A multispectral optical illumination system with precise
Contents
1. MAC mCherry pelt 2 GFP was subjected to blue illumination thus exciting ChR2 in the region containing the anterior sensory neurons thus initiating a reversal and it was subsequently illuminated with green light exciting MAC thus inhibiting signal transmission and halting the reversal Fig 6c and ref 12 http www nature com nmeth journal v8 n2 extref nmeth 1555 S9 mov ACKNOWLEDGMENTS We thank members of the Caenorhabditis Genetic Center W Schafer Y Tanizawa Medical Research Council Laboratory of Molecular Biology Cambridge UK and E Boyden Massachusetts Institute of Technology for reagents the US National Institutes of Health H L the Alfred P Sloan Foundation H L the Human Frontier Science Program Organization S J H the Deutsche Forschungsgemeinschaft grants GO1011 2 1 SFB807 P11 FOR1279 P1 EXC115 1 and the Schram Foundation A G for funding We also thank J Andrews and B Parker in the Georgia Institute of Technology School of Chemical and Biomolecular Engineering machine shop and D Woodyard in the glass shop AUTHOR CONTRIBUTIONS J N S designed and constructed the illumination system J N S and M M C characterized the system S J H and A G contributed to reagents and provided valuable discussions J N S M M C S J H A G and H L designed the experiments J N S and H L prepared the manuscript COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests Pub2. 160 mm OLIC objective lens infinity corrected RLP relay lens pair IP 160 mm back image plane SLM spatial light modulator SP sample plane M lt 1 Adapted from reference 12 inputs or closed loop automated analysis of images Fig 2 e g targeting neurons and muscles in C elegans Experimental design Choice of 3 LCD projector A few considerations must be taken into account when selecting a 3 LCD projector The main specifica tions 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 projec tor with the same reported brightness yet smaller LCD panels 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 protocol 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 back ground illumination To minimize the background illumination thus preventing unwanted excitation of th
3. alternative projectors can be determined through careful mea suring Supplementary Note 2 of the locations for filter insertion in PROCEDURE Step 9 The postmodification spectrum Fig 3c has much narrower spectral widths for each color allowing for highly defined multicolor excitation Modification of microscope optics for infinity corrected systems The epifluorescence 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 lens system and when imaging the amount of magnification is determined by the ratio of the focal lengths of two lenses M TL OL gt 1 To transfer the projector s primary image PPI Fig 1c to the sample plane Fig 1c an accessory tube lens ATL Fig 1c 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 FL FL which will yield M lt 1 or cause demagnification Tube lenses from different microscope manufacturers have different focal lengths Leica 200 mm Nikon 200 mm Olympus 180 mm a
4. at x25 Adapted from reference 12 Q O Measured Ideal oO 0 8 Q oO 0 6 aN O 0 4 Relative intensity Relative intensity N O 0 2 O O N S Q A Ideal Measured Ideal Measured A l N O N a O Oo o N O Oo ANTICIPATED RESULTS Characterization of the illumination v system 0 25 50 75 100 125 150 175 200 0 5 10 15 20 235 30 After insertion of the internal filters Projected spot size um Projected spot size um in the 3 LCD projector the spectrum of the three color planes red green blue are spectrally restricted Fig 3c on the basis of the specifications of the filters Supplementary Table 1 as measured using a spectrometer CCS100 Thorlabs The narrow bandwidth of the spectrum al lows for sufficient separation of wavelength to excite distinct optogenetic reagents These results are expected on the basis of the band pass values of the filters If other filters are chosen the modified spectra should reflect those filter specifica tions 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 they 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 8 bit integer 0 255 for each color and thus defines the relative intensity at that location
5. elegans To demonstrate the capabilities of the system for dynamic illumination of a sample a few experiments were conducted on C elegans specimens expressing optogenetic reagents The first experiment shows dynamic control of an illumination pattern 218 VOL 7 NO 2 2012 NATURE PROTOCOLS npg 2012 Nature America Inc All rights reserved PROTOCOL a J z Figure 6 Example application selected area illumination of C elegans a Frames from supplementary video 2 of reference 12 demonstrating direct muscle control of a paralyzed animal using patterned light b Sequential frames from supplementary video 3 of reference 12 showing a bar of light passing over the worm from the posterior to the anterior region as the animal is freely crawling Initially the worm is traveling forward however when the light reaches the anterior mechanosensory neurons expressing ChR2 middle frame the worm quickly reverses direction c Sequential frames from supplementary video 8 of reference 12 showing the multispectral dynamic capacity of the illumination system The worm is illuminated with blue light in the region of the anterior mechanosensory neurons which express ChR2 thus eliciting a reversal The worm is subsequently illuminated with green light in the region of the command interneurons which express the hyperpolarizing MAC thus halting the reversal Scale bars 250 um used for direct control of musc
6. gener ally expressed in a larger population of cells Although there are techniques for single cell expression including the use of Cre or FLP recombinases these can be unreliable or they may not allow for sufficient expression of optogenetic reagents Furthermore to investigate integration of distinct neural signals expression in multiple cells is required To fully realize the potential of the optogenetic reagents the toolbox must be expanded to include techniques for specific and localized optical targeting of excitable cells In addition because currently available optogenetic reagents cover a broad range of the optical spectrum the ability to have multispectral optical illumination is valuable In this protocol we present a procedure to modify a commer cially available three panel liquid crystal display 3 LCD projector and integrate it with most inverted epifluorescence microscopes for the purpose of patterned illumination on a sample as was shown previously for the optogenetic activation and inhibition of neurons and muscles in C elegans The protocol allows for fully reversible modification of the microscope system Once completed the illumination system is capable of multicolor illumination and it can be applied to both static and moving samples The illumina tion pattern is defined by a computer and sent to the projector as a second video output the image is then relayed from the projec tor to the microscope and
7. of 320 x 240 Both systems provide similar software user interfaces and options as well as subsequent data analysis capabilities Overview of the procedure The overall objective of the steps presented in this protocol is rela tively 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 epifluorescence port on a microscope and transfer a demagnified image to the sample plane Fig la A projector operates by shining light through a spatial light modulator SLM in this case an LCD thereby creating an image composed of hun dreds of thousands of individual pixels defined by the individually addressable SLM pixel elements The image formed at the 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 1b 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 epifluorescence optical train of an inverted microscope passing through the objective forming a demagnified image at the focal plane of the objective specimen plane Fig 1c d It is in this specimen plane that the object of inter est e g freely moving C elegans is located and illuminated The image projected onto the sample plane can be co
8. 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 NATURE PROTOCOLS VOL 7 NO 2 2012 215 PROTOCOL A CRITICAL STEP Step 24C i iv is a crucial calibration step that must be performed before using the main program Inaccurate alignment and calibration could cause mislocalized illumination This step must be performed on a regular basis e g daily in order to ensure accurate calibration of the system TROUBLESHOOTING 11 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 iii 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 iv Adjust the rotation of the camera such that the cross patterned lines are perfectly horizontal and vertical There are alignment marks on the image display to aid in this step vi Press 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 wi
9. which the MATERIALS REAGENTS Experimental C elegans with suitable expression of optogenetic reagents CAUTION All animal experiments must comply with relevant institutional and governmental animal care guidelines Blank unseeded nematode growth medium NGM plates EQUIPMENT General equipment Inverted fluorescence microscope infinity corrected or 160 mm e Motorized microscope X Y stage optional for moving samples e Stage insert for microscope stage capable of holding a 6 cm Petri dish oO Relative sensitivity Relative intensity PROTOCOL 1 0 O ChR2 l MAC 0 5 400 450 500 550 600 650 700 Wavelength nm 100 Blue post filter Green post filter Red post filter Blue pre filter Green pre filter Red pre filter 80 60 40 20 650 400 450 500 550 700 Wavelength nm worms backbone spline is to be divided The output parameters are saved to a text file and are described as follows e Velocity By using the previous and current position of the motorized stage the position of the animal within the field of view and the calibration of the camera micrometers per pixel the velocity of the animal is calculated e Two point angles The angle between two successive points is determined relative to 90 and normalized such that the expecta tion value of all angles is equal to zero Three point angles Similar to the two point angles except that the angle i
10. 2009 NATURE PROTOCOLS VOL 7 NO 2 2012 219 npg 2012 Nature America Inc All rights reserved PROTOCOL 10 ti 12 13 14 15 16 17 18 19 20 21 22 23 24 25 Guo Z V Hart A C amp Ramanathan S Optical interrogation of neural circuits in Caenorhabditis elegans Nat Methods 6 891 896 2009 Stirman J N Brauner M Gottschalk A amp Lu H High throughput study of synaptic transmission at the neuromuscular junction enabled by optogenetics and microfluidics J Neurosci Methods 191 90 93 2010 Stirman J N et al Real time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans Nat Methods 8 153 158 2011 Leifer A M Fang Yen C Gershow M Alkema M J amp Samuel A D T Optogenetic manipulation of neural activity in freely moving Caenorhabditis elegans Nat Methods 8 147 152 2011 Schultheis C Liewald J F Bamberg E Nagel G amp Gottschalk A Optogenetic long term manipulation of behavior and animal development PLoS ONE 6 e18766 2011 Fiala A et al Light induced activation of neurons in Drosophila using channelrhodopsin 2 J Neurogenet 20 115 116 2006 Schroll C et al Light induced activation of distinct modulatory neurons triggers appetitive or aversive learning in Drosophila larvae Curr Biol 16 1741 1747 2006 Suh G S B et al Light activation of an innate ol
11. Fig 5a The contrast ratio for each color was determined by the ratio of full on pixels set to 255 for that color Fig 5a to full off zero pixel value Fig 5a and for the modified Hitachi CP X605 projector the ratios are as follows red 896 1 green 463 1 and blue 605 1 Ratios were measured using a power meter PM100D Thorlabs These values are less than the manufacturer s stated contrast ratio of 1 000 1 as a result of 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 in order 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 decreases i e greater than a twofold decrease in contrast ratios after modifications are performed might be the slight incidental rotational misalignment in the polarizing filters occurring in PROCEDURE Step 8 Should this be the case these filters can be slightly adjusted the projector reassembled and contrast ratios remeasured however this is a time consuming process and care should be taken to avoid initial misalignment The contrast transfer function of the projector and microscope optics will spread the area of illumination over a larger area Th
12. al circuits and synaptic functions in C elegans In addition this technology can replace or supplement other techno logies used for illumination in other model systems including D melanogaster D rerio and cells in which region specific illumination of optogenetic reagents is beneficial We also envision NATURE PROTOCOLS VOL 7 NO 2 2012 207 npg 2012 Nature America Inc All rights reserved PROTOCOL that this 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 cre ate a system for patterned illumination the system can be used in place of existing techniques that use spatially defined illumina tion including enhancing resolution by reconstruction of samples using structured illumination technique and patterned photo crosslinking In addition the illumination intensity is sufficient to perform standard fluorescent imaging and the multispectral capability of the illumination system can allow for simultaneous multicolor fluorescence imaging When extremely fast shuttering lt 15 ms is not needed the projector can replace the excitation epifluorescence 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 shutteri
13. ated illumination patterns can be set with the Scheduled op tion see Read Me Program Overview in supplementary software from ref 12 xv To begin the segmentation Fig 2d 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 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 it with the play button Place the mouse cursor over the head of the animal and press the Enter button on the keyboard Follow the position of the head with the cursor as the video is played The encoded video will automatically be saved with the name of the original file plus HE xxii To analyze the completed video open the Complete Video Analysis program xxiii Enter the calibration value for micrometers per pixel at full resolution and select the binning of the camera used For the data obtained we measured 3 3 um per pixel and used a 2 x 2 binning thus providing a calibration of 6 6 um per pixel for our recorded videos xxiv Start the program with the
14. control of neuronal activity Nano Lett 7 3859 3863 2007 Grossman N et al Multi site optical excitation using ChR2 and micro LED array J Neural Eng 7 16004 2010 Delica S amp Blanca C M Wide field depth sectioning fluorescence microscopy using projector generated patterned illumination Appl Opt 46 7237 7243 2007 Itoga K Yamato M Kobayashi J Kikuchi A amp Okano T Cell micropatterning using photopolymerization with a liquid crystal device commercial projector Biomaterials 25 2047 2053 2004 Gradinaru V et al Targeting and readout strategies for fast optical neural control in vitro and in vivo J Neurosci 27 14231 14238 2007 Campagnola L Wang H amp Zyka M J Fiber coupled light emitting diode for localized photo stimulation of neurons expressing channelrhodopsin 2 J Neurosci Methods 169 27 33 2008 Schoenenberger P Grunditz A Rose T amp Oertner T G Optimizing the spatial resolution of Channelrhodopsin 2 activation Brain Cell Biol 36 119 127 2008 Stephens G J Johnson Kerner B Bialek W amp Ryu W S Dimensionality and dynamics in the behavior of C elegans PLoS Comput Biol 4 e1000028 2008 Brenner S Genetics of Caenorhabditis elegans Genetics 77 71 94 1974 Gray J M Hill J J amp Bargmann C I A circuit for navigation in Caenorhabditis elegans Proc Natl Acad Sci USA 102 3184 3191 2005 Holden Dye L amp Walker R J Anthelmint
15. d such that the FFL p of the lens pair and the 160 mm plane coincide and such that the back focal length BFL p of the lens pair and the primary projector image coincide Fig 1d System assembly The projector is mounted on a stable lab jack to provide z translational ability the ATL or RL and the projec tor must be centered along the optical axis of the epifluorescence port Fine adjustments to the location of the lenses and projector are made to ensure that the demagnified projector image and the object of interest e g C elegans are coincident When connected to a computer and set up as a dual monitor display the completed system will relay the image for the second monitor projector through the microscope which reduces it in size thus projecting it onto the sample General software We created custom software capable of automat ically tracking C elegans acquiring images identifying anatomi cal locations and directing the projector to illuminate the animal at the desired location color and intensity Fig 2 The software is written in LabVIEW and can be found along with a complete description in the online supplementary material of reference 12 http www nature com nmeth journal v8 n2 full nmeth 1555 html supplementary information The complete software con sists of four main programs which are briefly described below The software was written for a specific camera AVT Guppy anda specific motorized microscope sta
16. demagnified determined by the objec tive and the accessory optics Images for projection can be easily defined statically through programs such as Microsoft PowerPoint or they can be dynamic and more complex in design through the use of image processing techniques in MATLAB or LabVIEW The resolution of the generated optical pattern depends on the microscope objective selected and for example is better than 10 um using a X25 objective The temporal resolution and accuracy of the system is ultimately limited by the refresh rate of the projector 60 Hz the response time of the pixel elements and the lag time of the projector and is found to be 111 ms Supplementary Fig 1 and Supplementary Note 1 The high illumination intensity from a typical projector gt 4 mW mm with a x4 objective is sufficient for the activation of most optogenetic reagents Furthermore the intensity of illumination can be varied throughout the projec tion pattern by defining the 8 bit value of the pixel for each color at the desired location To fully demonstrate the capabilities of this multispectral system we show it applied to the dynamic optical activation of optogenetic reagents in freely moving C elegans where we simultaneously excite and inhibit specific cells Applications of the method One application of this illumination system and similar systems as shown in this paper and previously is for the dissections of various neur
17. dichroic and emission filters but without the excitation filter place the stage micrometer calibration slide on the microscope stage and bring the slide into focus iii Turn up the transmitted light intensity Use a piece of paper to find the position along the epifluorescence 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 IP som in Figure 1d iv Place the RLP 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 1d 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 FFL of the lens pair Fig 1c TROUBLESHOOTING npg 2012 Nature America Inc All rights reserved 214 VOL 7 NO 2 2012 NATURE PROTOCOLS npg 2012 Nature America Inc All rights reserved PROTOCOL Computer setup and system alignment TIMING 1 h 19 With the projector connected to the computer adjust the display settings in order 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 moni
18. e all the wires connecting to the main board as well as the three LCD panel connections Fig 4e Disconnect all wires taking note of where the wires were connected Unlatch the LCD panel cable connector and slide out the LCD panel cable from the main board Fig 4f There are three screws on the right side of the main board that need to be removed as well as an additional one at 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 be obtained before disconnecting in order to facilitate accurate reassembly later 7 Remove the screws on the cover of the dynamic iris Fig 4g Remove the cover and then slide out the dynamic iris unit Disconnect the green grounding wire 8 The cover of the main optical train of the projector must now be removed The Hitachi CP X605 has four screws holding down the cover Fig 4h that is to be removed as well as two plastic brackets Fig 4h that can be unlatched with a flathead screwdriver or spatula Remove the cover A CRITICAL STEP Connected to the optical train cover removed in this step are three polarizing filters Fig 4i that 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 alte
19. e effects of such spreading can be measured by defining a spot of known size projecting it through the illumination system and then measuring the width of the illumination where the intensity falls to 10 of the maximum value In this manner a resolution limit can be defined the smallest spot size that can be reliably defined By recording these measurements for this system Fig 5b d we find a limit of 14 um using a x4 objective and 5 um using a x25 objective 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 inches 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 PROCEDURE Steps 21 23 to ensure a high spatial resolution This is increasingly crucial as the magnification and numerical aperture of the objective increases By using the custom software for automated illumination of C elegans the temporal accuracy was measured and was found to be 70 ms for the projector alone and 111 ms for the complete system Supplementary Fig 1 and Supplementary Note 1 Measured spot size um width at 10 max intensity gt O Measured spot size um width at 10 max intensity N O o Selected area illumination of C
20. e 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 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 cus tom filters internally Fig 3a The action spectra of the optogenetic reagents previously used ChR2 ref 1 and MAC are shown in Figure 3b The spectrum of each color of the unmodified Hitachi CP X605 projector is quite broad Fig 3c and would thus cause considerable 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 maxi mize optogenetic activation and minimize cross activation 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 PROTOCOL Inverted Petri dish Motorized XY stage A Primary projector Image PDE Accessory tube lens infinity corrected or relay lens pair 160 mm and method of cutting of the filters used in this protocol for the Hitachi CP X605 are found in Supplementary Table 1 Filter sizes for
21. ed 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 TIMING 0 25 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 manufacturer s user s manual instructions and set as follows all keystone settings should be zero offset brightness contrast color and tint should be set to the middle position usually default 0 on the Hitachi CP X605 and the active iris should be turned off 16 Adjust the vertical and horizontal lens shift setting to a neutral zero offset position by following the manufacturer s user s manual instructions 17 Remove the projection lens by unscrewing it counterclockwise Assembly of the projector and microscope system TIMING 3 h 18 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 a 160 mm fixed tube length microscope B can be used for these steps A Assembly of projector and microscope system infinity corrected 1 Remove the epifluorescence optical train from the inverted fluorescence microscope Follow the manufacturer s user s manual for schematics and description A CRITICAL STEP All opt
22. emove the screws on the back of the projector case so that the internal circuit boards can be removed later There are ten such screws on the back of the Hitachi CP X605 projector that should be removed 3 Locate and remove the screws on the bottom of the projector these screws 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 4b holding the case together Remove the screws and save them for later reassembly 4 Return the 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 cables connecting the top control panel to the main circuit board two cables for the Hitachi CP X605 Disconnect these cables from the main unit Fig 4c The case cover can now be completely removed and set aside TROUBLESHOOTING 5 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 4d A CRITICAL STEP We suggest that a photograph of the projector and the location of the wires be obtained before disconnecting in order to facilitate accurate reassembly later 6 You will now be able to se
23. ere are some important distinctions Leifer et al use a single DMD from Texas Instruments and thus only single color illumination is used at a given time compared with the system described here and pre viously which can perform simultaneous spatially independent three color illumination Second Leifer et al use light 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 here and previously uses the native metal halide light source with the addition of custom band pass filters blue 430 475 nm 4 62 mW mm green 543 593 nm 6 03 mW mm and red 585 670 nm 5 00 mW mm By using the native metal halide light source of the projector no additional cost is incurred and the optical configuration of the system is simplified Finally the two systems differ in the software used for real time control and feedback and the closed loop opera tion 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 using the full resolution of the camera 1 024 x 768 We chose to use LabVIEW with Vision software for its ease of use for programming non experts our system operates with a closed loop temporal accuracy of 111 ms Supplementary Note 1 at a camera resolution
24. expensive and require substantial knowledge of optical components and design A further limita tion 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 considerable complexity due to multicomponent 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 implementa tion 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 compara ble commercial systems Such a light source is standard in fluores cent imaging and provides high brightness illumination across a broad spectrum Furthermore the protocol described here does not require an expert knowledge in optics engineering or physics to be able to assemble the equipment 208 VOL 7 NO 2 2012 NATURE PROTOCOLS Recently Leifer et al have described the use of a related sys tem for optical manipulation of C elegans Although it is similar in many ways to the system described here th
25. factory avoidance response in Drosophila Curr Biol 17 905 908 2007 Zhang W Ge W P amp Wang Z R A toolbox for light control of Drosophila behaviors through Channelrhodopsin 2 mediated photoactivation of targeted neurons Eur J Neurosci 26 2405 2416 2007 Arrenberg A B Del Bene F amp Baier H Optical control of zebrafish behavior with halorhodopsin Proc Natl Acad Sci USA 106 17968 17973 2009 Douglass A D Kraves S Deisseroth K Schier A F amp Engert F Escape behavior elicited by single Channelrhodopsin 2 evoked spikes in zebrafish somatosensory neurons Curr Biol 18 1133 1137 2008 Arrenberg A B Stainier D Y R Baier H amp Huisken J Optogenetic control of cardiac function Science 330 971 974 2010 Schoonheim P J Arrenberg A B Del Bene F amp Baier H Optogenetic localization and genetic perturbation of saccade generating neurons in zebrafish J Neurosci 30 7111 7120 2010 Umeda K Shoji W Ishizuka T amp Yawo H Transgenic zebrafish expressing an optimized channelrhodopsin variant under regulation of Gal4 UAS systems optogenetic stimulation of Rohon Beard neurons J Physiol Sci 60 S118 2010 Zhu P X et al Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the Tet system Front Neural Circuits 3 21 2009 Arenkiel B R et al In vivo light induced activation of neural circuitry in transgenic mice expre
26. ge Prior and would need to be modified for another camera or stage More detailed information 210 VOL 7 NO 2 2012 NATURE PROTOCOLS can be found in the Program Overview file and in comments within the programs that accompany reference 12 Projector Alignment software This program obtains parameters for a coordinate system transformation between the camera coor dinates Aale defining the object of interest within the field of view and the projector coordinates X Y defining the intended illumination pattern A grid of 20 solid circles is projected center positions X Y sequentially through the constructed optical sys tem These images are reflected off a highly reflective surface such as a front coated mirror and are imaged with the camera and then the locations of the projected circles are determined center positions X_ Y The scaling and offset parameters are determined and saved for use by the main program Color Illumination and Tracking software main program This program controls the acquisition of images real time tracking of the animal keeping within the field of view image processing to determine relative positions in the animal constructing the illu mination pattern based on user selectable inputs position color intensity and duration scaling the image and finally relaying it to the projector Within this program there are three main loops i image acquisition ii motorized stage contro
27. gents channelrhodopsin 2 ChR2 and MAC c Measured spectra for the red green and blue color planes before and after addition of the internal filters Panels a and c are adapted from ref 12 The spectra are adapted from refs 2 and 3 UHB ultra high pressure bulb LP long pass SP short pass from a camera coordinate to projector coordinates and is relayed to the projector for illumination of the object Fig 2g These loops each operate at 25 Hz and thus have a temporal resolution of 40 ms Head Encode software This is a simple program in which the user follows the position of the head of the animal with the cursor This position is encoded in the video The reason for encoding the posi tion of the head is to ensure in subsequent video analysis that the head rather than the tail is always identified accurately We have found that occasionally when reversing the curvature of the tail is similar to the head and can thus be erroneously labeled as the head if automated identification is used Complete Video Analysis software This analysis program uses the videos obtained in Color Illumination and Tracking that were encoded with the position of the head in Head Encode and extracts a number of detailed parameters The options within this program are the threshold values for defining the animal a conversion factor micrometers per pixel for converting camera pixel measurements into micrometers and the number of equal segments into
28. ic drugs in WormBook ed The C elegans Research Community Published online doi 10 1895 wormbook 1 143 1 02 November 2007
29. ical components should be handled with care Save all optical components noting the locations from which they came for later reassembly if necessary 11 Place the ATL in the epifluorescence optical path near the filter cube centering it along the optical axis 111 Remove the transmitted light optical filter With the filter cube in place with the dichroic and emission filters but without the 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 iv Turn up the transmitted light intensity By using a piece of paper find the position along the epifluorescence optical path where the image of the micrometer comes into sharp focus This should be at the back focal plane of the ATL v Place the projector such that the primary projector image coincides with the location of the focal plane of the ATL determined in step 18A iv Fig 1c TROUBLESHOOTING B Assembly of the projector and microscope system 160 mm 1 Remove the epifluorescence optical train from the inverted fluorescence microscope Follow the manufacturer s user s manual for schematics and description A CRITICAL STEP All optical components should be handled with care Save all optical components noting the locations from where they came for later reassembly if necessary 11 Remove the transmitted light optical filter With the filter cube in place with the
30. ication ATL for infinity corrected microscope 1 inch mounted achromatic e High brightness and high contrast LCD projector Hitachi doublet Thorlabs cat no AC254 series match focal length closely with cat no CP X605 or similar microscope tube length Leica 200 mm Nikon 200 mm Olympus e Custom filters for insertion internal to the projector Supplementary Table 1 180 mm and Zeiss 165 mm e Anti static mat Desco cat no 45010 PROCEDURE Modification of the LCD projector TIMING 2 5 h CAUTION All steps in this section should be performed with the projector unplugged and after at least 30 min if the projector was previously switched on as the bulb can be very hot It is also suggested that one should work on an anti static mat 1 Begin by removing the frame around the projector lens in order to remove the 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 4a and twisting the lens counterclockwise Carefully set the lens aside A CRITICAL STEP Be careful when handling the projector lens to ensure that it is not damaged or scratched it will be used later A CRITICAL STEP For some projectors the lens assembly cannot be removed For those projectors this step can be omitted 2 R
31. l and iii illumi nation control Fig 2a These functions are contained in separate processing loops to increase speed and operate in a closed loop in order to accurately maintain illumination of the desired locations as the animal changes body posture and location First the image is acquired using a digital camera Fig 2b and thresholding this image results in a binary image Fig 2c from which the center of mass can be calculated From this current position the offset of the animal is calculated and a command is sent to the motorized stage to recenter the object The binary image of the animal is thinned to a single pixel backbone and segmented based on user selectable parameters Fig 2d e e g six equally spaced segments Within the program the user can select which segments to illuminate as well as the color intensity and the duration of illumination Fig 2f This illumination pattern Fig 2f is scaled and offset to translate npg 2012 Nature America Inc All rights reserved 475 nm SP filter add a 600 nm LP filter add 568 50 nm filter add UHB mercury lamp Primary projector image plane Relay zoom lens Focusing diverging projection lens to be removed Figure 3 Modifications of the 3 LCD projector to limit the spectral width of the RGB colors a Internal filters are added to the 3 LCD projector thus narrowing the band pass for each RGB color b Action spectra for the optogenetic rea
32. le contractions in a paralyzed worm The worm strain ZX299 lin 15 n765ts zxEx22 pmyo 3 ChR2 H134R YFP lin 15 expresses ChR2 in the muscle cells therefore when illuminated with blue light the muscles will contract Animals are immobilized with ivermectin 0 01 mg ml solution which hyperpolarizes motor neurons but leaves muscles fully functional By dynamically altering the illumination pattern on the basis of the shape of the animal and optically activating muscle contraction the animal can be optogenetically controlled Fig 6a and ref 12 http www nature com nmeth journal v8 n2 extref nmeth 1555 S3 mov A second experiment demonstrates the ability to dynamically alter projection patterns at high resolution in a moving animal Using animals expressing ChR2 in the gentle touch sensory neurons AQ2334 lite 1 ce314 Is123 pmec 4 ChR2 punc 122 RFP a bar of light was scanned along the body of the animal while it crawls freely When the illumination reaches either the anterior or posterior sensory neurons the animal will reverse or accelerate respectively Fig 6b and ref 12 http www nature com nmeth journal v8 n2 extref nmeth 1555 S4 mov A final demonstration uses the full capacity of the system for dynamic multicolor illumination A strain expressing excitatory ChR2 in the gentle touch sensory neurons and inhibitory MAC in the command interneurons ZX899 lite 1 ce314 Is123 pmec 4 ChR2 punc 122 RFP zxEx621 pglr 1
33. lished online at http www natureprotocols com Reprints and permissions information is available online at http www nature com reprints index html Nagel G et al Channelrhodopsin 2 a directly light gated cation selective membrane channel Proc Natl Acad Sci USA 100 13940 13945 2003 Zhang F et al Multimodal fast optical interrogation of neural circuitry Nature 446 633 639 2007 Chow B Y et al High performance genetically targetable optical neural silencing by light driven proton pumps Nature 463 98 102 2010 Papagiakoumou E et al Scanless two photon excitation of channelrhodopsin 2 Nat Methods 7 848 854 2010 Andrasfalvy B K Zemelman B V Tang J Y amp Vaziri A Two photon single cell optogenetic control of neuronal activity by sculpted light Proc Natl Acad Sci USA 107 11981 11986 2010 Nagel G et al Light activation of channelrhodopsin 2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses Curr Biol 15 2279 2284 2005 Liewald J F et al Optogenetic analysis of synaptic function Nat Methods 5 895 902 2008 Mahoney T et al Intestinal signaling to GABAergic neurons regulates a rhythmic behavior in Caenorhabditis elegans Proc Natl Acad Sci USA 105 16350 16355 2008 Liu Q Hollopeter G amp Jorgensen E Graded synaptic transmission at the Caenorhabditis elegans neuromuscular junction Proc Natl Acad Sci USA 106 10823 10828
34. ll be saved vii Pick a worm and place it onto a blank 6 cm NGM plate viii Allow the worm to freely crawl for 25 min on the plate in order to allow it to recover from the mechanical disturbance of picking and adjust to the lack of food ix Open the main program Color Illumination and Tracking and start it with the play button TROUBLESHOOTING x Select the location and name of the video to be saved when prompted xi 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 worm and center it within the field of view and bring it into focus xii Adjust the bright field illumination intensity such that the binary image is an accurate representation of the worm Fig 2c xiii With the worm in the center of the field of view select the TRACK button to begin automated tracking of the animal xiv 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 In addition within this block set the values 0 255 of the individual red green and blue lights 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 settings can be adjusted with the slide bar set to Simple More complic
35. mple 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 we suggest that this be done once in a while weekly in order to ensure proper alignment Small offsets in the axial location of the lens and projector from the ideal locations Fig 1c d will make only slight alterations in the amount of demagnification Example applications methods of illumination control 24 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 multicolor static pattern generation or predefined pattern generation and projection with no feedback and option C uses custom software for real time automated illumination of samples that may vary in space and time A Simple illumination using a mouse pointer TIMING 0 10 h 1 Place the sample on the microscope and bring it into focus i1 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 ill
36. n motorized stage repositioning and automated illumination control b Acquired bright field image of C elegans c Binary image after applied thresholding d The binary image is thinned to a single pixel backbone representing the AP axis of the animal and segmented according to user selectable parameters number and location The locations for segmenting are based along the relative path length of the backbone where the head is 0 and the tail is 1 e Resulting segmentation of the binary image f Color pattern generated based on user selectable options including segment number color RGB intensity 0 255 for each color and illumination duration g The resulting multicolor illumination pattern projected onto the moving C elegans The image is falsely colored based on the intended illumination pattern Scale bar 250 um the specimen is placed slightly in front of the front focal plane FFL 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 1d should be placed 160 mm from the nosepiece open ing However owing to mechanical restrictions this is usually not possible Therefore the primary projector image must be trans mitted to the plane 160 mm from the nosepiece opening This is accomplished by using a relay lens RL Fig 1d consisting of a 1 1 matched RL pair RLP The RLP should be locate
37. n for stage used See also supporting documentation of reference 12 TIMING Steps 1 13 Modification of the LCD projector 2 5 h Steps 14 17 Adjustment of the projector settings 0 25 h Step 18A Assembly of the projector and microscope for infinity corrected microscope 3 h Step 18B Assembly of the projector and microscope for a 160 mm microscope 3 h Steps 19 23 System alignment 1 h Step 24A Simple illumination using a mouse pointer 0 10 h Step 24B Static or predefined dynamic illumination using Microsoft PowerPoint 0 25 h Step 24C Selected area illumination of C elegans using custom software 0 25 h NATURE PROTOCOLS VOL 7 NO 2 2012 217 npg 2012 Nature America Inc All rights reserved PROTOCOL oO Figure 5 Characterization of the completed illumination system a Relative intensity as a function of color pixel value 0 255 for each RBG color plane b Ideal 59 6 um and measured 68 5 um width of a defined projection pattern using a x4 objective This demonstrates spatial spread in illumination as a result of the contrast transfer function of optical components Width was measured at the point where the intensity drops to 10 of the maximum value 0 50 100 150 200 250 4100 50 0 50 400 c Measured spot size using a x4 objective Pixel value 8 bit Position um This shows a resolution limit of 14 um at x4 d Measured spot size using a x25 objective This shows a resolution limit of 5 um
38. nd Zeiss 165 mm The ATL to be inserted should be chosen to best match the focal length of the tube lens of the microscope manu facturer in this way the power of the objective closely matches the amount of demagnification The distance between the ATL and the PPI should be equal to the focal length of the ATL Fig 1c The distance between the ATL projector combination and the objective lens is not as critical however it is generally recommended that this distance be kept as short as possible Modification of microscope optics for 160 mm fixed tube length systems Although the 160 mm fixed tube length microscopes are an older style they are more than adequate for the purpose of construct ing this multispectral illumination system and can often be found more inexpensively In a 160 mm fixed tube length microscope NATURE PROTOCOLS VOL 7 NO 2 2012 209 npg 2012 Nature America Inc All rights reserved PROTOCOL a b Motorized stage control Illumination Image aquisition ge aq control 4 BN 2 N fp Threshold Image processing ae Calculation of illumination segments Projected image A Center of l 4 mass X Y A CSELEEELELLLEI te e r S SEDER RGEEEE at Figure 2 Custom software for the real time illumination of freely behaving C elegans a Three independent loops each operating at 25 Hz control image acquisitio
39. ng system Finally 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 Comparison with other methods Many of the existing techniques for optogenetic illumination are performed by positioning optical fibers in the vicinity of the target 8 by statically focused laser illumination or by static shadowing of illumination regions These methods are frequently imprecise or are performed in static samples thus limiting their applicability Current state of the art illumination systems involve the use of two photon microscopy light emitting diode LED arrays digital light processing DLP mirrors or commer cially available LCD projectors to spatially restrict light and they have the ability to dynamically alter the illumination pattern These techniques allow for a high degree of light localization to target individual neurons or groups of neurons or muscles and they can form 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 laboratories and custom constructed DLP based systems are both
40. npg 2012 Nature America Inc All rights reserved PROTOCOL A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents Jeffrey N Stirman Matthew M Crane Steven J Husson Alexander Gottschalk amp Hang Lu School of Chemical and Biomolecular Engineering Georgia Institute of Technology Atlanta Georgia USA Interdisciplinary Program in Bioengineering Institute of Biosciences and Bioengineering Georgia Institute of Technology Atlanta Georgia USA Johann Wolfgang Goethe University Institute of Biochemistry Frankfurt am Main Germany Frankfurt Institute for Molecular Life Sciences Johann Wolfgang Goethe University Frankfurt am Main Germany Correspondence should be addressed to H L hang lu gatech edu Published online 12 January 2012 doi 10 1038 nprot 2011 433 Optogenetics is an excellent tool for noninvasive activation and silencing of neurons and muscles Although they have been widely adopted illumination techniques for optogenetic tools remain limited and relatively nonstandardized We present a protocol for constructing an illumination system capable of dynamic multispectral optical targeting of micrometer sized structures in both stationary and moving objects The initial steps of the protocol describe how to modify an off the shelf video projector by insertion of optical filters and modification of projector optics Subsequent
41. nstructed through programs such as Microsoft PowerPoint or other graphic illustrators for simple static patterns or for patterns that change with time in a predefined manner These projected images would be suitable for immobilized animals or cells or objects that vary slowly over time as there is no real time feedback For freely behaving animals or for dynamic events one must use soft ware that can provide and process real time feedback Custom programs can be written in LabVIEW MATLAB or C which can dynamically alter the illumination patterns based on user npg 2012 Nature America Inc All rights reserved Figure 1 Optical configuration of the system and a Sample plane components a Final optical configuration for the Cm system The epifluorescence optics are replaced by C mount an accessory tube lens infinity corrected or relay lens pair 160 mm and a modified 3 LCD projector b Optical configuration of the projector in the original unmodified state c Optical configuration of the constructed illumination system for an infinity corrected microscope d Optical configuration of the constructed illumination system for a 160 mm microscope ATL accessory tube lens BFL back focal length of the RPL FFL front focal length of the RLP FL focal length of the ATL PDL projector diverging lens PI projected image M gt 1 PPI projector primary image PZL projector zoom lens OL somm objective lens
42. or for solutions Dim image Bulb is near the end of its life Check the projector for bulb hours and refer to the user s Color is missing absent Color or image is striped Disconnected LCD panel cable Shifted or broken filter Unsecured loose LCD panel cable manual on replacing Disassemble the projector to ensure that LCD panel cables have been securely reattached Disassemble the projector and check all inserted filters if they have shifted or possibly broken Replace if necessary Disassemble the projector to ensure that LCD panel cables have been securely reattached 18A v Insufficient space to Accessory tube lens focal plane Extend the accessory tube lens to the rear of the position the projector located within the body of the microscope allowing the focal plane to be located microscope outside the body of the microscope 18B v Insufficient space to Relay lens focal plane located Select a matched lens pair of greater focal length position the projector within the body of the micro scope 21 Image never focuses Incorrectly positioned lenses Check that all lenses are located as described in Figure 1 24C 1 Program gives error upon Different camera from what the Alter the LabVIEW code to communicate with the specific 24C ix starting program was written for camera used See also supporting documentation of reference 12 Different motorized stage from Alter the LabVIEW code to communicate with the specific what the program was writte
43. play button xxv When prompted select the 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 xvi xvii xviii xix xx xxi npg 2012 Nature America Inc All rights reserved NA NAA NAA NAAA NAA TROUBLESHOOTING Troubleshooting advice can be found in Table 1 216 VOL 7 NO 2 2012 NATURE PROTOCOLS npg 2012 Nature America Inc All rights reserved TABLE 1 Troubleshooting table PROTOCOL Step Problem Possible reason Solution 4 Case cover will not slide off Not all screws have been Check both the back and the bottom of the projector to removed ensure all necessary screws have been removed 9 Filters will not fit Improperly sized filter Measure the opening at the location for the filters and check dimensions of the custom filters Alter the filters as necessary to fit 14 Projector will not turn on Internal disconnected cables If all cables were not correctly connected when reassembling the projector then unit will not power up Take the projector apart and ensure that all cables are connected Other errors associated with the Check error blinking codes and consult the user s manual project
44. red 212 VOL 7 NO 2 2012 NATURE PROTOCOLS npg 2012 Nature America Inc All rights reserved PROTOCOL WLLL PPLE ELLE Th Pritrt eee PELE EEE Eth late Figure 4 Disassembly and insertion of custom optics into the 3 LCD projector 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 the 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 locations for red green and blue filter insertion L Removal of the diverging projection lens from the zoom lens system 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 4j Locations of the insertion of the custom filters Supplementary Note 2 are indicated with boxes in Figure 4k Insert the precut optical filters dimensions for filters for the Hitachi CP X605 can be found in Supplementary Table 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 wi
45. s determined between three successive points and is relative to 180 e Average angles For both the previous angles measured the average of the absolute value of all the angles determined along the worm is found This gives some indication of the overall amount of bending of the worm e Length Measurements of the length of the worm are made in micrometers using the user input conversion factor e Head to tail distance The straight line distance from the worm s head to tail is made Illustration program Microsoft PowerPoint alternatively use LabVIEW with vision MATLAB with image processing toolbox or custom software e Computer capable of dual video output Intel 13 1 6 GHz processor with 3 GB RAM or better e Stage micrometer calibration slide Amscope cat no MR100 e Camera AVT Guppy F 033 Edmund Optics or similar e Support lab jack 10 x10 inches VWR cat no 14233 368 e High temperature epoxy e Electrical tape NATURE PROTOCOLS VOL 7 NO 2 2012 211 npg 2012 Nature America Inc All rights reserved PROTOCOL Microscope optics RLP for 160 mm fixed tube length microscope Achromatic Doublet Pair e Dichroic mirror Semrock cat no FF662 FDi01 25x36 Thorlabs cat no MAP10100100 A e Emission filter Thorlabs cat no FB650 40 e Low magnification objective x4 to X10 for whole animal imaging Bright field filter Edmund Optics cat no NT66 096 Equipment for projector modif
46. ssing channelrhodopsin 2 Neuron 54 205 218 2007 220 VOL 7 NO 2 2012 NATURE PROTOCOLS 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Aravanis A M et al An optical neural interface in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology J Neural Eng 4 143 S156 2007 Ayling 0 G S Harrison T C Boyd J D Goroshkov A amp Murphy T H Automated light based mapping of motor cortex by photoactivation of channelrhodopsin 2 transgenic mice Nat Methods 6 219 224 2009 Cardin J A et al Targeted optogenetic stimulation and recording of neurons 7n vivo using cell type specific expression of Channelrhodopsin 2 Nat Protoc 5 247 254 2010 Huber D et al Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice Nature 451 61 64 2008 Wang H et al High speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin 2 transgenic mice Proc Natl Acad Sci USA 104 8143 8148 2007 Macosko E Z et al A hub and spoke circuit drives pheromone attraction and social behaviour in C elegans Nature 458 1171 1175 2009 Davis M W Morton J J Carroll D amp Jorgensen E M Gene activation using FLP recombinase in C elegans PLoS Genet 4 e1000028 2008 Wang S et al All optical interface for parallel remote and spatiotemporal
47. steps involve altering the microscope s epifluorescence optical train as well as alignment and characterization of the system When fully assembled the illumination system is capable of dynamically projecting multispectral patterns with a resolution better than 10 pm at medium magnifications Compared with other custom assembled systems and commercially available products this protocol allows a researcher to assemble the illumination system for a fraction of the cost and can be completed within a few days INTRODUCTION Recently there has been considerable interest in optically targeting optogenetic reagents for noninvasive excitation and inhibition of cultured cells and neurons and muscles in small model organ isms such as the nematode Caenorhabditis elegans the fruit fly Drosophila melanogaster the zebrafish Danio rerio and the mouse Optogenetic reagents are light gated ion channels and pumps and when expressed in excitable cells neurons and muscles illuminating them with the appropriate wavelength of light causes depolarization e g Channelrhodopsin 2 or ChR2 ref 1 or hyperpolarization e g Halorhodopsin or NpHR MAC and Arch 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
48. th electrical tape A CRITICAL STEP All optical components should be handled with care TROUBLESHOOTING 10 Once the filters have been successfully placed and secured Fig 4k the projector can be reassembled by reversing Steps 4 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 and the projector can potentially be damaged 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 in which 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 4l These are stops for the projection focus lens preventing it from being fully unscrewed Loosen these screws until the diverging projection lens can be fully rotated counterclockwise and off the zoom lens assembly NATURE PROTOCOLS VOL 7 NO 2 2012 213 PROTOCOL 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 remov
49. tor and should be set to use the full resolution of the projector Hitachi CP X605 1 024 x 768 The desktop should also be set to use a solid black background thus not projecting any unwanted images to the sample 20 Place a piece of fluorescent paper or slide glass on the microscope stage and bring it into focus through the eyepieces or camera 21 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 TROUBLESHOOTING 22 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 A blank NGM plate also 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 23 With the projector on and projecting an image make further adjustments of the X Y and Z positions 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 crucial step to ensure that the projected image is focused on the sa
50. umination system as well as qualitatively assess the reaction of the sample B Static or predefined dynamic illumination using Microsoft PowerPoint TIMING 0 25 h 1 Create a new presentation in Microsoft PowerPoint Set the background of the slides to solid fill with black as the color 11 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 i11 To create a time series sequence of patterns create patterns for each time point and use the Custom Animations option to determine the transition times iv Place the sample on the microscope and bring it into focus v 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 TIMING 0 25 h 1 Open the Beamer Alignment program and start it with the play button see ref 12 A CRITICAL STEP These steps describe the use of the custom software written for our specific camera and motorized Stage To adapt it to
Download Pdf Manuals
Related Search