from a larger filter by a professional glass cutter The specifications dimensions and method of cutting of the filters used in this protocol for the Hitachi CP X605 are found in Table 3 1 Filter sizes for alternative projectors can be determined through careful measuring of the locations for filter insertion The procedure to modify the projector optics and add internal filters are discussed in more detail in Appendix B and are pictorially shown in Fig 3 4 64 Table 3 1 Dimensions and specifications of the custom filters for insertion in to the modified Hitachi CP X605 214 Color Red Green Blue Specifications 600 nm long pass 568 50 nm band pass 475 nm short pass Red Company UQG Optics U K Chroma U S A
163. pulse in the anterior and another set with a simultaneous anterior and posterior pulse with the same intensity The behavioral response based on illumination modes Fig 5 11d show how the combined probability of reversals decreases while the probability for no response increases when the posterior sensory 115 neurons PLML R are excited This suggests that the signals from the anterior and posterior sensors are integrated at all times to produce the proper behavior Additionally certain combinations of anterior and posterior illumination intensities appeared to be conflicting sensory signals and resulted in conflicting commands as the animals quickly alternated between forward and reverse locomotion Supplementary Video 7 of Reference 117 100 90 80 70 60 50 40 30 20 10 Hi NR E sip wer Gr of animals Rep NEP Illumination scheme Figure 5 11 Distributions among the four response states for anterior illumination alone or simultaneous anterior posterior illumination at the same intensity 1 17mW mm n 40 for each Adapted from REFERENCE 117 5 5 Simultaneous Multi Color Illumination Because many of the currently available light sensitive proteins used in optogenetics 124 125 237 are spectrally distinct an illumination system that can be used to illuminate at different wavelengths would be valuable For instance ChR2 is activated in 116 the blue region while NpHR 124 and MAC 125 which bot
164. r 2011 Stephens G J et al Dimensionality and dynamics in the Behavior of C elegans Plos Comp Bio 2008 4 4 p e1000028 Liewald J F et al Optogenetic analysis of synaptic function Nat Methods 2008 Holden Dye L and R J Walker Anthelmintic drugs in WormBook T C e R Community Editor WormBook Goodman M B Mechanosensation in WormBook T C e R Community Editor WormBook White J et al The structure of the nervous system of the nematode Caenorhabditis elegans Philos Trans R Soc Lond B Biol Sci 1986 314 1165 p 1 340 Macosko E et al A hub and spoke circuit drives pheromone attraction and social behaviour in C elegans Nature 2009 458 7242 p 1171 1175 Kaplan J and H Horvitz A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans Proc Natl Acad Sci USA 1993 90 6 p 2227 2231 Brockie P J and A V Maricq Jonotropic glutamate receptors genetics behavior and electrophysiology in WormBook T C e R Community Editor WormBook Park S J M B Goodman and B L Pruitt Analysis of nematode mechanics by piezoresistive displacement clamp Proc Natl Acad Sci USA 2007 104 44 p 17376 Liu Q G Hollopeter and E Jorgensen Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction Proc Natl Acad Sci USA 2009 106 26 p 10823 10828 Mellem J E et al Action potentials contribute to neuronal signaling in C elegans
165. r 4 quarter this might not have the requisite resolution if the neurons expressing ChR2 are located in close proximity to one another or if the position of the neurons change as the animal moves One solution is to fluorescently label the neurons of interest with a soluble fluorescent protein and thereby through fluorescent imaging we can precisely define the locations of the neurons This could be accomplished by co express soluble mCherry and ChR2 in the same neurons and continuously fluorescently image the mCherry signal The fluorescent signal will specifically define the intended targets The projector based illumination system can then illuminate one or more of the neural targets This can both increase processing speed and increase the resolution of the system Because the illumination system is already capable of multi spectral illumination at high intensities excitation of the mCherry would present no challenge The main system alteration is to include simultaneous imaging of a red channel mCherry and bright field for whole animal behavioral recording Fig 6 1 131 7 i kz Far red transmitted x ye light Inverted petri dish containing worm Dichroic 1 Projector or other spatial light modulator Dichroic 2 Camera 1 for O fluorescent imaging Camera 2 for transmitted light imaging used for whole animal behavioral recording Figure 6 1 Schematic representation of a system for the simultaneous imaging of fluorescen
166. r multiple worms were not analyzed We found an average of 6 6 worms could be analyzed per loading cycle Loading isolation and release time averaged 13 seconds and after worm isolation our experiments lasted 7 seconds 2 seconds with no illumination and 5 seconds with blue illumination This averages to 3 loading cycles per minute and an average of 6 6 single worms per loading cycle yielding an over all rate of about 20 animals per minute The experimental loading efficiency depends on a number of factors including worm density and driving fluidic pressure which typically varied in the range of 1 5 5 psi and 300 1200 animals mL There is a trade off between the number of channels with single worms versus multiple worms and the loading time and loading efficiency We found high speed and efficiency of loading Fig 2 4 was best achieved with an injection pressure of 2 5 psi and worm density of 900 38 animals mL The worm density was initially estimated visually and was later confirmed and tested with a custom worm densitometer Chapter 2 5 3 Using our current scheme the rate of processing animals is already two orders of magnitude faster than manual approaches We tested wild type and mutants expressing ChR2 in the motor neurons innervating the body wall muscles zx s3 is a worm strain carrying ChR2 in the GABAergic neurons which act to release an inhibitory transmitter 208 at the neuromuscular junction NMJ of body
167. r even single neurons can be illuminated in freely moving animals We note that however neurons could be activated not only by illuminating cell bodies but also processes This has to be considered if a region harboring the cell of interest also contains dendrites from other neurons expressing ChR2 Thus one should choose promoters as restricted in their expression pattern as possible and consider using combinatorial techniques for expression such as the cre lox system However scenarios can be envisioned where it may be beneficial if neuronal processes can be illuminated rather than cell bodies e g when the cell body of interest is spatially too close to other ChR2 postive cells that should not be activated while the processes of the particular neuron run in a region that is free of other cells or processes Currently we aimed at particular cells using the knowledge of their relative position in the animals body which required to illuminate regions larger than just one cell body such as to ensure illumination of the neuron at all times Nevertheless our system has the potential of higher precision if the cell of interested could be labeled with a fluorescent protein and 125 the fluorescence could be followed The software could be trained to shine light at the desired neuron and not at others also expressing the fluorescent protein 126 CHAPTER 6 THESIS CONTRIBUTIONS AND FUTURE WORK 6 1 Thesis Contributions This thesis
168. ransmission Furthermore they found sub threshold depolarization of neurons was controllable with ChR2 Similarly later in the same year Landmesser s and Herlitze s group demonstrated ChR2 was sufficient to drive neural spiking and synaptic transmission 131 They also demonstrated for the first time in whole animal preparations chic embryos that ChR2 could drive 19 neural sctivity and induce a behavoral response A short time later the Gottschalk group published a paper that in addition to showing ChR2 could alter the depolarization state of excitable cells demonstrated two additional important features 122 First they introduced a ChR2 varient that had a single amino acid substitution H134R This variant was demonstrated to have a larger stationary current and is a gain of function variant ChR2 gf Secondly they demonstrated cell specific expression of ChR2 in the nematode C elegans using cell or tissue specific promoters There are several advantages First this allowed to test the function of ChR2 directly in muscle cells of C elegans showing that when expressed in muscles and illuminated with blue light muscle cells depolarize and cause contraction of musculature in the animal Secondly they expressed ChR2 specifically in neurons of C elegans responsible for gentle touch mechanosensation and showed that ilumination of blue light could indeed cause specific excitation of these neurons rapidly leading to a behavoral respo
169. ray such that the i 0 element becomes the N 1 element and all others are reordered such that iginai N 1 iinitial Calculate Segments Timing Results Thin and Segment Flip HT Width Rate Hz J15 25 Figure 4 8 Control panel used to initiate the thinning and segmenting operations as well as user control of the assignment of the location of the head The location of the head is then maintained by subsequently comparing the two terminal ends of the most recent spline array to the previously annotated and ordered spline array assigning the head location to the end point that has the smallest distance to the previous head location In this way once the assignment is made it is maintained throughout This procedure can fail at times when the animal curls upon itself as in an omega turn however then the user can simply correct using the Flip HT button Now that the location of the head is determined the remaining relative locations can be assigned First the overall length as measured in pixels must be determined The absolute length at each element is found by i Li gt V e Xni tOn Yna n 1 and is defined for i gt 0 The total length is found with i N and the relative length and hence location along the AP axis 0 to 1 is found by dividing the L value by Ly Each xi yi coordinate of the spline is then associated with a relative position along the AP axis The overall length of a young adul
170. re the complete connectome or wiring pattern of all neurons has been reconstructed through serial electron microscopy images 62 This connectome has also seen recent improvements through both additional electron micrographs and computational analysis 64 The wiring diagram of C elegans is a powerful tool for assessment of neural function and association of neural state with behavior If a neuron is identified or implicated in a particular response or behavior other neurons involved in that process can be inferred from the connectivity diagram The connections between neurons are either through gap junctions approximately 900 or through chemical Synapses approximately 6400 additionally there approximately 1500 connections between the nervous system and the musculature the neuromuscular junction NMJ 62 As it with the cell lineage and cellular localization the location of synapses is also highly stereotypical among isogenic animals showing over 75 reproducibility among animal 65 In addition to the basic behaviors such as locomotion feeding foraging and defecation C elegans demonstrates a wide repertoire of more complex behaviors including mating omega turns pirouettes and sex specific behaviors C elegans also demonstrates a number of sensory related behaviors including nociception olfaction propioception mechanosensation and chemosensation 66 72 The circuits responsible for the sensory functions and behaviors are ofte
171. re 3 6 Figure 3 7 Figure 3 8 Figure 3 9 Figure 4 1 Figure 4 2 Figure 4 3 Figure 4 4 Figure 4 5 Figure 4 6 Figure 4 7 Figure 4 8 Figure 4 9 Final optical configuration for the system Optical configuration of the system and components Schematic of final configuration of the modified projector Disassembly and insertion of custom optics into the 3 LCD projector Spectral and intensity characterization of the illumination Measuring the uniformity of illumination across the entire projector image Measuring the effect of lens contrast transfer function on illumination spatial spread Measuring the limits of spatial resolution Measurement of temporal accuracy and resolution Cross pattern used for projector alignment A grid of 20 points is sequentially projected and imaged Software modules for performing imaging stage movements and image processing Front panel of the LabVIEW custom program Live image acquisition and binary image creation The stage only tracks the object within the field of view Custom software for processing the acquired images ultimately creating illumination pattern for the real time illumination of freely behaving C elegans Control panel used to initiate the thinning and segmenting operations User panel for illumination control Figure 4 10 Example of the segmentation of a thinned image into quarters Figure 4 11 This pane displays the illuminati
172. re as we employ would be rate of the image processing display loop 40 ms 3 3 4 2 Temporal Illumination Accuracy The temporal accuracy can be defined as the time it takes for the image to actually appear relative to the time the image was sent to the projector This is affected by the pixel response time rise time At ise Fig 3 9e as well as a known phenomena although not reported by manufacturers of both DLP and LCD projectors monitors and TVs known 75 as input lag or display lag The lag time Atjag Fig 3 9e is mostly due to the image pre processing which occurs within the electronics of the display The lag time was measured as the time it takes from the time the image was sent to the projector until the time the intensity is 5 of the maximum and the rise time was measured as the time it takes to transition from 5 to 95 of the maximum intensity Therefore the overall temporal accuracy is the sum of the lag time plus rise time or the time from image output to reach 95 of the maximum intensity Measuring this over the range 40 50 75 100 125 150 250 500 750 1000 and N 100 for each yielded an average of 70 ms N 1000 with a standard deviation of 7 ms This would be the temporal accuracy of the projector alone Taking into account the automated software for animal tracking image processing and automated illumination would further increase this by 16 ms for image acquisition and 25 ms for image process
173. require an expert in optics engineering or physics to be able to assemble the equipment Recently Leifer et al have described the use of a similar system for optical manipulation of C elegans 143 Although similar in many ways to the system described here there are some important distinctions Leifer et al use a single DMD from Texas Instruments and thus only single color illumination is used at a time compared to the system described in this chapter which can perform simultaneous spatially independent 3 color illumination Secondly Leifer et al use light 143 from either a blue laser 473 nm 5 mW mm or green laser 532 nm 10 mW mm providing spectrally narrower and slightly higher intensity in comparison the system described in this chapter uses the native metal halide light source with the addition of custom bandpass filters blue 430 59 nm 475 nm 4 62 mW mm green 543 nm 593 nm 6 03 mW mm and red 585 nm 670 nm 5 00 mW mm By using the native metal halide light source of the projector no additional cost is incurred as well as simplifying the optical configuration of the system Finally the two systems differ in the software used for real time control and feedback and the closed loop operation speed By using the C programming language optimizing the code and using Intel s Integrated Performance Primitives Leifer et al were able to achieve a closed loop temporal accuracy of 20 ms while
174. rge scale integration multiplexing 25 chamber and channel isolation On chip valves also allow for greater ability to control fluid flow and distribution of reagents and specimens The general flow diagram of creating a microfluidic device and soft lithography is shown below Fig 1 6 Flow Layer Control Layer 74 74 z lt 7 x Xx Design Masks in AutoCAD 7 U7 7X High resolution printing 7X xt 7 lt 7 lt x I Fabrication of masters SU8 a a E and or AZ photoresist on Si wafer Pour spin coat PDMS on master i Ei pa Ez and partially cure Replica molding Peel PDMS from master Align and bond two layers Punch holes and bond to glass slide D Figure 1 6 Process for microfluidic device fabrication Masters are created using photolithography PDMS is then molded on the masters and cured Individual PDMS layers are aligned and bonded and the final device is bonded to a microscope slide 26 1 3 2 Microfluidics in C elegans research Because of its small size C elegans is ideally suited for microfluidic manipulation Microfluidics has largely been applied to studying C elegans in two ways 1 altering the chemical and physical environment around the animal and 2 positioning the animals for imaging and sorting The initial research harnessing the power of microfluidics for C elegans research was performed in the laboratory of Dr Cornelia Bargmann b
175. rm 58 any pattern for complex illumination schemes In addition the illumination patterns can change dynamically and the system can be automated to allow for continuous illumination even in moving targets However the commercially available single chip DLP system two photon and LED based methods may be cost prohibitive to many labs and require substantial knowledge of optical components and design A further limitation of the two photon LED array and single DLP based systems is that they are generally limited to single color illumination If more than a single color is used then it must be achieved by rapid switching between colors and thus it is not truly simultaneous this adds significant complexity due to multi component synching and adds substantial cost In contrast the 3 LCD projector based system presented here has three independent light paths for red green and blue which allow for true simultaneous illumination The off the shelf availability of 3 LCD projectors makes the system presented in this protocol affordable and feasible for implementation in most laboratories By using the native metal halide light source of the projector no additional cost is incurred and the final system is one to two orders of magnitude cheaper than comparable commercial systems Such a light source is standard in fluorescent imaging and provides high brightness illumination across a broad spectrum Furthermore the protocol described here does not
176. rmative it is a read out of the ability to synthesize release or bind neurotransmitter additionally the shape of the length time series plot can be indicative of neurotransmitter 21 recycling Defects in synaptic transmission alter this behavioral output and can therefore be observed and quantified Current methods of performing OptloN investigations are quite labor intensive individual animals picked and are placed on a blank agar plate the plate is then placed on a microscope stage and manually tracked while the animal is illuminated with blue light Videos of this process are acquired and either manually or automatically processed measuring the relative body length as a function of time Although already a powerful new technique OptIoN s current drawbacks of low throughput manual manipulation of animals variation in animal analysis and long data processing time need to be overcome to make it a widely applicable tool in neurogenetics 1 2 4 Techniques for optogenetics illumination Most optogenetic experiments are currently done using either whole field illumination 167 by positioning an optical fiber directly in the vicinity of the neurons 168 169 or by focusing light onto neurons in immobilized animals 137 Thus the illumination is either spatially non specific or it can only be applied to larger or non moving animals The expression of transgenes in a subpopulation of cells is routine in C elegans but precise single
177. rolling and standardizing the force with which animals are stimulated Micro force transducers have been fabricated to allow control of forces 233 While precise the micro force systems are technically demanding particularly when used on behaving animals and when needed to apply in anatomically different positions simultaneously Using light to drive ChR2 the stimulus intensity which translates into signal strength in neurons 234 235 can be easily controlled over a wide range with spatial specificity and in a variety of illumination and intensity profiles Changing the light intensity in optogenetic experiments normally requires changing the lamp voltage or introducing neutral density filters which change the light intensity over the entire field of view With the illumination method described here one can easily control the local intensity by varying the pixel values Fig 3 5b First we show that illumination using graded intensities elicits differential behaviors when stimulating the second anterior quarter of pmec 4 ChR2 animals with blue light We recorded the animals responses to 0 29 1 17 and 4 67 mW mm illumination intensities and reordered whether different stimulation strengths produce behaviors with different probability distributions We grouped the behavior of all the animals illuminated at different intensities into four categories a robust large reversal R defined as reversals with three or more headswings 236 a
178. rum even extending into the infrared making multiplexing and co localization studies possible 48 51 The transparent nature of C elegans makes the use of GFP especially easy and powerful In 1994 Martin Chalfie et al published a paper in Science describing the use of GFP for analysis of gene expression 52 Gene expression analysis had previously been performed with the LacZ X Gal reporter system 53 54 In this system the LacZ gene which encodes f galactosidase is placed downstream from a genetic regulator known as the promoter The promoter element is a sequence of DNA located upstream from the gene of interest and recruits transcription factors that associate with the RNA polymerase thus enabling transcription Along with other regulatory elements the promoter is a critical regulator of genetic translation whereby the protein is only expressed in certain cells and tissues The LacZ X Gal reporter system allows for visualization of cells and tissues where the promoter is active and hence gene of interest is actively expressed B galactosidase can hydrolyze X Gal 5 bromo 4 chloro indolyl galactopyranoside into galactose and 5 bromo 4 chloro 3 hydroxyindole After the latter dimerizes and is oxidized it forms a blue product which can easily be visualized In this way the LacZ X Gal system can be used to visualize specific cells and tissues expressing the gene of interest under the control of the promoter The main drawback to thi
179. s Neuron 1989 3 4 p 473 485 Sulston J E and J G White Regulation and cell autonomy during post embryonic development of Caenorhabditis elegans Developmental Biology 1980 78 2 p 577 597 Bargmann C I and H R Horvitz Chemosensory neurons with overlapping functions direct chemotaxis to multiple chemicals in C elegans Neuron 1991 7 5 p 729 742 Bargmann C I and H R Horvitz Control of larval development by chemosensory neurons in Caenorhabditis elegans Science 1991 251 4998 p 1243 1246 Schnabel R Autonomy and nonautonomy in cell fate specification of muscle in the Caenorhabditis elegans embryo a reciprocal induction Science 1994 263 5152 p 1449 1452 Harbinder S et al Genetically targeted cell disruption in Caenorhabditis elegans Proceedings of the National Academy of Sciences of the United States of America 1997 94 24 p 13128 13133 Goodman M B et al Active Currents Regulate Sensitivity and Dynamic Range in C elegans Neurons Neuron 1998 20 4 p 763 772 Raizen D M and L Avery Electrical activity and behavior in the pharynx of Caenorhabditis elegans Neuron 1994 12 3 p 483 495 Rogers C M et al Regulation of the pharynx of Caenorhabditis elegans by 5 HT octopamine and FMRFamide like neuropeptides Journal of Neurobiology 2001 49 3 p 235 244 Cook A C J Franks and L Holden Dye Electrophysiological recordings from the pharynx in WormBook T C e R Comm
180. s we expect the animal to have little to no biological variation In the analysis of the length of animals before illumination Fig 2 5 we find a standard deviation 2 08 and we believe this is primarily due to experimental noise fluctuations in illumination contrast and variations in length due to motion artifacts The increase of 1 in the standard deviation after the onset of the illumination is due to biological variation and noise this variation cannot be decreased If it were possible to decrease the experimental noise to zero then o 1 and we would only need 4 animals per population for true difference of means 2 5 a 0 05 and power of 0 8 showing a drastic decrease in the required 53 number of animals However it is highly unlikely to be able to decrease the experimental noise significantly First the animals must remain somewhat unrestrained to be able to freely contract or relax and therefore there will always be some motion artifacts Secondly we rely on the contrast difference between the animals and the media aqueous solution for imaging because this difference in refractive index between the animals and the aqueous solution is low the contrast will remain somewhat low and always introduce some noise in imaging Techniques for increasing the contrast such as phase contrast imaging or dark field imaging are possible and might decrease the experimental noise however these remain challenging in microfluidics
181. s after modifications are performed might be due to slight incidental rotational misalignment in the polarizing filters occurring in 69 Step 8 Appendix B Should this be the case these filters can be slightly adjusted the projector reassembled and contrast ratios re measured however this is a timely process and care should be taken to avoid initial misalignment 3 3 2 Illumination Distribution across the Field of view Spectral power measurements light intensity at 460nm 568nm and 620nm were made at the object plane using a PM100D with S121C sensor power meter Thorlabs In order to accurately define the intensity we first measured the variation of intensity across the demagnified projector image at the object specimen plane To do this we projected a spot 20 pixels in diameter and scanned this spot across the XY plane while measuring the power After subtraction of the background light intensity blank projector image we normalized the values to 1 A heat map of this scan is seen in Figure 3 6a along with line scans taken across the center of the image Fig 3 6b c From these plots we observe the central region of the projected light is uniform within 6 of the maximum Because we actively track the animal and keep it within the center of the field of view we never illuminate outside this region We then measured the power illuminating the full area of the projector Dividing this value by the full area of illumination 3 0
182. s involved in the mechanosensory circuit AVA AVB AVD and PVC These measurements will give a baseline in response to chemical stimuli One can then combine the chemical stimulation with optogenetic stimulation of the anterior mechanosensory neurons responsible for escape response and monitor the calcium transients in the same 136 interneurons I expect a modulation of the calcium response based on the addition of an opposing sensation attractive vs repulsive as the interneurons in the two sensory modalities have some overlap Because one would use the blue light activated ChR2 the new red calcium indicator RCaMP Looger Janelia Farm could be simultaneously used To monitor Ca fluorescence and optogenetically stimulate neurons one would use a similar illumination scheme described earlier I have outlined a few techniques for use in dissection of neural circuits in C elegans Other model organisms such as D melanogaster as well as cultured cells are ideally suited for similar studies of neural circuits as outlined in this section The future directions outlined will yield new technologies for optogenetic illumination and by combining those with calcium imaging and microfluidics very sophisticated experiments can be performed dissecting neural circuits and sensory integration in C elegans Using the technologies described in this thesis and the follow up technology and experiments described in this chapter would give new insight into the
183. s method is that the tissue must be fixed and permeabilized for efficient staining This is where the advantages of GFP become readily apparent By placing the DNA sequence encoding GFP downstream of the regulatory promoter GFP can be expressed and visualized in specific cells 52 This was demonstrated in C elegans when GFP was expressed in touch sensitive neurons under the control of the promoter for the mec 7 gene 52 Needing no additional co factors and efficiently folding at room temperatures after expression GFP can be visualized using fluorescent microscopy 43 49 50 52 This means the animals do not need to be fixed and visualization can be performed in living animals GFP can also be fused to proteins and thus can be used to monitor protein trafficking and localization 53 55 with the knowledge that the fusion product might have different diffusion characteristics or trafficking than the endogenous protein The green fluorescent protein has found wide spread use in biology and has transformed experimental biology The three Nobel Prizes demonstrate the utility of C elegans for studies in basic biology The relative simplicity of the animal yet the conservation of biological principles across species have allowed the animal to become a widely utilized organism There are other useful features of C elegans both genetically and physiologically that need be mentioned to motivate the use of this animal in this dissertation
184. s using structured illumination and ChR2 expression First we tracked 103 animals expressing ChR2 in the cholinergic motor neurons ZX460 While the animals were moving forward we illuminated the head with blue light 430 475 nm at regular intervals This produced a dorsal coiling effect 226 when the head was illuminated and resulted in an animal moving in a triangle Fig 5 la In the second experiment we controlled the muscles of neuronally paralyzed animals that express ChR2 ZX299 124 using structured illumination Ivermectin 0 01 mg ml solution a nematocidal agonist of glutamate gated Cl channels which causes neuronal hyperpolarization was delivered to the animals this eliminates the activities of motor neurons which are known to express ivermectin sensitive channels while muscles remained excitable 227 and were controlled with the light pulses Partitioning the paralyzed animal into four quadrants Dorsal Anterior Dorsal Posterior Ventral Anterior and Ventral Posterior and exciting the muscles in alternating patterns we were able to produce S shaped body postures suggestive of locomotion patterns during crawling Fig 5 1b Although non quantitative these experiments demonstrate that illumination of optically controllable cells can be well defined easily controlled and dynamically alterable using the projector system LSS kW kd Figure 5 1 Sequential frames from acquired videos showing qualitative behaviora
185. shooting table 153 ix LIST OF FIGURES Figure 1 1 Organization of a neural circuit Figure 1 2 Mechanosensory circuit of C elegans Figure 1 3 Structure of ChR2 Figure 1 4 Action spectrum of two common optogenetic reagents Figure 1 5 Dye filled image of a microfluidic device Figure 1 6 Process for microfluidic device fabrication Figure 1 7 Microfluidics for precise control of the external environment Figure 1 8 Schematic of a microfluidic device for screening and sorting of C elegans Figure 2 1 Microfluidic chip used in this investigation Figure 2 2 Index matched solution greatly decreases contrast between flow and control layers Figure 2 3 Schematics of computer data processing Figure 2 4 Histogram of the worm loading efficiency Figure 2 5 Contraction and relaxation of C elegans muscles under photoactivation of motor neurons Figure 2 6 Nicotine 30 mM induced contraction and ChR2 induced relaxation Figure 2 7 Device for increased throughput Figure 2 8 Schematic of the automated robotic system Figure 2 9 Measurement of concentration based on light scattering Figure 2 10 Example of a length time series plot from the preliminary RNAi screen Figure 2 11 Master microfluidic control box Figure 2 12 Power analysis Page 10 16 18 19 24 26 28 29 34 35 37 38 40 41 42 43 45 47 49 53 Figure 3 1 Figure 3 2 Figure 3 3 Figure 3 4 Figure 3 5 Figu
186. small molecule enhancer of regeneration Proceedings of the National Academy of Sciences of the United States of America 107 43 p 18342 18347 Zeng F C B Rohde and M F Yanik Sub cellular precision on chip small animal immobilization multi photon imaging and femtosecond laser manipulation Lab on a Chip 2008 8 5 p 653 656 Guo S X et al Femtosecond laser nanoaxotomy lab on achip for in vivo nerve regeneration studies Nature Methods 2008 5 6 p 531 533 Ben Yakar A and F Bourgeois Ultrafast laser nanosurgery in microfluidics for genome wide screenings Current Opinion in Biotechnology 2009 20 1 p 100 105 Ben Yakar A N Chronis and H Lu Microfluidics for the analysis of behavior nerve regeneration and neural cell biology in C elegans Current Opinion in Neurobiology 2009 19 5 p 561 567 Thorsen T S J Maerkl and S R Quake Microfluidic large scale integration Science 2002 298 5593 p 580 584 Ottesen E A et al Microfluidic digital PCR enables multigene analysis of individual environmental bacteria Science 2006 314 5804 p 1464 1467 Unger M A et al Monolithic microfabricated valves and pumps by multilayer soft lithography Science 2000 288 5463 p 113 116 Xia Y N and G M Whitesides Soft lithography Annual Review of Materials Science 1998 28 p 153 184 Hulme S E et al Lifespan on a chip microfluidic chambers for performing lifelong observation of C elega
187. small reversal r defined as reversals with less than three headswings 236 a slowing or pausing response but no reversals SI P and no measurable responses NR Regardless of the illumination intensities we observed that these four categories always exist and are distinguishable Fig 5 8 the number of animals showing NR SI P r and R are 28 14 35 and 43 respectively Grouping the behavioral responses by the illumination intensities Fig 5 9 112 we show that the low intensity stimulation produces a higher probability of no response and slow response in the animals while the animals are much more likely to reverse upon stimulation at higher intensities Fig 5 9b This suggests that the illumination intensities and hence depolarization state of the neuron affect the sensory neuron responses and ultimately modulate the distribution of the behavioral responses 500 400 N Ww o So o oO i rill pint Velocity um s 8 Do a o oO o oO 300 400 Time s Figure 5 8 Velocity plots from pooled data from animals receiving different illumination intensities 117 NR No Response SI P indicates a slowing or pausing of the animal with no negative velocity r is a small reversal and R is a large reversal The number of animals showing NR SI P r and R are 28 14 35 and 43 respectively Error bars s e m 113 a o 100 90 80 70 i NR 60 Bi sip 50 E 40 H
188. spline starting from one end of the spline The final array is determined by adding the X position of the contour point to the X offset of the corresponding element of the spline the same is done for the Y coordinates The array is a composed of cluster of two elements X and Y sequentially ordered along the length of the contour xcontour A xf fset a contour Ayse l i 88 The 0 array element i 0 is one terminal end of the contour and the other terminal end is defined by i N 1 where N is the number of pixels in the original binary thinned image 8 Anatomical locations of neural cell bodies are given in relative coordinates along the AP axis http wormatlas org neuronalwiring html with 0 head and 1 tail To determine the appropriate locations along the AP axis the array of clusters must be ordered such that the 0 element corresponds to the head of the animal Many automated methods have been evaluated to determine which end of the animal is the head however these usually fail especially when the animal reverses and the tail looks very similar to the head To most accurately determine the location of the head we employ manual annotation The program assigns one end of the thinned image as the head and the other as the tail and places a small dot in the location of the 0 element If this incorrectly labels the tail then the user can select the Flip HT button Fig 4 8 which will then reverse the ar
189. st be performed before using the Main Program Inaccurate alignment and calibration could cause mislocalized illumination These steps must be performed on a regular basis e g daily to ensure accurate calibration of the system ii ili iv v vi vii TROUBLESHOOTING Place a highly reflective material on the microscope stage this can be a front coated silver mirror or a blank NGM plate Bring the front surface of the reflective material into focus by focusing the microscope on an imperfection or dust on the surface A window will open on the secondary monitor projector displaying a cross pattern Adjust the location of the projector such that the cross pattern is located roughly in the center of the field of view of the microscope Adjust the rotation of the camera such that the cross pattern lines are perfectly horizontal and vertical There are alignment marks on the image display to aid in this step Repeat steps 24C ii and iii until the cross pattern is centered and horizontal vertical Hit continue to initiate calibration At this step a sequence of 20 solid circles will be projected and the corresponding location will be recorded The calibration parameters for translation from camera coordinates to projector coordinates will be saved Pick an animal onto a blank 6 cm NGM plate 149 viii ix x xi xii xiii xiv xv Allow the animal to freely crawl for approxi
190. stems bringing the technology to any lab The biggest improvement to the system would be to increase the spatial accuracy This would best be accomplished by increasing the temporal accuracy by decreasing the time delay There are a few methods to reduce this time delay and therefore increase the temporal accuracy 1 using a high speed camera 2 optimizing the software image processing thereby reducing the processing time and 3 some newer models of projectors offer a gaming mode which greatly reduces image pre processing and can reduce the input lag by up to 66 Other system improvements or adding system functionality are discussed in Chapter 6 79 CHAPTER 4 SOFTWARE FOR SELECTED AREA ILLUMINATION OF FREELY MOVING C ELEGANS AND BEHAVIORAL ANALYSIS This chapter describes the custom software written for control of the optical illumination system described in Chapter 3 for the purpose of multi spectral optical illumination of neural and muscular targets in freely moving C elegans Software modules were written for control of motorized microscope stage camera acquisition and the projector control Additional modules were used for analysis of worm posture and identification of anatomical locations along the anterior posterior axis of the animal used for defined illumination of targets All modules were integrated into a single program for tracking and illumination Additional programs were written for alignment of the projector sys
191. t animal is usually around 150 pixels and hence each point along the AP axis represents about 0 67 of the total 4 3 3 2 Segmentation The location of segmentation is controlled via user input Fig 4 9 and segmentation is initiated by selecting the Thin and Segment button Fig 4 8 Figure 4 9 User panel for illumination control including animal segmentation properties color intensity and duration of illumination 90 Within the control panel Fig 4 9 the user can select the number of segments S and the locations of the segmentation lines as defined by the AP axis position red box Fig 4 9 The first leftmost numeric control is always set to zero and the next ones define the location of separation along the thinned image In the example in Figure 4 9 the segments are chosen to equally separate the animal into quarters Fig 4 10 Backbone P j Figure 4 10 Example of the segmentation of a thinned image into quarters The white dot indicates the head the red line defines the AP axis and the yellow lines segment the animal In Figure 4 7 the animal is divided into sixths and hence 6 segments S 6 would be entered along with the following values for segmentation 0 0 167 0 333 0 500 0 667 0 833 The segmentation lines according to the segmentation parameters yellow lines Fig 4 7 4 10 are found by first matching the element in the length array Li that most closely matches the desired
192. t equally among the two populations control and mutant is found 212 Fig 2 12a If we fix the power at 0 8 and vary the discriminative ability difference of means we find as the difference decreases the n value drastically increases 212 Fig 2 12b Therefore if we wish to keep the discriminative ability high difference of means 1 5 to 2 5 then the number of animals analyzed must remain between 25 and 50 52 Absolute Difference 0 i 0 0 10 20 30 40 50 O 20 40 60 80 100 120 140 n value per population n value per population Figure 2 12 Power analysis showing the number of animals that must be analyzed for high discriminative ability a Power as a as a function of n value For a given a 0 05 o 2 94 and desired difference of means 2 5 we find n 23 for a power of 0 8 b Keeping power 0 8 a 0 05 and o 2 94 we see that significantly more animals are required for smaller difference of means There are two ways to increase the power of statistical analysis increasing the sample size and decreasing sigma standard deviation If we keep the power 0 8 then the number of animals required for analysis can be decreased by decreasing the standard deviation The spread in data standard deviation comes from two sources experimental noise and biological noise It is difficult to distinguish the two and separate their contributions Prior to illumination the animal is not subjected to any stimulus and thu
193. t gated glutamate receptor Neuron 2007 54 4 p 535 545 Zhang F et al Multimodal fast optical interrogation of neural circuitry Nature 2007 446 7136 p 633 U4 Chow B Y et al High performance genetically targetable optical neural silencing by light driven proton pumps Nature 2010 463 7277 p 98 102 Zemelman B V et al Selective photostimulation of genetically ChARGed neurons Neuron 2002 33 1 p 15 22 Nagel G et al Channelrhodopsin 1 A light gated proton channel in green algae Science 2002 p 2395 2398 Nagel G et al Channelrhodopsin 2 a directly light gated cation selective membrane channel Proceedings of the National Academy of Sciences of the United States of America 2003 p 13940 13945 Sineshchekovy O A K H Jung and J L Spudich Two rhodopsins mediate phototaxis to low and high intensity light in Chlamydomonas reinhardtii Proceedings of the National Academy of Sciences of the United States of America 2002 99 13 p 8689 8694 Nagel G et al Channelrhodopsin 2 a directly light gated cation selective membrane channel Proceedings of the National Academy of Sciences of the United States of America 2003 100 24 p 13940 13945 164 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 Li X et al Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodop
194. t the intended video The first frame of the video will be displayed Place the mouse cursor over the position of the head and hit the enter key Follow the location of the head with the cursor until the program is complete A video will be saved in the same location with the name of the original video plus HE and the location of the cursor for each frame is encoded in the video file Continue ero eblblkbeLenhi No Illumination 640x240 2X 8 bit image63 127 45 To start click the play button A dialog box will then appear asking for video Select video and it will load The first frame will be displayed Place the mouse cursor over the head and hit the enter key Follow the location of the head until the program is done A video will be saved in the same location with the name of the original video plus HE Figure 4 13 Front panel for encoding the position of the head in the video file After starting the program the user follows the position of the head with the cursor 96 4 6 Complete Video Analysis This program is used to analyze the videos for multiple parameters These parameters are saved in a text file which can be opened and analyzed in Microsoft Excel or other data analysis programs 4 6 1 Using the Program Upon starting the program Fig 4 14 a dialog box will appear prompting the user to select the videos for analysis choose the HE video from the last program one can
195. t usage and precious or expensive reagent consumption can be minimized 172 The advantages of microfluidics have been exemplified when culturing cells on chip 171 173 177 performing biochemical assays 178 183 and manipulating and imaging multicellular organisms such as C elegans 99 142 184 195 Another major advantage to using microfluidics is the ability to automate the system There has also been an increasing push to parallelize and multiplex experiments on a single chip through large scale valve integration 196 With both automation and parallelization microfluidics has the potential to perform massive biological experimentation and screening with decreased time manpower and cost 177 197 24 1 3 1 Microfluidic device design and fabrication As microfluidics was born from the microelectronics industry many of the first microfluidic devices were constructed from relatively hard materials such as silicon ceramic materials or glass Currently the more popular choice for device fabrication both for its ease of use rapid prototyping and use of on chip valves is polydimethylsiloxane PDMS 198 199 PDMS is an elastomeric material that is highly deformable It is typically made from a mixture of two components in a 10 1 ratio a base component and a cross linking component After mixing the two liquid components curing occurs at room temperature with elevated temperatures greatly decreasing the curing times The use o
196. technologies to other laboratories In Chapter 3 we set out to develop an inexpensive flexible yet powerful tool for optogenetic illumination The main limitation of current techniques is that they are spatially non specific To overcome this limitation we modified a commercially available 3 LCD projector and integrated it with an inverted epi fluorescent microscope The system was designed to demagnify an image that is created by a computer transferred to the projector and illuminates a sample at the imaging plane The construction of the completed illumination system is fairly simple to perform using the details of the protocol outlined in this thesis and is at least 10x cheaper than other described systems It demonstrated a high spatial resolution capable of producing a spot size of 5 um at 25x magnification The systems main limitation is in the temporal accuracy which is limited to 111 ms and thus ultimately limits the spatial accuracy when dealing with a moving target The system also represents a significant advance in optogenetic illumination technologies because it can simultaneously illuminate any 128 targets within the field of view with independent full control over the illumination color intensity and duration No other systems have previously demonstrated such control and flexibility while remaining economical In order to control the designed illumination system dynamically and have the ability to target neurons and
197. tem and analysis of acquired videos All programs were written in LabVIEW 2009 utilizing the Vision Development Module and Matlab scripts were implemented in some of the modules requiring Matlab The programs discussed in this chapter can be found at http www nature com nmeth journal v8 n2 full nmeth 1555 html supplementary information The rest of the chapter describes the details of the programs and how to use the programs 4 1 General Computer Setup The main purpose of this software is to relay an image to the projector to accurately illuminate samples placed at the imaging plane e g moving C elegans Intended illumination patterns are sent to the projector through a second video port The computer is configured to have two displays one for the main monitor and the second is for the projector with display resolution set to the naive resolution of the projector 1024x768 for the Hitachi CP X605 It is important to configure the computer to have dual display by extending the desktop rather that mirrored Within the LabVIEW programs images 80 are transferred to the projector by displaying the desired image in a window offset to coordinates such that is located entirely on the second monitor i e the projector For example in our LabVIEW software all images are offset by X 1913 and Y 30 The main monitor has an X resolution of 1920 and there is an 8 pixel border around the window This gives an X position of 1921 8 1913 The Y
198. the manipulation of optogenetic reagents The system is capable of multi spectral illumination in definable patterns with the ability to dynamically alter the intensity color and shape of the illumination The illumination system is controlled by a set of software programs introduced in aim three and is demonstrated through a set of experiments in aim four where we selectively activate and inhibit specific neural nodes expressing optogenetic reagents in freely moving C elegans With the ability to target specific nodes in a freely moving animal we can correlate specific neural states to behavior allowing for the ability to dissect neural circuits Taken together the developed technologies for optogenetic researchers will allow for experimentation with previously unattainable speed precision and flexibility XV CHAPTER 1 INTRODUCTION This dissertation utilizes optogenetic and microfluidic methods applied to C elegans in order to increase the available tools for researchers in neurobiology To understand the motivation and application of the developed tools this chapter introduces the three main components of the thesis and challenges with the current technologies C elegans optogenetics and microfluidics 1 1 C elegans as a model organism In the late 1960 s Sidney Brenner began using the small nematode Caenorhabditis elegans for his research in developmental biology and the genetic basis of behavior 1 2 Dr Brenner had a
199. ther limited to a single imaging channel or compressively immobilized the animals and were therefore not suited for our particular purpose We designed a relatively simple microfluidic device capable of loading up to 16 animals into individual imaging channels The channels are sized to be slightly wider than young adults therefore allowing the animals relatively 127 free behavior contraction or elongation The device is designed for rapid loading unloading and generally loading single animals per channel Supporting software was written and successfully demonstrated capability of fully controlling and automating the microfluidic device Furthermore additional software could extract loaded animals and automatically measure the length of the animals as a function of time illumination To further enhance the application of microfluidics for large scale screens we also demonstrated for the first time the integration of microfluidics and traditional high throughput screening technologies such as a robotic liquid handler Although the combined system was not completely automated we still manually washed animals from culture plates this was a significant step in increasing the processing capabilities of microfluidics The additionally developed supporting hardware such as the device to measure the worm concentration and the microfluidic control box also greatly increase the ability to perform screens and the ease of transferring microfluidic
200. ticle Analysis subVIs which we have chosen to not output to the saved text file The following can also be easily found using this VI bounding rectangle area perimeter angle moments of inertia hydraulic radius see IMAQ Vision for LabVIEW User Manual and IMAQ Vision Concepts Manual for more information Additionally within the video the illumination parameters are encoded These are read from the avi file and saved to the final data txt file for each frame The final data txt files saves all the data for each frame and the order of the columns is time illumination level length of animal velocity average 2 pt angles number of 2 pt angles 2 pt angles average 3 pt angles number of 3 pt angles 3 pt angles head to tail distance and amplitude On the front panel Fig 4 14 these calculated parameters are displayed in tabbed windows Also at the bottom of the screen is a color map of both the 2 point and 3 point bending angles In this image time is along the X axis and the Y axis is the location along the animal segments The color indicates the bending angle black is 0 degrees and intensities of the red and blue indicate angle the more intense the color the deeper the angle The video analysis program is not limited to the system discussed in Chapter 3 one can acquire video on any system such as a dissecting microscope Assuming the contrast is sufficient on blank agar plates for example this program can
201. tly labeled neurons and optogenetic illumination Blue light for ChR2 is provided by the projector Chapter 3 and can be spatially pattered Green light for mCherry excitation is also provided by the projector Transmitted light uses a far red source The red mCherry emission and transmitted light are split by a downstream dichroic and are imaged separately onto CCD cameras As a separate technology I suggest the construction of an illumination system capable of multi spectral illumination across a wide field of view gt 2 cm The main difficulty of such a system is maintaining sufficient intensity required for optical activation of optogenetic reagents as well as spatial resolution for targeting distinct sections of C elegans or D melanogaster This system can be constructed either by modifying existing microscopes to relay a projector image to the sample plane at a slight magnification or by constructing a customized microscope system This system would have decreased resolution compared to the system described in Chapter 3 but would be 132 able to target multiple independent animals simultaneously while still maintain the ability to at least segment the animal into anterior and posterior halves This system would allow population assays to be performed rapidly accruing significant amount of data with few experiments 6 2 2 Combine optogenetics and behavioral recording The goal of this section is to outline a few experiments and tec
202. to pursue and complete the task of obtaining my PhD iv TABLE OF CONTENTS Page ACKNOWLEDGEMENTS iv LIST OF TABLES ix LIST OF FIGURES x SUMMARY XIV CHAPTER 1 Introduction 1 1 1 C elegans as a model organism 1 1 1 1 C elegans Development 2 1 1 2 RNAi 3 1 1 3 Tissue cell and protein visualization in C elegans 4 1 1 4 Transgenic animals C elegans nomenclature and genetic maintenance 7 1 1 5 The neuroanatomy and neurophysiology of C elegans 9 1 2 Optogenetics 17 1 2 1 Channelrhodopsin 2 17 1 2 2 Optogenetics and neuroscience 19 1 2 3 Optogenetics in C elegans research 21 1 2 4 Techniques for optogenetics illumination 22 1 3 Microfluidcs 23 1 3 1 Microfluidic device design and fabrication 25 1 3 2 Microfluidics in C elegans research 26 1 4 Thesis Outline 30 2 Microfluidics machine vision and lab automation for high throughput optogenetic screening 2 1 Motivation and overview 2 2 Microfluidic Device Design Fabrication and Operation 2 3 Image acquisition and analysis 2 4 Results 2 5 Automated Robotic Liquid Handling and Integration 2 5 1 Higher throughput device 2 5 2 Integration of microfluidic device and liquid handler 2 5 3 Measuring worm concentration 2 5 4 Preliminary RNAi screen 2 6 C elegans culture 2 7 Master microfluidic control box 2 7 1 Components 2 8 Conclusions 2 8 1 Limitations and considerations 3 Design construction and characterization of a multi modal optical illumination system 3 1 Mot
203. tor cortex by photoactivation of channelrhodopsin 2 transgenic mice Nature Methods 2009 6 3 p 219 224 Cardin J A et al Targeted optogenetic stimulation and recording of neurons in vivo using cell type specific expression of Channelrhodopsin 2 Nature Protocols 2010 5 2 p 247 254 Mahoney T et al Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans Proceedings of the National Academy of Sciences of the United States of America 2008 105 42 p 16350 16355 Liu Q G Hollopeter and E Jorgensen Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction Proceedings of the National Academy of Sciences of the United States of America 2009 106 26 p 10823 10828 Leifer A M et al Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans Nature Methods 8 2 p 147 U71 Lindsay T H T R Thiele and S R Lockery Optogenetic analysis of synaptic transmission in the central nervous system of the nematode Caenorhabditis elegans Nature Communications 2 Narayan A G Laurent and P W Sternberg Transfer characteristics of a thermosensory synapse in Caenorhabditis elegans Proceedings of the National Academy of Sciences of the United States of America 108 23 p 9667 9672 Petzold B C et al Caenorhabditis elegans Body Mechanics Are Regulated by Body Wall Muscle Tone Biophysical Journal 100 8 p 1977 1985
204. uidic flow properties to distribute the worms when a worm occupies a channel the resistance in that channel increases and flow is diverted to other channels Animals are highly restricted in this device providing sufficient immobilization for high magnification imaging and laser ablation 28 A significant number of other microfluidic devices have been developed for imaging animals with high throughput 142 186 189 192 194 195 These devices are similar in operation usually employing a two layer PDMS design where the on chip valves provide mechanical barriers stopping the animals in precise location for imaging 142 186 189 203 and other optical manipulations such as laser axotomy 192 194 and neural laser ablations 204 Additional valves provide a mechanism to divert animals into appropriate channels for sorting A simple microfluidic device used for imaging and sorting is shown in Figure 1 8 a Ag n b c Le Kos s Figure 1 8 Schematic of a microfluidic device for screening and sorting of C elegans a Overview of the device showing the flow layer white containing the animals and the control layer red that form the valves b Cross section of a valve area The top layer is pressurized depressing in into the flow layer creating a partially closed section c Illustration of the completed device with access holed punched d Device with pins inserted to introduce the animals flow layer or
205. und of the slides to solid fill with black as the color Draw the desired geometrical shape Set the RGB color of the object by right clicking the object select Format shape and then select Solid Fill under the Fill tab Under Color select More colors and the Custom tab In this window the specific values for the Red Green and Blue intensities can be set For example Zhang et al used a ring of blue light B 255 G 0 R 0 to confine D Melanogaster larvae expressing ChR2 in nociceptive neurons 148 To create a time series sequence of patterns create patterns for each time point and use the Custom Animations option for determination of the transition times Place the sample on the microscope and bring it into focus To project the created objects or animations set the presentation to display on the secondary monitor and begin the slide show C Selected area illumination of C elegans using custom software 117 TIMING 0 25 h i Open the Beamer alignment program and start with the play button 148 CRITICAL STEP These steps describe using the custom software written for our specific camera and motorized stage In order to adapt it to other cameras and stages a few alterations must be made to the software These are discussed in more detail in the supporting documentation of the software 117 A CRITICAL STEP Steps 24C iv are critical calibration steps that mu
206. unity Editor WormBook Richmond J E Electrophysiological recordings from the neuromuscular junction of C elegans in WormBook T C e R Community Editor WormBook Kerr R et al Optical imaging of calcium transients in neurons and pharyngeal muscle of C elegans Neuron 2000 26 3 p 583 594 Shyn S I R Kerr and W R Schafer Serotonin and G o modulate functional states of neurons and muscles controlling C elegans egg laying behavior Current Biology 2003 13 21 p 1910 1915 Suzuki H et al In vivo imaging of C elegans mechanosensory neurons demonstrates a specific role for the MEC 4 channel in the process of gentle touch sensation Neuron 2003 39 6 p 1005 1017 Kahn Kirby A H et al Specific polyunsaturated fatty acids drive TRPV dependent sensory signaling in vivo Cell 2004 119 6 p 889 900 Kimura K D et al The C elegans thermosensory neuron AFD responds to warming Current Biology 2004 14 14 p 1291 1295 Shimozono S et al Slow Ca2 dynamics in pharyngeal muscles in Caenorhabditis elegans during fast pumping Embo Reports 2004 5 5 p 521 526 162 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 Hilliard M A et al In vivo imaging of C elegans ASH neurons cellular response and adaptation to chemical repellents Embo Journal 2005 24 1 p 63 72 Chronis N M Zimmer and C I Bargmann Microf
207. upernatant and adding the appropriate volume of M9 buffer For each animal population this was done and the final animal solution was transferred to the multi well plate This was done for 6 24 populations of animals at a time a a Relative Worm b 2 Concentration 5 4 12 worms mL S12 2 10 1500 2 10 8 900 8 D6 450 Ze a 3 3 9 150 g 4 2 2 a 8 0 3 0 0 10 20 3 40 50 60 70 80 0 300 600 900 1200 1500 Time s Worm Concentration worms mL Figure 2 9 Measurement of concentration based on light scattering a Measure of scattering as a function of time indicating rate of worm settling to the bottom of the centrifuge tube b Quantification of worm density by measuring the amount of light scattered into the detector at t 60s Control of the Gilson 215 liquid handler was achieved by custom software in LabVIEW Serial commands were sent to the Gilson 215 by ActiveX commands controlled in LabVIEW The off chip pinch valves and on chip valves were controlled by 45 the master control box see Section 2 7 operated via the LabVIEW program The LabVIEW program for automated analysis was similar to that discussed in section 2 3 2 5 4 Preliminary RNAi screen In order to test the system and C elegans genetic constructs we performed a preliminary RNAi screen for synaptic transmission with Sabastian Wabnig Dr Gottschalk lab Goethe University This screen utilized both cholineregic and gabaergic anim
208. uracy 3 3 4 1 Temporal Illumination Resolution Analogous to the measurements made for spatial resolution Fig 3 7 3 8 the temporal resolution was measured Fig 3 9 Due to the pixel response time time it takes for the LCD pixel elements to change polarization states as the applied value is changed from 0 to 255 and back again and the refresh rate of the projector it is expected that a measured pulse of light from the projector will be less than the ideal or intended pulse duration To test the temporal resolution we placed a Switchable Gain Amplified Silicon Detector PDA100A Thorlabs at the focal plane of the objective The detector was set to a gain of 40 dB and analog voltage measurements were acquired with an analog data acquisition unit USB 6221 National Instruments Measurements were recorded in a LabVIEW program written to acquire measurements from the DAQ card in a loop operating at 2 KHz In a separate loop images were sent to the projector in predefined pulse durations The on of the pulses were simple blue squares of value 255 and the off was a black image 50 duty cycle The duration of the pulse was measured and characterized by the full width half maximum Atgpwym of the pulse Fig 3 9a d performed over a range of 40 ms to 1000 ms As expected the measured pulse width is less than the intended pulse duration and would be ultimately limited by the refresh rate of the projector 17 ms or for custom softwa
209. ure dependent 3 4 Drs Sulston and Horvitz took on the task of tracking the complete cell lineage among the developing post embryonic animal 5 During this time the animal develops from 671 cells L1 to a total of 959 somatic cells in the adult hermaphrodite 302 of these are neurons and 95 are body wall muscle cells 6 They found that this development is stereotypical among animals and they could map all final cells to precursor cells Later Dr John Sulston mapped the cell lineage from the single cell fertilized egg stage to the L1 larval stage 7 This completed the complete lineage mapping and is still the only complete mapping of cell division and lineage analysis of an animal In itself the cell lineage mapping is an enormous task and finding But additionally this led the researchers and specifically Robert Horvitz to study and understand the mechanisms of cellular apoptosis which had previously been reported 8 10 During the study of cellular division and differentiation they found that there were 131 cells that existed in the L1 hatched larva that did not give rise to daughter cells or survive to adulthood they underwent programmed cell death apoptosis 5 7 11 In both of these studies these researchers their collaborators students and postdocs found and characterized a number of mutant animals In discovering mutants characterizing the nature of the mutation and eventually finding the gene responsible C el
210. urnal of Comparative Physiology a Sensory Neural and Behavioral Physiology 1991 169 1 p 59 67 Sweeney S T et al Targeted expression of tetanus toxin light chain in Drosophila specifically eliminates synaptic transmission and causes behavioral defects Neuron 1995 14 2 p 341 351 Hedgecock E M and R L Russell Normal and mutant thermotaxis in nematode Caenorhabditis elegans Proceedings of the National Academy of Sciences of the United States of America 1975 72 10 p 4061 4065 Mori I and Y Ohshima Neural regulation of thermotaxis in Caenorhabditis elegans Nature 1995 376 6538 p 344 348 Zimmer M et al Neurons Detect Increases and Decreases in Oxygen Levels Using Distinct Guanylate Cyclases Neuron 2009 61 6 p 865 879 Albrecht D R and C I Bargmann High content behavioral analysis of Caenorhabditis elegans in precise spatiotemporal chemical environments Nat Meth 2011 8 7 p 599 605 Gray J M J J Hill and C I Bargmann A circuit for navigation in Caenorhabditis elegans Proceedings of the National Academy of Sciences of the United States of America 2005 102 9 p 3184 3191 Moulder G L et al alpha Actinin Is Required for the Proper Assembly of Z Disk Focal Adhesion Like Structures and for Efficient Locomotion in Caenorhabditis elegans Journal of Molecular Biology 2010 403 4 p 516 528 Qadota H et al PKN 1 a Homologue of Mammalian PKN Is Involved in the Regulation of Musc
211. use this resolution is sufficient to distinguish the anterior and the posterior sensors and it ensures illumination of the relevant cell bodies in all animals When the last quarter was illuminated with blue light thus exciting PLML R neurons we observed the expected velocity increase Fig 5 5b Conversely when the second quarter of the body was illuminated exciting AVM and ALML R neurons we observed a large velocity decrease followed by a reversal Fig 5 5b These behaviors were robust and reproducible between animals as can be seen in individual animal traces Fig 5 6 This demonstrates that the optical illumination system and software is well suited to investigate the neural basis of behavior and is capable of eliciting cell specific behaviors a b pmec 4 ChR2 600 ALML R PLML R 400 f N ie iil sit i i puit it o AVM PVM N 400 Velocity um s71 Time s Figure 5 5 Optical stimulation of anterior posterior mechanosensory neurons Adapted from REFERENCE 117 a Illustration of the positions of neurons expressing ChR2 in pmec 4 ChR2 transgenic worms b Average velocity plots of pmec 4 ChR2 animals under illumination conditions shown as a blue bar above n 13 posterior illumination n 15 anterior illumination Error bars s e m 109 a 1000 800 Vp ve Ng hi be A aN aE ie ai ian aul phy re RUA Velocity um s 1000 1200 0 1 2 3 4
212. using the full resolution of the camera 1024 x 768 143 We chose to use LabVIEW with Vision software discussed in Chapter 4 for its ease of use for non programming experts and our system operates with a closed loop temporal accuracy of 111 ms at a camera resolution of 320 x 240 Both systems provide similar software user interfaces and options as well as subsequent data analysis capabilities 3 1 3 Overview of the Procedure The overall objective of the steps presented in this protocol is relatively simple to take an image created by a projector and instead of enlarging it and projecting it onto a screen to relay the image through the epifluorescent port on a microscope and transfer a demagnified image to the sample plane Fig 3 1 60 Sample plane Camera C mount coupler Inverted petri dish Motorized XY stage 1 Primary Projector Image 662nm Dichroic Accessory tube lens Infinity corrected or Relay lens pair 160 mm Figure 3 1 Final optical configuration for the system Adapted from 117 The epifluorescent optics are replaced by an accessory tube lens infinity corrected or relay lens pair 160 mm and a modified 3 LCD projector A projector operates by shining light through a spatial light modulator SLM in this case an LCD and thereby creating an image composed of hundreds of thousands of individual pixels defined by the individually addressable SLM pixel elements The image formed at the
213. video s HE avi to be analyzed The data based on the video will be saved to a text file with the extension data txt The data order of the columns is time illumination level length of animal velocity average 2 pt angles number of 2 pt angles 2 pt angles average 3 pt angles number of 3 pt angles 3 pt angles and head to tail distance TROUBLESHOOTING See Table 1 for troubleshooting advice 151 e TIMING Steps 1 13 Modification of the LCD projector 2 5 hrs Steps 14 17 Adjustment of the projector settings 0 25 hrs Step 18A Assembly of projector and microscope for infinity corrected microscope 3 hrs Steps 18B Assembly of projector and microscope for 160 mm microscope 3 hrs Steps 19 23 Alignments of the system 1 hr Step 24A Simple illumination using a mouse pointer 0 10 hr Step 24B Static or pre defined dynamic illumination using PowerPoint 0 25 hr Step 24C Selected area illumination of C elegans using custom software 0 25 hr 152 TABLE B 1 Troubleshooting table Step 4 14 18 A v 18 B v 21 24 C i Problem Case cover will not slide off Filters will not fit Projector will not turn on Dim image Color is missing absent Color or image is striped Insufficient space to position the projector Insufficient space to position the projector Image never focuses Program gives error upon start Possible reason Not all screws have been removed M
214. was measured for the time of reversal gray box and the maximum A L was found These values A Lmax were compared between wild type and mutants Another aspect of the research presented in this thesis was the transfer of technology In many of the projects we undertake in Dr Lu s laboratory we work closely with collaborators around the United States and internationally As we develop 156 technologies to advance the state of the art in neuroscience and aid our collaborators in their research they often wish to have the technology fully accessible and available in their own lab As such much of our time is spent insuring the hardware and software we develop is easily used by others often non engineers and non experts in microfluidics imaging or programming I have been very grateful to work with Dr Gottschalk and his research group and have the opportunity to visit his lab Frankfurt Germany twice each for a month During these visits I was responsible for transferring the technologies discussed in Chapters 2 5 and establishing the systems along with training students in the lab on proper usage and troubleshooting Over the past few years of developing and transferring the technology I have had significant interaction with the collaborators discussing alterations to the hardware and software to make it more user friendly and accessible The final software represents multiple iterations adding additional functionality and making the
215. was previously on as the bulb is very hot It is also suggested that one works on an anti static mat 1l 2l 3l 4l Begin by removing the frame around the projector lens to be able to remove lens For the Hitachi CP X605 there are two screws on the bottom of the frame and two additional screws that can be found by opening the lens shift cover on the top of the case that must be unscrewed After removing the frame remove the entire zoom lens by pressing up on the lens release latch Fig 3 4a and twisting the lens counter clockwise Carefully set the lens aside A CRITICAL STEP Use care when handling the projector lens to ensure it is not damaged or scratched as it will be used later A CRITICAL STEP for some projectors the lens assembly is not able to be removed For those projectors this step can be omitted Remove the screws on the back of the projector case so that the internal circuit boards can later be removed There are ten such screws on the back of the Hitachi CP X605 projector to be removed Locate and remove the screws on the bottom of the projector which connect the main body and the top of the projector case The Hitachi CP X605 has nine screws on the bottom of the projector Fig 3 4b holding the case together Remove the screws and save for later reassembly Return projector to the upright position Carefully begin to lift off the top portion of the case Angle the cover back and look inside to locate connector ca
216. worms are located during active tracking of the animals This demonstrates that the uniformity of the projected illumination intensity is within 6 of the maximum Knowing the calibration factors one can also correct compensate during illumination if desired b Variation of intensity across the X axis at Y 384 Dotted red lines represent central region c Variation of intensity across the Y axis at X 512 Dotted red lines represent central region 3 3 3 Spatial Resolution and Accuracy There are a number of factors involved in determination of system resolution and accuracy We will address each individually below 71 3 3 3 1 Imaging Resolution The limits of resolution follow the well known equation Resolution r 0 61 NA where is the wavelength of the light used for imaging and NA is the numerical aperature of the objective utilized We use a 4x objective with NA 0 1 and image using 650 nm light This yields a limit of resolution of about 4 um Our camera is measured to have 6 6 um pixel at 320x240 and using the Rayleigh criteria needing 2 pixels to define a spot this yields a camera resolution of 13 2 um Thus we are camera limited when imaging 3 3 3 2 umination Spatial Resolution A 4x objective was used for all experiments At this magnification the theoretical size limit of illumination is 3um corresponding to one pixel on the projector However due to contrast transfer functions of optical lenses and imagi
217. xperiments that would directly follow up on the work I have discussed in this thesis 6 2 1 Optimize the methods for optogenetic illumination The objective of this section is to enhance the methods for optogenetic illumination increase the spatial and temporal resolution and develop technologies to perform high resolution illumination of multiple animals simultaneously The methods for illumination discussed in this thesis targeting neurons in freely moving animals are based largely on anatomical features and thus relying on pre defined stereotypical locations of the neurons This leads to some uncertainty about the extent and precision of illumination and hence uncertainty about the excitation of optogenetic reagents Additionally these methods though performed at low magnifications 4x are still limited to single animals The engineering methods suggested in this section seek to fill the gaps of the optogenetic 130 illumination methods presented in this thesis allowing for targeted illumination of neurons based on locating the neurons directly and developing technologies to perform population based assays The technologies outlined in this section are highly transferrable and well suited for studies in D melanogaster larvae and cultured cells as well A current limitation of the illumination system Chapter 3 is the accuracy when illuminating intended targets Although we can target specific sections of the animal e g 1 quarte
218. y thinning process bwmorph BW thin inf can be found in Reference 224 Originally this algorithm was written and executed completely in the LabVIEW environment However we found that it takes roughly twice the processing time to complete the operation in LabVIEW than performing the operation within the Matlab script The thinning process is proportional to the total number of pixels that compose the binary image Therefore to reduce the processing time we rescale the original live image size 640x480 to 320x240 which results in a total thinning time per image of about 15 ms This single pixel backbone determines the anterior posterior AP axis of the animal After determination of the single pixel backbone we then need to determine the length of this backbone and from that determine the relative locations along the AP axis The newly created single pixel thinned binary image I is processed by the Get Points on Contour subVI This subVI takes a binary image input and determines the XY coordinates along a single contour of the image Furthermore this subVI fits a cubic spline to all the contour points thus finding a smooth curve that minimizes the error between the fit curve and the descretized points along the contour The final output array of the subVI indicates the XY coordinates of the original points along the contour and the XY offset of those points to the best fit cubic spline The array is ordered sequentially along the
219. y Dr Hang Lu In this initial work the external environment was manipulated and the behavior of animals introduced into the environment was observed In one study olfactory learning in C elegans was investigated by observing the preferential migration of animals toward a particular food source bacterial strain 185 The animals are introduced into a central arena made of PDMS which is surrounded by several channels terminating with a chamber containing various forms of bacteria Fig 1 7a Animals will travel to those areas to which it is attracted and avoid those to which they have an aversion The microfluidics provides a highly controlled manner of defining the olfactory cues that guide C elegans In another study a gradient of oxygen was established in a PDMS device Fig 1 7b 184 The animals will migrate to the area of the device with their preferred O concentration Animals deficient in O2 sensation can similarly be examined in the device Again microfluidics provides a means of establishing a precisely defined external environment not possible by traditional means Another powerful use of microfluidics applied to C elegans neuroscience was developed by Nikos Chronis again in Dr Bargmann s laboratory In this study they combined microfluidics and Calcium imaging 99 By using laminar flow in a microfluidic channel they could precisely define the concentration and duration of a chemical stimulus flowing over the nose of an animal
220. y thesis committee for valuable guidance and advice on shaping the structure and content of this thesis I thank Dr Philip Santangelo whom I have known since entering the graduate program He has given me valuable advice on a number of both research and non research related issues Finally I express my great gratitude to my long distance committee member and close collaborator Dr Alexander Gottschalk Through his expertise in C elegans neuroscience optogenetics and experimentation and our constant communication we have had a highly successful collaboration I thank all current and past members of the Lu lab I make special note of the two founding members of the lab Kwanghun Chung and Edward Park both who provided a great deal of teaching and troubleshooting and without both I feel the lab and all successive members would not have been as successful Of all the great members of the lab I would especially like to express my thanks to Matthew Crane During my time here he has provided valuable assistance and guidance both experimentally and educationally More importantly he has been a great sounding board and friend I express my great gratitude and love to all my family members and most especially my parents who have given me much needed support through all my many years of school Finally I would like express my thanks and love to my fianc e Gina Cremona who has given me love support and advice over the past several years allowing me

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