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Design of a Coordinated System for Real-time 3

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1. 3 0 0 Description of Final Implementation In order to divide the imaging responsibilities evenly across the available processing resources features in the control application have been divided into four synchronous sequential modules and most image processing functions have been moved into an isolated fifth module to reduce processing load The following section describes the responsibilities of these five modules organized from the highest to least priority module 3 1 0 Camera Module The camera module is responsible for all functions relating to communication with the Hamamatsu camera This module initializes the image stream and manages exposure settings Figure 7 Once the camera is configured properly and the data stream is formed this module supervises the image buffers to ensure that if a frame is dropped it is caught and saved before it falls out of the 10 image buffer Also should a frame be dropped as is common in an uncontrolled operating environment like Microsoft Windows XP the camera module allocates itself additional resources by temporarily disabling nonessential functions within the camera module and the communication module with which it shares a processor During recording the camera module interfaces with the recording module to coordinate stage motion DSU cube emission filter state and Sutter emission filter state If the stage needs more time to move than the time between frames or if the DSU cube n
2. Figure 3 Astrocyte network exhibiting calcium wave via Fluor 4 AM calcium indicator after mechanical stimulation Fluor 4 AM is a cell membrane permeable fluorescent dye that binds allosterically at four sites per fluorophore with Ca This dye is the best available reversible calcium marker with excitation at 497nm and emission at 521nm 2 In the Silva lab Fluor 4 AM is used as a single wavelength calcium indicator and the emission filter used to detect binding is designed for the FITC fluorophores which has similar excitation emission spectra excitation at 494nm and emission at 520nm First primary spinal cord astrocytes taken from rat pups are incubated for about 30 minutes with a membrane permeability ester Once the membrane permeable Fluor 4 AM enters the cells this ester charges the dye to prevent it from leaking back out The intensity of the emission of the Fluor 4 AM calcium indicator varies in proportion to the calcium concentration Figure a AO es Endoplasmic Reticulum Figure 4 Functional diagram of Fluor 4 AM calcium marker image courtesy of Diana Yu Silva Research Group 1 1 3 Current project Ratiometric Calcium wave Imaging The Silva lab also employs the radiometric method of imaging the calcium based signaling of glial networks This method involves FRET Fluorescence Resonance Energy Transfer in a molecule designed to bind calcium FRET occurs whe
3. I would also like to acknowledge the support and continued valued input from Nicholas George of Olympus America Inc I am forever indebted to my parents Doug and Lori and my sister Sarah for their unwavering support and understanding as well as Kourtney King and Will Fisher from whom I have received an invaluable store of wisdom patience and encouragement when it was most required vil ABSTRACT OF THE THESIS Design of a Coordinated System for Real time 3 D Image Construction via Confocal Microscopy by Nathan Roy Shepard Master of Science in Bioengineering University of California San Diego 2007 Professor Gabriel Silva Chair In order to facilitate the imaging of signaling networks and to fully utilize the capabilities of confocal microscopy it is desirable to build a system to coordinate several hardware components of an imaging platform This document describes the integration of hardware with a confocal microscope and the design of a custom control application to optimize image sampling and 3 D image reconstruction of signaling networks By implementing advanced timing controls and minimizing switching delays speed and accuracy are optimized and the sampling space and time resolution are maximized The control application reliably demonstrated successful 3 D imaging at the maximum frame rate supported by the camera viii 1 0 0 Introduction In order to make a meaningful short term contribution to long term
4. IMAQdx BER e magdx BER gt ImaQdx BETA e maQdx aioa IMAQdx E p 77 P ctiveattribute ValueDBL__ gt ActiveAttribute gt ValueString PactiveAttribute 0 01 gt Increment 13 Shutter Mode MaxDBL__ o MinDBL gt Minimum 10 JHPOutOFRnaAct Source a Shutter time Type loJ Kmsec Time CtRef E Oldval z i Shutter time NewvYal E Shutter time msec pel Shutter time Le b El a tee a Ea Figure 7 LabVIEW Camera Module featuring Image Format change This section of the camera module responds when the user modifies the Image Format control changing the recorded image resolution and frame rate The previous capture is closed and a new capture named cam0 left is started using new values for exposure timeout 20000ms and shutter mode relative Finally the exposure control on the user interface is modified to reflect the new maximum and minimum exposure times as determined by the camera bottom 3 2 0 Communication Module The communication module is responsible for maintaining all of the communication protocols to all of the hardware other than the IEEE 1394 camera Therefore it is the second highest priority module 3 2 1 Prior Stage Communication with the stage is achieved via an RS232 serial protocol The communication module regularly queries the stage horizontal position X Y and records 1t to the user interface but
5. 12 38 The most important function of the recording module is managing the memory during the recording process The legacy imaging control wrote all the acquired data to RAM Random Access Memory in real time during acquisition causing severe memory shortages in long or detailed recording To reduce this problem the recording module follows behind the camera module as it is recording and saves images in RAM to disk when extra CPU Central Processing Unit cycles are available This allows the camera module to record images at full speed without dropping frames when the machine is low on resources but it also allows the recording module to move the data over to disk when resources are available to do so This way a fraction of the RAM is required for an acquisition and the only limit to acquisition length is the space available for storage on the hard drive Also the data is almost immediately saved in permanent storage so that if there is a failure during recording the data is not lost 39 fi Snap Record Value Change Ma Record E E a CA 4 Pattern Centers ELEN f PDisabled Snap E pS Figure 12 LabVIEW Recording Module featuring capture setup This section of the recording module responds immediately after the Record button has been pressed while the user is in 3 D imaging mode Once activated this algorithm moves and initializes the pre calculated image l
6. DSU cube has severe implications of the light path that stimulated further design improvement 2 6 4 Profiles Because the image changes so drastically between DSU cubes the exposure settings in the legacy system needed to be drastically adjusted each time the filter was changed These camera setting changes were not only time consuming but inconsistent and difficult to repeat without extensive documentation for each experiment Thus a system was designed to memorize not only exposure settings but also light intensity settings for each combination of objective DSU cube IX condenser image format florescence bright field mode prism orientation and confocal disk orientation Because of the need to memorize and recall these and other important user settings between uses it became desirable to save several dozen setting to a configuration file at the end of each session Because settings for different experiments are very different the profile feature was added to allow for multiple groups of settings to be saved and loaded for the use of different users or experiments 23 2 6 5 IX Condenser In the bright field path the IX condenser is controlled via a filter wheel attached to the scope A simple algorithm was developed to control this wheel Similar to the DSU cube exposure and light intensity settings for each condenser filter are memorized and reset when each condenser is selected 2 6 6 Neutral Density Filter The adju
7. Diana Interview with Nathan Shepard Silva Research Group June 8 2007 Yu Diana Interview with Nathan Shepard Silva Research Group Sep 7 2007
8. Time Systems with Multicore Technology NI Developer Zone Video url http sine ni com apps utf8 nievn ni action display_offerings_by_event amp event_id 29803 amp event _subtype WEB_EVENT_DEMAND amp site NIC amp I US Aug 10 2007 2 National Instruments Develop High Performance Real Time Systems with Multicore Technology NI Developer Zone Video url http sine ni com apps utf8 nievn ni action display_offerings_by_event amp event_id 29803 amp event _subtype WEB_EVENT_DEMAND amp site NIC amp I US Aug 10 2007 National Instruments Image Analysis and Processing NI Developer Zone url http zone ni com devzone cda tut p id 3470 Feb 1 2006 4 0 0 Final Workflow This section describes the workflow involved in performing the following functions in the new imaging software Imaging is divided into four different types still single frame imaging still movie imaging 3 D single cube imaging and 3 D movie imaging 4 1 0 Still Imaging Still imaging refers to imaging that does not require movement of the objective or the stage during acquisition 4 1 1 Single Frame Single frame acquisition refers to a single image capture of a motionless subject at one particular X Y Z coordinate This is the most simple image workflow Figure 15 Figure 16 43 44 7 r mm Be se 2975 N 4 ol d 4000 b j gt p t oD CITED b O H 1 Cikk to center CTRL click Lo measure y Ashot E
9. research efforts already underway it is important to understand the nature and purpose of that research The Silva Research Group at UCSD is concerned with understanding how the Central Nervous System CNS processes information using the neural retina as a CNS model This understanding is desirable to work towards the functional clinical regeneration of the CNS To these ends the lab utilizes confocal microscopy to study the physiology of neuronal and glial networks in the CNS 1 1 0 Interest in a Custom Imaging System Until February 2007 members of the Silva Research Group had been using a control software called ImagePro to coordinate the confocal imaging system The age of the software caused many hardware compatibility problems leading to frustrating failures and unnecessary data losses Most importantly however the legacy imaging system was unable to adapt to the needs of the research members because its implementation was rigid and inflexible New imaging procedures stimulated interest in a more customizable imaging system and the integration of additional imaging hardware further stimulated effort toward this project The following sections describe the purpose and procedures of the imaging experiments underway in the Silva lab during the development of the new imaging system 1 1 1 Current project Up regulation of GFAP and Vimentin Preretinal macular gliosis is a condition in which a thin membrane grows in front of the m
10. secondary screen 41 Figure 15 Front panel user interface primary screen oooooonocononnnonanonanononcnonocanaconn ccoo anno 44 Figure 16 Detailed real time observation image secondary SCreenN oooococnnnccccnonccinnnnnos 45 Figure 17 User interface primary screen during movie recording oooococononcconnnonnnonananns 47 Figure 18 User interface both screens immediately after image Capture ooooonnoccccnnn 47 Figure 19 3 D image space with 2x2 tiled images compiled as a combination of Z axis depth 4 slices and time 15 timestamps oooocoonnoccccnoncncnoncnononanonnnanononcnonncncnnnacinnnss 50 vi Acknowledgements I would like to gratefully acknowledge the support and supervision of Dr Gabriel Silva throughout this project I would like to thank the members of the Silva research group for their advice and input during the design and debugging stages of development Specifically I would like to thank Marie Davidson Diana Yu Marius Buibas Smita Pathak Matt Li Erin Barron David Kupec Krystal Chiao and Christopher MacDonald for their advice support and constructive discussions during the development I would like to thank Jorge Noguera at National Instruments office for his enthusiastic support and advice regarding both the software and hardware design I am also grateful for the consultation of Brent Runnells at National Instruments in regard to controlling the high speed camera in the early stages of development
11. sensitivity Control Joystick X axis invert Control Joystick Y axis invert Control DSU cube select Control Objective turret select Control IX Condenser select Control Bright field shutter open close Control Bright field intensity control volts Control RS322 COM port baud rate select Indicator RS232 COM port busy light Lambda DG 4 e Control Fluorescence shutter open close e Control Fluorescence intensity PRIOR Stage e Control Automatic calibrate and move to 0 0 0 position Control Manual jump to coordinate Control Automatic save recall position bookmarks Control Maximum acceleration limit Control Maximum speed limit e Indicator Current Position Hamamatsu ORCA ER Camera e Control Frame rate select User Interface Module e Feature Recall bright field intensity setting e Feature Recall fluorescence intensity setting e Control Run Stop Reset button e Control Quit Exit button e Indicator Real time IEEE 1394 Image Recording Module e Control Record button e Control Movie length sec e Control Compression filter select e Control Quality 0 100 compression select Control Save image file routine 1 2 0 01 07 2007 IX 81 Scope e Control RS232 COM port select PRIOR Stage 55 56 e Control RS232 COM port select Lambda DG 4 e Control RS232 COM port select Hamamatsu ORCA ER e Control Video mode select including both Format 7 amp standard IEEE 1394 formats Control Shutt
12. this is the module s only autonomous function The primary function of this module is interrupt based command referral to the stage Different modules within the application will write instructions to the stage communication module 33 and trigger a low priority interrupt Once triggered the stage communication module sends the command or command set to the stage If more than one command has been queued between interrupts it sends both commands together sequentially Finally if a response from the stage is required by the command sent the stage communication module records this response to an output buffer Figure 8 Figure 8 LabVIEW Communication Module featuring PRIOR stage current X Y position acquisition This Timeout section of the control module runs each time 50ms passes without user input or control changes This particular section of code the fourth of four timeout routines starts by reading the RS232 buffer input from the PRIOR stage and parsing the results into an array of ASCII American Standard Code for Information Interchange inputs bottom Once this is complete the routine scans through each input to find position indicators analyses them and reports the results to the current position indicator on the user interface panel top left to right 3 2 2 Olympus IX 81 Inverted Microscope The communication with the scope is managed through an RS232 serial
13. 004 Nov 21 11 1671 82 PRIOR Inc Prior Stage Controller Instructions for use 2000 Runnells Brent Interview with Nathan Shepard National Instruments Nov 29 2007 Silva Research Group Research www silva ucsd edu research html University of California San Diego Accessed 2 21 07 Sommer W Cui X Erdmann B Wiklund L Bricca G Heilig M Fuxe K The spread and uptake pattern of intracerebrally administered oligonucleotides in nerve and glial cell populations of the rat brain Antisense Nucleic Acid Drug Dev 1998 Apr 8 2 75 85 Straub SV Nelson MT Astrocytic calcium signaling the information currency coupling neuronal activity to the cerebral microcirculation Trends Cardiovasc Med 2007 Aug 17 6 183 90 Stringer JL Repeated seizures increase GFAP and vimentin in the hippocampus Brain Res 1996 Apr 22 717 1 2 147 53 Sutter Instrument Company News and Downloads url http www sutter com news software_downloads html 2003 Tack J Smith TK Calcium imaging of gut activity Neurogastroenterol Motil 2004 Apr 16 Suppl 1 86 95 Review MacDonald Chris Interview with Nathan Shepard Silva Research Group Jan 26 2007 Manning TJ Jr Sontheimer H Recording of intracellular Ca2 Cl pH and membrane potential in cultured astrocytes using a fluorescence plate reader J Neurosci Methods 1999 Sep 15 91 1 2 73 81 Martinez Contreras A Huerta M Lopez Perez S Garcia Estrada J Luquin S Beas Zarate C Astrocytic
14. 006 National Instruments Image Analysis and Processing NI Developer Zone url http zone ni com devzone cda tut p id 3470 Feb 1 2006 National Instruments New Features in LabVIEW 8 5 NI Developer Zone url http www ni com labview85 upgrade htm Aug 10 2007 National Instruments Synchronizing Motion Vision and Data Acquisition NI Developer Zone url http zone ni com devzone cda tut p id 4214 Feb 1 2006 Nguyen SM Lieven CJ Levin LA Simultaneous labeling of projecting neurons and apoptotic state J Neurosci Methods 2007 Apr 15 161 2 281 4 Epub 2006 Dec 20 Olympus International Instructions X81 Motorized Inverted Research Microscope Tokyo Japan 2004 Olympus International X2 Application Note N Olympus Development Document April 4 2005 Olympus International X2 Host I F Specifications N Olympus Development Document April 4 2005 Olympus International IX2 ZDC Application Note N Olympus Development Document April 4 2006 66 Olympus International U ZPCB Application Note N Olympus Development Document April 4 2006 Olympus International U ZPCB Host I F Specifications N Olympus Development Document April 4 2006 Winship IR Plaa N Murphy TH Rapid astrocyte calcium signals correlate with neuronal activity and onset of the hemodynamic response in vivo J Neurosci 2007 Jun 6 27 23 6268 72 Yu Diana Interview with Nathan Shepard Silva Research Group Jan 26 2007 Yu
15. 2 147 53 10 Sommer W Cui X Erdmann B Wiklund L Bricca G Heilig M Fuxe K The spread and uptake pattern of intracerebrally administered oligonucleotides in nerve and glial cell populations of the rat brain Antisense Nucleic Acid Drug Dev 1998 Apr 8 2 75 85 ll Sommer W Cui X Erdmann B Wiklund L Bricca G Heilig M Fuxe K The spread and uptake pattern of intracerebrally administered oligonucleotides in nerve and glial cell populations of the rat brain Antisense Nucleic Acid Drug Dev 1998 Apr 8 2 75 85 13 Nguyen SM Lieven CJ Levin LA Simultaneous labeling of projecting neurons and apoptotic state J Neurosci Methods 2007 Apr 15 161 2 281 4 Epub 2006 Dec 20 Lee GM Rasch EM Thornthwaite JT Cytophotometric comparisons of DNA levels in neuronal and glial cells of the cerebellum a comparative study Cell Biochem Funct 1984 Oct 2 4 225 36 Lin J Cai W Effect of vimentin on reactive gliosis in vitro and in vivo analysis J Neurotrauma 2004 Nov 21 11 1671 82 5 Nakazawa T Takeda M Lewis GP Cho KS Jiao J Wilhelmsson U Fisher SK Pekny M Chen DF Miller JW Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin Invest Ophthalmol Vis Sci 2007 Jun 48 6 2760 8 Straub SV Nelson MT Astrocytic calcium signaling the information currency coupling neuronal activity to the cerebral microcirculation Trends Cardiovasc
16. 2 0 Imaging Requirements and Criteria The primary imaging needs of the lab members revolve around the use of fluorophores and quantum dot nanotechnology to image neuronal and glial cells Examples of experimental acquisitions using the confocal microscope include recording movies of network response to various stimuli still images of protein locations within networks and 3 D three dimensional representations of injured tissue To achieve the best results during these experiments the operator must have quick and full access to all of the controls on one screen as well as live feedback in the form of hi res image display and real time rudimentary image processing techniques A method of real time feedback motion control is also desirable for imaging a large 3 D space with a time varying signal The process of exposing the image and moving the subject must be optimized and coordinated to provide the highest possible sampling frequency for the Operator and for experimental data collection A real time 3 D representation of the image plane with several overlaid wavelengths corresponding to different fluorophores is desirable for analysis and viewing Each wavelength filter must have different corresponding optical and exposure settings to optimize its layer for overlay Finally the image capture time must be kept as low as possible to maximize the sampling space Several common experiments required the quick and precise return to specifi
17. 3 Figure 15 Front panel user interface primary screen 10 co a IE tte A Open pen mndar out ees PEyeplece Flour gt DAPI 350 4700m aoso f OPEN FITC 495 520 g Mirror a a IQdot605 TRITC 557 Polarized for frre K 292 binning 16 bit 16 3 Ips Rone m Open tmege tevin 45 Figure 16 Detailed real time observation image secondary screen First the optical settings of the light path are set via the optical settings controls above the low resolution control image on the front panel of the user interface Figure 15 Once set correctly the light intensity settings to the right of the control image should be set and the appropriate shutter opened Then exposure and camera settings should be set found below the control image The histogram indicator and large observation image on the secondary monitor are useful in optimizing the image quality This process should be repeated for each DSU filter that the user needs to image Once image channel settings have been set the desired DSU filter channels should be selected in the Image Channels list control Also the color mapping of the imaging 46 channel may be set using this control To optimize system performance during recording the large observation image may be closed Once the controls are properly set the user presses the Snap button and waits for the recording to complete using the Recording pro
18. Lambda Once the communication protocol between the DG 4 and the control algorithm was established the command set for controlling the mirror and intensity settings was installed and incorporated into the application 2 6 0 Olympus IX 81 Scope 2 6 1 Interaction with Olympus International The most time consuming process in the integration of the scope hardware was obtaining proprietary command information for Olympus This process began in Nov 2006 and an incomplete command set was delivered in Jan 2007 The complete command set did not arrive until May 2007 2 6 2 Objective The first scope hardware control to be implemented was the objective turret control The turret controls which objective is currently in the light path as well as the 21 focus of that objective The z axis focus axis of the turret is controlled primarily by the Jog Wheel attached to the scope but it is also incorporated into the exact position control and bookmark system described in the stage section As an added benefit the mouse scroll wheel was enabled as a z axis control providing another means of focusing the objective In order to safely switch objectives the turret must first be lowered the objective switched and then the turret may rise to its previous location In observation of various imaging experiments a possible workflow improvement in the objective control became apparent Each time the objective was changed in the legacy system the
19. Manual 2001 4 PRIOR Inc Prior Stage Controller Instructions for use 2000 46 Lambda Inc Lambda 10 3 Sutter Instrument User Manual 2005 Y Buibas Marius Interview with Nathan Shepard Silva Research Group Jan 26 2007 48 National Instruments Event Driven Programming in LabVIEW NI Developer Zone url http zone ni com devzone cda tut p id 3331 Feb 1 2006 Davidson Marie Interview with Nathan Shepard Silva Research Group Nov 6 2006 National Instruments Are National Instruments Products Compatible with Windows XP 64 bit for AMD s x86 64 Technology NI Developer Zone url http digital ni com public nsf allkb C4ECOC87B6D70A A886256E61006568C8 Mar 24 2004 gt National Instruments Develop High Performance Real Time Systems with Multicore Technology NI Developer Zone Video url http sine ni com apps utf8 nievn ni action display_offerings_by_event amp event_id 29803 amp event _subtype WEB_EVENT_DEMAND amp site NIC amp I US Aug 10 2007 National Instruments New Features in LabVIEW 8 5 NI Developer Zone url http www ni com labview85 upgrade htm Aug 10 2007 4 3 Davidson Marie Interview with Nathan Shepard Silva Research Group Jan 26 2007 Bovik Alan C Handbook of Image and Video Processing Academic Press June 2005 pp 377 419 15 2 0 0 History of Work This section summarizes the research and development process required to implement the new confocal microscopy imaging syste
20. Med 2007 Aug 17 6 183 90 7 Winship IR Plaa N Murphy TH Rapid astrocyte calcium signals correlate with neuronal activity and onset of the hemodynamic response in vivo J Neurosci 2007 Jun 6 27 23 6268 72 Winship IR Plaa N Murphy TH Rapid astrocyte calcium signals correlate with neuronal activity and onset of the hemodynamic response in vivo J Neurosci 2007 Jun 6 27 23 6268 72 Bertram CM Baltic S Misso NL Bhoola KD Foster PS Thompson PJ Fogel Petrovic M Expression of kinin B1 and B2 receptors in immature monocyte derived dendritic cells and bradykinin mediated increase in intracellular Ca2 and cell migration J Leukoc Biol 2007 Jun 81 6 1445 54 Epub 2007 Feb 27 2 Yu Diana Interview with Nathan Shepard Silva Research Group Sep 7 2007 21 Bertram CM Baltic S Misso NL Bhoola KD Foster PS Thompson PJ Fogel Petrovic M Expression of kinin B1 and B2 receptors in immature monocyte derived dendritic cells and bradykinin mediated increase in intracellular Ca2 and cell migration J Leukoc Biol 2007 Jun 81 6 1445 54 Epub 2007 Feb 27 2 Bertram CM Baltic S Misso NL Bhoola KD Foster PS Thompson PJ Fogel Petrovic M Expression of kinin B1 and B2 receptors in immature monocyte derived dendritic cells and bradykinin mediated increase in intracellular Ca2 and cell migration J Leukoc Biol 2007 Jun 81 6 1445 54 Epub 2007 Feb 27 2 Hailer NP Wirjatijasa F Roser N Hischebeth GT Korf HW Dehghani F Astro
21. Snap Instead of pressing Snap the user must define a movie length in the Length sec control Once this is entered a compression filter may be selected from the menu Finally the user presses Record Movie to begin the acquisition The user may wait for the Recording progress bar to finish or press Stop recording to end the capture and save the acquired data Once complete a set of timestamped images in 3 D space are compiled and saved to disk Figure 19 1G 2007 09 07 01 07 36F 100 TR 300 al e FITC 495 520 lcd 26 2007 09 06 23 51 03 F 100 TR 300 Qdo160s 06 2007 09 08 13 04 33 F 100 TR 300 bs 2007 09 08 13 06 05 100 1R 300 9 2007 08 29 09 19 42 no primary FITC TR 2007 08 29 09 17 34 no primary FITC TR 2007 09 05 12 45 43 FITC TRITC 300ms y exa Contrast Stretch FITC 495 520 asez gt 3 j El ea om ro Figure 19 3 D image space with 2x2 tiled images compiled as a combination of Z axis depth 4 slices and time 15 timestamps 5 0 0 Discussion This section describes desirable and feasible additions to the existing imaging system The scope of these additions is limited to a one year implementation time frame 5 1 0 Real time 3 D Rendering By adding processing hardware integrated closely with the acquisition control PC it will be possible to render 3 D representations of the image space including overlaid fluorescence wavelengths in real time Limitat
22. UNIVERSITY OF CALIFORNIA SAN DIEGO Design of a Coordinated System for Real time 3 D Image Construction via Confocal Microscopy A Thesis submitted in partial satisfaction of the requirements for the degree Master of Science in Bioengineering by Nathan Roy Shepard Committee in charge Professor Gabriel Silva Chair Professor Michael Heller Professor Maryann Martone 2007 Copyright Nathan Roy Shepard 2007 All Rights Reserved The Thesis of Nathan Roy Shepard is approved Chair University of California San Diego 2007 111 Table of Contents Signature Patene 4h eae E ee ee aa aa 111 MA A Io ede RO ee iv List of Figure iia A ii vi e A A n a a a E a a E subelement vii Abstractor the THESIS iia ated asec tif A a a viii 100 Introd ct oN a a See A de RE Aaaa at 1 1 1 0 Interest in a Custom Imaging System oooccnnncccnonocononoccnoncnonnnnnconnnncnnnnnonnnncrnnncnonnncno 1 1 1 1 Current project Up regulation of GFAP and Vimentin s es 1 1 1 2 Current project Flour 4 Calcium wave Imaging oooonocccnoccnonnconncnoncc onncannnnnn nono 4 1 1 3 Current project Ratiometric Calcium wave IMaglN8 coooonnnccnoncnoncncnncnnannnonnnnns 5 1 2 0 Imaging Requirements and Criteria cooococnnocccnonoconononcnnncnonnnnnnnnnnncnnnnnonnncnonncninnnnnss 6 1 3 0 Available Hardware Components 0 0 cscccesssccessceceseceessececesncecssccecnsececsceeescens 8 1 4 0 Lab VIEW Development Softw atte lt div
23. acula This can occur spontaneously or as a result of surgery retinal injury or detachment or simply as a result of aging In order to test various drugs ability to reduce gliosis in astrocytes it is desirable to increase gliosis in Mueller cells expose these cells to various anti gliotic drugs and measure the reduction in the rate of gliosis using intermediate filament proteins such as GFAP glial fibrillary acidic protein and Vimentin The Silva lab uses immortalized rat Mueller cells of the cell line rMC 1 derived from primary retinal glial cells of rat pups and astrocytes from the spinal cords of rat pups as host cells The first step of the process is ICC immunocytochemistry in which the cells are exposed to a primary anti GFAP antibody made in mouse that binds the GFAP Then the cells are exposed to a secondary fluorescent anti mouse protein made in goat attached to a FITC fluorescein isothiocyanate fluorophore This fluorophore excites at 495nm and emits at 520nm Figure 1 To further test gliosis the amount of Vimentin another prominent intermediate filament protein in glia is also measured Some cells are exposed to an anti Vimentin antibody made in rabbit followed by an anti rabbit antibody made in goat attached to a TRITC Rhodamine derivative fluorophore This dye is excited at 557nm and emits at 576nm Figure 2 Quantum dot technology is also being adapted in order to test gliosis at deeper level
24. age Figure 18 47 EN peaked pro a yo x 7 n a p 3406 os ie E polen 20 m1 EEN pelas D gt EUER S507275 J 7 i tal o rasi me SE A A y pi Caibe atr A f Mims Mi b EE m Eyepiece Flour EJ LLL AA DAPI 350 47001 J 4 FITC 495 520 F e TRITC 557 5760 a Qdot605 Polarized Sone 100 Q Men image tania 4 D gt ALTO 4 A gt HE a cea AAN Figure 17 User interface primary screen during movie recording E lt lt M Figure 18 User interface both screens immediately after image capture 4 2 0 3 D Imaging 3 D acquisition requires that the subject be moved between frames In order to accomplish this an array of points describing the image space must be defined so that the 48 control algorithm knows where and when to move the stage and how to reconstruct the image after it has been captured Also for proper image reconstruction the confocal disk must be in the light path during the acquisition 4 2 1 Single Image Set Unlike the still imaging procedure the first step in 3 D imaging is to define the limits of the image space The limits are defined by eight X Y Z coordinates that make up a 3 D rectangle In order to specify the dimensions of this rectangle the user must define two opposite corners To do this the user moves the subject to one of the corners in each of the three axes either by using the joystick and the jog wheel controls or by clicking on the control i
25. al small issues 4 3 3 09 04 2007 IX 81 Scope e Control Neutral density filter select Sutter 10 3 Filter Switch e Feature Further optimization of high speed switching implemented 4 3 4 09 08 2007 Image Toolbox e Feature Memory leak fixed Feature Redesign of control layout Control Build movie Control Post processing gain adjust Control Post processing brightness adjust Control Channel to measure e Indicator Brightness measure References Ahuja TK Wu SH Intrinsic membrane properties and synaptic response characteristics of neurons in the rat s external cortex of the inferior colliculus Neuroscience 2007 Mar 30 145 3 85 1 65 Epub 2007 Jan 29 Bertram CM Baltic S Misso NL Bhoola KD Foster PS Thompson PJ Fogel Petrovic M Expression of kinin B1 and B2 receptors in immature monocyte derived dendritic cells and bradykinin mediated increase in intracellular Ca2 and cell migration J Leukoc Biol 2007 Jun 81 6 1445 54 Epub 2007 Feb 27 Bovik Alan C Handbook of Image and Video Processing Academic Press June 2005 pp 377 419 Buibas Marius Interview with Nathan Shepard Silva Research Group Jan 26 2007 Buibas Marius Interview with Nathan Shepard Silva Research Group June 6 2007 Buibas Marius Interview with Nathan Shepard Silva Research Group May 23 2007 Damodaran TV Bilska MA Rahman AA Abou Doni MB Sarin causes early differential alteration and persistent overexpression in mRNAs codin
26. alcium imaging with dual excitation dyes Biophys J 1998 Oct 75 4 2025 9 2 Yu Diana Interview with Nathan Shepard Silva Research Group Sep 7 2007 Yu Diana Interview with Nathan Shepard Silva Research Group Sep 7 2007 Yu Diana Interview with Nathan Shepard Silva Research Group Sep 7 2007 35 Straub SV Nelson MT Astrocytic calcium signaling the information currency coupling neuronal activity to the cerebral microcirculation Trends Cardiovasc Med 2007 Aug 17 6 183 90 36 Leybaert L Sneyd J Sanderson MJ A simple method for high temporal resolution calcium imaging with dual excitation dyes Biophys J 1998 Oct 75 4 2025 9 37 Lambda Inc Lambda 10 3 Sutter Instrument User Manual 2005 38 Buibas Marius Interview with Nathan Shepard Silva Research Group Jan 26 2007 32 Davidson Marie Interview with Nathan Shepard Silva Research Group Jan 26 2007 4 Leybaert L Sneyd J Sanderson MJ A simple method for high temporal resolution calcium imaging with dual excitation dyes Biophys J 1998 Oct 75 4 2025 9 4l Manning TJ Jr Sontheimer H Recording of intracellular Ca2 Cl pH and membrane potential in cultured astrocytes using a fluorescence plate reader J Neurosci Methods 1999 Sep 15 91 1 2 73 81 Davidson Marie Interview with Nathan Shepard Silva Research Group Jan 26 2007 Buibas Marius Interview with Nathan Shepard Silva Research Group Jan 26 2007 4 Lambda Inc Lambda DG 4 User
27. alled late in the development of the control application When an image set is loaded each emission spectrum channel that was recorded with the image is available for display 26 and any combination of channels may be overlaid Any color may be selected for each channel and a histogram control provides a means of weighting the intensity of each channel A final histogram indicator allows for quick analysis of the final image before it is saved 2 9 1 Image Processing Tools The Image Toolbox provides several options for displaying and modifying image sets Tiled images are stitched together z stack images are displayed with a vertical scroll to move through the z axis Images recorded with timestamps are displayed with a horizontal scroll to move through time Color movies can be built from prerecorded post processed timestamped image sets Zoom is provided as well as various histogram shaping algorithms including a full scale contrast stretch and a histogram equalizer Post processing gain and brightness may be modified to generate the best quality and most appealing output image A burnt in scale is available in any color and length and it may be placed in any corner of the image Once the image is modified it may be saved with the original image set as a convenient bmp image file type and the saved image will be loaded each time the image set is opened in the Image Toolbox A thresholding function was added in April 2007 to allow
28. an 26 2007 Silva Research Group May 23 2007 Silva Research Group June 6 2007 1 George Nicolas Olympus RS232 Commands Personal communication via e mail Jan 8 2008 101 MacDonald Chris Interview with Nathan Shepard Silva Research Group Jan 26 2007 National Instruments Synchronizing Motion Vision and Data Acquisition NI Developer Zone url http zone ni com devzone cda tut p id 4214 Feb 1 2006 Conclusion In spite of the quality and variety of the commercial systems available for professional image capturing the specificity and flexibility necessary for the Silva Research Group demand a custom robust software package Although the possible features and benefits that could be included in an imaging system are limitless the Scope Controller software described in this document has demonstrated performance suitable for the desired imaging applications without requiring additional hardware resources to do so The modularity and flexibility of this design lends this application to further development expansion and integration with future hardware Through an understanding of the goals and requirements of the researchers in the group this short term development project has contributed to their long term efforts towards an understanding of the neural retina 54 Appendix Version History Version Release Date New Features Changes 1 0 0 12 05 2006 IX 81 Scope e Control Joystick enable Control Joystick
29. an error required an extensive amount of development time as the camera is highly unique and poorly documented Once the 18 camera s video feed was installed properly control of peripheral attributes such as brightness gain gamma and shutter time were necessary These attributes except for shutter time were not controlled in ImagePro so an algorithm to automatically optimize them was written at the behest of lab members Another problem with the camera integration needed to be addressed Frequently both in the legacy system and in the new software internal registers in the camera would attempt simultaneous read write operations and the camera would freeze up stalling at unpredictable times and necessitating a program restart or in the case of the legacy system a complete system power cycle and cold start To address this issue code was developed to catch the error within 15 frames of the failure and reset internal camera registers to ensure continued performance This code also protects these vulnerable registers from simultaneous read write operations during recording limiting user control during this time but ensuring an uninterrupted imaging sequence Based on observation of lab imaging work an important feature of the new software was immediate conversion of image spatial data to real world units Using a calibration slide an algorithm was designed to automatically calibrate each combination of objective and image
30. and microglia cells reactivity induced by neonatal administration of glutamate in cerebral cortex of the adult rats J Neurosci Res 2002 Jan 15 67 2 200 10 Media Cybernetics Inc Image pro Plus Start up Guide for Windows Silver Spring MD 2004 65 Nakazawa T Takeda M Lewis GP Cho KS Jiao J Wilhelmsson U Fisher SK Pekny M Chen DF Miller JW Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin Invest Ophthalmol Vis Sci 2007 Jun 48 6 2760 8 Nakazawa T Takeda M Lewis GP Cho KS Jiao J Wilhelmsson U Fisher SK Pekny M Chen DF Miller JW Increased neurogenesis and astrogenesis from neural progenitor cells grafted in the hippocampus of GFAP Vim mice Stem Cells 2007 Jul 12 National Instruments Are National Instruments Products Compatible with Windows XP 64 bit for AMD s x86 64 Technology NI Developer Zone url http digital ni com public nsf allkb C4ECOC87B6D70AA886256E61006568C8 Mar 24 2004 National Instruments Develop High Performance Real Time Systems with Multicore Technology NI Developer Zone Video url http sine ni com apps utf8 nievn ni action display_offerings_by_event amp event_id 2980 3 amp event_subtype WEB_EVENT_DEMAND amp site NIC amp 1 US Aug 10 2007 National Instruments Event Driven Programming in LabVIEW NI Developer Zone url http zone ni com devzone cda tut p id 3331 Feb 1 2
31. ansmit light Indicator PRIOR Stage real time data receive light Indicator Lambda DG 4 real time data transmit light Indicator Lambda DG 4 real time data receive light 2 2 0 01 30 2007 User Interface Module Feature Left click on image to center Feature Ctrl click on image to measure distance Feature Remember last saved data path in profile Feature Saved profile files moved to Profiles folder Feature Removed debugging tools and nicely compiled application Recording Module Feature No Compression option Feature Recorded frames saved to hard disk and removed from RAM in real time during acquisition Feature Automatic avi movie built at completion of recording 3 0 0 02 03 2007 User Interface Module Feature Switching objective turret moves down before and after to protect objectives Feature Stop Reset does not change profile settings Feature Joystick Jog wheel sensitivity settings remembered per objective Feature Auto calibrate pixel nm for each objective image format combination Feature Auto set shutter time Feature Bookmark remembers Z axis position Control Recall settings button Recording Module Feature Increase maximum exposure time 200ms to 800ms 58 3 1 0 02 04 2007 User Interface Module e Feature Redesign of control indicator layout on front panel e Feature Robustness testing debugging several small issues 3 2 0 02 09 2007 User In
32. c locations after moving away so a system of position bookmarks was desirable A method to either save or discard images immediately after acquisition was also requested Also a native application for quick image viewing and manipulation is helpful to analyze image quality and diagnose exposure problems on the fly preventing the recording of bad or unnecessary data and ensuring the quality of successful image recording Finally a means of controlling the entire imaging system using only a mouse and keyboard was desirable to eliminate complications arising from cumbersome hardware controls this also allows for remote operation of the imaging system All of these features must be implemented on a user level platform application that caters to user preferences tendencies and desired features with careful resource management in mind so as to avoid straining the relatively few memory and processor resources available on the average desktop computer This application must be able to quickly manage a high volume of incredibly large image data sets without showing delay in any user tasks or losing any recorded data From a compatibility standpoint the application must not interfere with any other installed software or permanently change and hardware settings already in place The imaging system is designed to carefully control all of the connected hardware and remember all of the operator s previous settings and preferences while leaving all
33. communication protocol through the Olympus IX2 UCB hardware control module This protocol is handled in the same way as the stage protocol except that commands to the scope must not be sent simultaneously Rather commands must receive 34 acknowledgement of completion or error from the IX2 UCB module before another command is sent Figure 9 This prevents damage to the stepper motors in the scope by only moving one at a time The hardware controlled by the scope includes the objective turret the bright field path the IX condenser and DSU cube and the condenser wheel Scope VISA RN ame tt al iow Pine Pe Bytes at Port R Gu i fs LTE Hwait scope ooo Figure 9 LabVIEW Communication Module featuring Scope Port This section of the communication module responds whenever any part of the application places a command for the confocal microscope in queue Once activated this routine indexes through the scope command queue and writes the commands to the appropriate RS232 port For some potentially damaging operations such as switching the objective turret this routine is ordered to wait for the scope to fully complete the previous command before sending the next 3 2 3 Lambda DG 4 The DG 4 fluorescent light generator is controlled via an RS232 serial connection managed by a similar protocol to the stage and scope control modules The difference in this communication channel is that it is a one way cha
34. cytic factors protect neuronal integrity and reduce microglial activation in an in vitro model of N methyl D aspartate induced excitotoxic injury in organotypic hippocampal slice cultures Eur J Neurosci 2001 Jul 14 2 315 26 Ahuja TK Wu SH Intrinsic membrane properties and synaptic response characteristics of neurons in the rat s external cortex of the inferior colliculus Neuroscience 2007 Mar 30 145 3 851 65 Epub 2007 Jan 29 2 Ahuja TK Wu SH Intrinsic membrane properties and synaptic response characteristics of neurons in the rat s external cortex of the inferior colliculus Neuroscience 2007 Mar 30 145 3 851 65 Epub 2007 Jan 29 2 Tack J Smith TK Calcium imaging of gut activity Neurogastroenterol Motil 2004 Apr 16 Suppl 1 86 95 Review 7 Yu Diana Interview with Nathan Shepard Silva Research Group Sep 7 2007 14 2 Leybaert L Sneyd J Sanderson MJ A simple method for high temporal resolution calcium imaging with dual excitation dyes Biophys J 1998 Oct 75 4 2025 9 Manning TJ Jr Sontheimer H Recording of intracellular Ca2 Cl pH and membrane potential in cultured astrocytes using a fluorescence plate reader J Neurosci Methods 1999 Sep 15 91 1 2 73 81 2 Leybaert L Sneyd J Sanderson MJ A simple method for high temporal resolution calcium imaging with dual excitation dyes Biophys J 1998 Oct 75 4 2025 9 Leybaert L Sneyd J Sanderson MJ A simple method for high temporal resolution c
35. divided into four general types as specified by a combination of two binary states Imaging may be either still or 3 D meaning that the subject plane may lie motionless still throughout the acquisition or the subject may move between frames creating a 3 D reconstruction of the sampling space Regardless of dimensionality an acquisition may be either a snap or a movie meaning that each combination of filters and subject positions may be sampled once to create a snapshot of the image space or the entire space may be sampled repetitively with timestamps to mark the progression creating a movie For 3 D image acquisition timing plays an important role in the speed and quality of the capture Movement of the subject is coordinated to coincide with camera shutter closure to maximize the frame rate of the camera Figure 6 12 123 45 6 7 8 9 1011121314 1516 17 18 1920 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 Camera Shutter Camera Tx Rx A A A A Stage Move H AM f Figure 6 Timing diagram for 3 D image acquisition Silva Research Group Research www silva ucsd edu research html University of California San Diego Accessed 2 21 07 2 Davidson Marie Interview with Nathan Shepard Silva Research Group Nov 6 2006 3 Nakazawa T Takeda M Lewis GP Cho KS Jiao J Wilhelmsson U Fisher SK Pekny M Chen DF Miller JW Increased neurogenesis and astrogenesis from neural progenit
36. dt da 10 1 5 0 Theory of Implementation ico 3sas sesccacsseiecerssassaccvessdeedasastan sacs socaey sotecetuacsdaataaeseacs 11 20 0 History f Work A A O 16 21 0 Preliminary A es gaishe a Madan Se dhGt se tte sao sea a E 16 Zed A E des Rate 16 DED O A AMINO TA acs Se al loed id taate e a Sader ses rte ois a osoa lucas Gud Detach 17 2 4 0 New Development Environment ccscccesseccesnseceeneeceeneeceeneecesaeeceeneeeeeeeeesaes 19 230 Fl rescent AA TIN 2 do a e 20 20 0 Olympus 1X28 PS cod 20 2 6 1 Interaction with Olympus International 0 eee cece ceeeeeseeeeneecnaeenteeeeeeeeaees 20 DG DIODE CU VE dd 20 DID SC Ne SA A A i de BE lee 21 ZA ARP EOUIIGS A ON 22 SAN A O aire ots abr RE cae 23 2 6 6 Neutral Density Filtet cin rss 23 OA GSS Be BEA O NI 23 20 0 Joystick Jos Wheel a ae 23 2 69 Confocal Disk usina 24 2 6 10 Multi Purpose Hardware User Interface ooooococnnncccnnocccnoncncnnncnononccononanonnnnnos 24 2 1 0 Image Quality Controli oinn A ee eee ie 24 2 8 0 Zastack A a a a n a a RE a a 25 229 0 Image Toolbar aeo e iS 25 2 9 1 Image Processing Tools eiii dali ia etica dnd 26 29 2 Im ge Meas rements suncen icense a E A TRER 26 2 10 0 High speed Emission Filter Switch ici 27 3 0 0 Description of Final Implementation oococonocononocononoccnononononnnncnnnnnonnncncnnnnccnnncncnnncnnnns 30 3 1 0 Camera Module tap 30 3 2 0 Commun Cation Module 520 22 ceed ced ee ede E a REE eases 32 A A E AEA E E TET 32 3 2 2 Olym
37. e adjustments became very short 30ms as the filter would be in place and ready to image the next frame in that time In order to meet these criteria the image capture module had to be redesigned to process the incoming image data during the next image exposure time instead of between frames and during a Sutter channel acquisition to bypass some unnecessary functions and features Adding together the frame delay of the camera 60ms and the switch time of the Sutter 75ms the two longest exclusive processes or the acquisition the theoretical frame rate limit is 7 4Hz I was able to 28 optimize the capture to a frame rate of 4 7Hz indicating mechanical settling data transmission and software processing delays totaling 77ms frame Also whenever the Sutter emission filter is changed the DSU cube is automatically switched to the position containing the dichroic mirror and likewise the Sutter filter is automatically set to the open position whenever the DSU cube value is changed Exposure settings are still saved for all position of the DSU cube as well as for all positions of the Sutter instrument Since the adding the dichroic mirror to one of the six available positions in the DSU cube required the removal of the TRITC filter a switch was added to allow for its quick exchange with the mirror when necessary A new Emission Filter control was added to the control screen and two new channels were added to the master applicatio
38. eeds to changes states approximately a 1580ms process then the camera module delays the camera exposure until the stage and DSU cube are in position If exposure 30 31 settings need to be changed for use of the high speed Sutter filter switch nonessential functions are bypassed and image processing is delayed until the following frame exposure time in order to increase capture frame rate Additionally the camera module is responsible for monitoring the camera for instability and failure The Hamamatsu specifically has a tendency to fail when IEEE 1394 DCAM control registers attempt to simultaneously perform read and write operations Every 15 frames the camera module tests for this failure in order quickly correct it and reinitialize the image stream During recording this module prevents any write operations to these registers to avoid failure during image capture Unfortunately this prevents user modifications to the camera settings during recording Lastly the camera module is responsible for the display of the low resolution control image as well as the high resolution real time observation image This responsibility includes real time histogram analysis of the acquisition and display of that histogram as well as any pertinent histogram shaping saturation warnings 32 000000000000000 0000000000000000000 d o 0 2 p 2200000000000000 000000000000000001 Jaj 2 Image Format Value Change vh paoia a
39. er time usec Control Gain Control Brightness Control Zoom constant e Indicator Frames per second User Interface Module e Feature Recall shutter time setting Feature Recall gain setting Feature Recall brightness setting Control Show large true image button Indicator Recording Saving progress bar e Indicator Status Help text Recording Module e Indicator Expected movie file size Communication Module e Feature IX 81 Scope command queue e Feature Lambda DG 4 command queue 2 0 0 01 15 2007 IX 81 Scope e Control Jog wheel enable e Control Jog wheel sensitivity e Control Focus e Control Auto focus e Control Z axis microns revolution Recording Module e 3 D recording pattern settings o Control Number of Z axis slices o Control Slice depth nm o Control X Y axis grid size o Indicator Frames cube o Indicator Physical cube size nm o Indicator Entire cube refresh rate Hz 2 1 0 01 28 2007 Datel Systems PC e Feature Dedicated development hard drive IX 81 Scope e Control Light prism path select Hamamatsu ORCA ER Camera e Feature Frame rate regulation 57 Feature Dropped frame prevention User Interface Module Feature Save customized settings preferences to profile Feature Load previously saved profile Communication Module Indicator IX 81 Scope real time data transmit light Indicator IX 81 Scope real time data receive light Indicator PRIOR Stage real time data tr
40. for a binary image to be recorded based on a set of thresholds for each channel 2 9 2 Image Measurements For comparison purposes the normalized brightness of a particular imaging channel may be easily measured and compared with other images in the same set as well 27 as outside images An original histogram shows the number of pixels at each level of intensity and a final histogram shows the RGB Red Green Blue intensities of the output image Further analysis tools are easy to implement as they become desirable 2 10 0 High speed Emission Filter Switch In May 2007 the Silva Group invested in a Sutter 10 3 instrument capable of switching emission filters at speeds on the order of magnitude of the camera frame rate 16Hz The purpose of the switch was to image time variant calcium waves using both a cyan and a yellow emission filter on every other frame In terms of hardware control this presented an interesting challenge In previous imaging scenarios the limiting factor in image acquisition had been the mechanical movement time of the stage 500ms or the mechanical switch time of the DSU cube 1600ms If no mechanical change was required that meant that no exposure adjustments were required and the program would allow the camera to free run capture at the maximum frame rate When using the Sutter instrument pre set exposure adjustments would be necessary between each frame however the time allotted to make thes
41. format so that a suitable conversion from pixels to nanometers could be performed in real time during image acquisition Once this algorithm was complete reference scales were added to the user interface By analyzing computer resource performance during image capture on the legacy system it was determined that the ImagePro software was recording image to RAM Random Access Memory during the image capture process and only moving them into permanent storage when the user chose to save the images at the end of the imaging 19 session This led to two problems with the legacy system First image data for long movie sequences was very large gt 1 Gigabyte so RAM space would fill during long or detailed captures and cause the system to crash Second when the system did freeze a frequent occurrence any unsaved data would be lost To compensate for these problems I implemented an algorithm in the recording module that uses excess CPU cycles to save image data recorded in RAM to disk in a temporary file structure in real time This is a low priority algorithm lagging behind the real time recording algorithm to ensure that frames are not dropped Thus it does fall behind on very long or detailed captures or when the operating system requires more resources for unrelated tasks However all data is almost immediately saved to disk only a fraction of available RAM is used and the option to save the data in a user defined format is available i
42. g for glial fibrillary acidic protein GFAP and vimentin genes in the central nervous system of rats Neurochem Res 2002 May 27 5 407 15 Davidson Marie Interview with Nathan Shepard Silva Research Group Jan 26 2007 Davidson Marie Interview with Nathan Shepard Silva Research Group May 23 2007 Davidson Marie Interview with Nathan Shepard Silva Research Group Nov 6 2006 George Nicolas Olympus RS232 Commands Personal communication via e mail Jan 8 2008 Hailer NP Wirjatijasa F Roser N Hischebeth GT Korf HW Dehghani F Astrocytic factors protect neuronal integrity and reduce microglial activation in an in vitro model of N methyl D aspartate induced excitotoxic injury in organotypic hippocampal slice cultures Eur J Neurosci 2001 Jul 14 2 315 26 Hamamatsu Photonics K K IEEE1394 Digital CCD Camera C4742 80 12AG Instruction Manual December 2003 Lambda Inc Lambda DG 4 User Manual 2001 Lambda Inc Lambda 10 3 Sutter Instrument User Manual 2005 63 64 Lee GM Rasch EM Thornthwaite JT Cytophotometric comparisons of DNA levels in neuronal and glial cells of the cerebellum a comparative study Cell Biochem Funct 1984 Oct 2 4 225 36 Leybaert L Sneyd J Sanderson MJ A simple method for high temporal resolution calcium imaging with dual excitation dyes Biophys J 1998 Oct 75 4 2025 9 Lin J Cai W Effect of Vimentin on reactive gliosis in vitro and in vivo analysis J Neurotrauma 2
43. get f 2007 04 21 09 57 15 binding site 100 un Tools Histogram Shaping Burmin Scale MTI Scale Length um TEN Ti start y pedire ry ls saoe Figure 13 Image Toolbox user interface 41 Windows Professional Figure 14 Image Toolbox display of a saved image secondary screen National Instruments Synchronizing Motion Vision and Data Acquisition NI Developer Zone url http zone ni com devzone cda tut p id 4214 Feb 1 2006 2 National Instruments Synchronizing Motion Vision and Data Acquisition NI Developer Zone url http zone ni com devzone cda tut p id 4214 Feb 1 2006 8 Hamamatsu Photonics K K IEEE1394 Digital CCD Camera C4742 80 12AG Instruction Manual December 2003 PRIOR Inc Prior Stage Controller Instructions for use 2000 5 Olympus International IX2 Application Note N Olympus Development Document April 4 2005 86 PRIOR Inc Prior Stage Controller Instructions for use 2000 7 Olympus International 1X2 Host I F Specifications N Olympus Development Document April 4 2005 42 Lambda Inc Lambda DG 4 User Manual 2001 Lambda Inc Lambda DG 4 User Manual 2001 n Lambda Inc Lambda 10 3 Sutter Instrument User Manual 2005 National Instruments Synchronizing Motion Vision and Data Acquisition NI Developer Zone url http zone ni com devzone cda tut p id 4214 Feb 1 2006 National Instruments Develop High Performance Real
44. gress bar above the control image as a reference When imaging is complete the image is displayed the light shutters are closed to prevent photo bleaching and the user is given options for saving the image 4 1 2 Multiple Timestamped Frames Imaging a movie that does not require movement of the stage or objective utilizes the maximum acquisition time of the camera To set up the acquisition first the user sets the optical settings above the control image Next the light intensity and shutter settings should be configured these controls are to the right of the control image Once set the exposure and camera settings below the control image should be set These steps should be repeated for each DSU cube that the user would like to image Once set the imaging channels should be selected in the control above the recording controls Finally the length of the recording should be entered in the Length field in the recording control This will be the maximum length of recording although the recording may me manually stopped at any point during acquisition Once these settings have been entered the user pressed the Record Movie button and waits for the acquisition to complete using the Recording progress bar above the control image as a reference Figure 17 When imaging is complete the last acquired image is displayed the light shutters are closed to prevent photo bleaching and the user is given options for saving the im
45. he standard Olympus IX 81 Scope command set Although simple they require a special instruction set to operate safely This instruction set is proprietary and has not yet been delivered by Olympus For the purposes of the Silva lab In Out states are sufficient and the confocal disk is accompanied by a hand switch to set this state manually As this is a minor inconvenience it has not been a design priority 2 6 10 Multi Purpose Hardware User Interface The Olympus IX 81 s hardware console includes an array of buttons both on the scope itself and on the accessory panel An algorithm was designed to handle user input via these buttons nonspecific inputs that can be modified to perform any desired function Several of the buttons were previously labeled for use with the legacy system and those with labels were associated to their former tasks 2 7 0 Image Quality Control In an effort to improve the quality of recorded images a live histogram indicator was installed in March 2007 along with various warning lights to indicate overexposure underexposure and undersampling At this point the algorithm to automatically set exposure settings was severely modified to increase the quality and speed of the exposure time calculation Also during the time immediately after exposure when the image is being saved the recorded image is displayed so that the user can review the quality of the 23 recording before saving During this time bo
46. inates could be manually entered I also noticed that because the camera records an inverted image plane from the eyepiece the joystick control was non intuitive when using the eyepiece for observation or centering Therefore I implemented an algorithm that automatically inverts the joystick axes when the eyepiece is in use As an additional feature a click to move algorithm was written to allow the user to local select and image a region of interest by clicking on the image display itself instead of manually positioning the stage As an added feature a new algorithm allowed for two points to be entered via mouse click and the distance between the two selected X Y Z coordinates to be measured using the calibrated pixel nanometer calculation 2 3 0 Camera In order to utilize all available functionality from the Hamamatsu Camera I scheduled a meeting with Brent Runnells a specialist in IEEE 1394 imaging at National Instruments Brent shared advice about minimizing the processing time between frames and coordinating the motion of the stage between captures National Instruments also loaned me a camera for three weeks to design an optimal algorithm The most significant challenge in controlling the Hamamatsu camera was utilizing both the standard IEEE 1934 video formats supported by the camera as well as the optimized Format 7 formats used by ImagePro Implementing these formats in such a way that switching between them did not cause
47. ional 1X2 Host I F Specifications N Olympus Development Document April 4 2005 6 Yu Diana Interview with Nathan Shepard Silva Research Group Jan 26 2007 Olympus International 1X2 Host I F Specifications N Olympus Development Document April 4 2005 6 Olympus International Instructions IX81 Motorized Inverted Research Microscope Tokyo Japan 2004 Olympus International IX2 ZDC Application Note N Olympus Development Document April 4 2006 Davidson Marie Interview with Nathan Shepard Silva Research Group May 23 2007 7 Davidson Marie Interview with Nathan Shepard Silva Research Group May 23 2007 72 Olympus International U ZPCB Host I F Specifications N Olympus Development Document April 4 2006 73 Olympus International U ZPCB Application Note N Olympus Development Document April 4 2006 Davidson Marie Interview with Nathan Shepard Silva Research Group May 23 2007 75 Davidson Marie Interview with Nathan Shepard Silva Research Group Jan 26 2007 76 Y y Diana Interview with Nathan Shepard Silva Research Group June 8 2007 77 Olympus International 1X2 Host I F Specifications N Olympus Development Document April 4 2005 ES Lambda Inc Lambda 10 3 Sutter Instrument User Manual 2005 72 Hamamatsu Photonics K K IEEE1394 Digital CCD Camera C4742 80 12AG Instruction Manual December 2003 8 Y y Diana Interview with Nathan Shepard Silva Research Group June 8 2007
48. ions of onboard processing power make this a negative functionality to include on the highly time dependant acquisition control hardware However a high speed full duplex communication channel between the acquisition and rendering hardware could combine an elegant user interface with a detailed image display and analysis 5 1 1 Communication with External Rendering Hardware The communication channel would be established via a high speed Ethernet between the PCs The protocol for this connection is very simple and it is fast enough to transfer image data at the maximum capture speed of the Hamamatsu camera A detailed command set for the acquisition software has been developed to make remote commands and queries available for use by the rendering hardware should a user interface on the rendering machine become desirable 5 1 2 Graphical User Interface for Image Manipulation Several user interface possibilities have been considered for use both on the rendering PC and on the acquisition PC however an ideal solution would be to replace 51 52 the observation image on the secondary screen Figure 16 with an interactive 3 D model computed and managed on the rendering PC The specific rules of user interaction are highly customizable and further user criteria and feature request information should be collected 5 2 0 Real time Network Nodal Analysis By incorporating a simplified version of network analysis software that ha
49. l Timing of imaging channel switch between frames end of sequence Image Toolbox e Feature Robustness testing debugging several small issues 4 2 4 04 10 2007 Image Toolbox e Feature Robustness testing debugging several small issues 4 2 5 04 17 2007 Recording Module e Optimized timing of 3 D imaging processes Image Toolbox e Feature More accurate color conversion of each channel e Control Bit depth binary range 4 2 6 04 29 2007 Image Toolbox e Feature Redesign of control layout e Control Auto color channels 4 2 7 05 22 2007 Sutter 10 3 Filter Switch e Control Emission filter e Control 10 3 RS232 COM port e Indicator CFP cyan fluorescence protein and YFP yellow added to imaging channels list Image Toolbox e Feature Image set tiling e Control removed X axis e Control removed Y axis 4 2 8 07 04 2007 Image Toolbox e Feature Left click image to zoom in e Feature Right click image to zoom out 4 2 9 07 21 2007 Sutter 10 3 Filter Switch e Feature Optimized high speed switching implemented Hamamatsu ORCA ER Camera e Feature Memory leak in shutter time error handler fixed 4 3 0 08 21 2007 Sutter 10 3 Filter Switch e Feature Robustness testing debugging several small issues 62 4 3 1 08 22 2007 Recording Module e Feature Support for quickly replacing DSU cube filters e Feature File save for TRITC channel fixed 4 3 2 08 29 2007 Image Toolbox e Feature Robustness testing debugging sever
50. ll settings feature 3 2 4 Sutter 10 3 High speed Emission Filter Switch The Sutter instrument is controlled via an RS232 connection similar to the other modules however due to lack of resources it is setup through an emulated serial port through a USB Universal Serial Bus to Serial RS232 hardware converter Due to the speed of the switch commands sent to the Sutter instrument are given the highest possible priority Many other program functions are bypassed when a command is 36 written to the Sutter instrument and once the switching begins the other three modules are synchronized to prepare for the next exposure 3 3 0 User Interface Module The user interface module is actually the main function of the application Because its timing is determined in large part by user input its processing priority is low however this module controls the actions and parameters of the other three modules This module is responsible for all of the controls on the front panel with the exception of the recording controls as well as the hardware buttons on the microscope body itself and the attached button array Figure 11 Because of the diversity of the controls managed by the user interface module this algorithm is interrupt driven This program structure allows for minimal use of the processor when the user is not making changes The module does have a brief maintenance loop that checks hardware status and communication protocols if the
51. m 2 1 0 Preliminary Research On Nov 11 2006 during a meeting with Dr Gabriel Silva to discuss the current projects underway in the lab the need for implementation of a new custom software designed to coordinate confocal microscopy was reviewed I accepted the responsibility of implementing a new system as my thesis project Starting on Nov 21 2006 I began observing microscopy procedures and experiments in the lab in order to gather usage information and to better understand the specific needs of the lab members I observed various microscopy experiments throughout this project using both ImagePro and the new software Based on my observations in the lab I choose to utilize the LabVIEW programming environment to optimize the speed and performance of the available hardware Development of the control application started on Nov 25 2006 2 2 0 Stage Controller The first challenge was to implement a motion control algorithm for horizontal position of the stage I observed that the preferred control of the stage was the joystick attached to the scope although it was frequently desirable to move to an exact X Y coordinate or return to an exact X Y coordinate used previously Therefore I implemented the joystick as the primary controller with an enable disable switch but I 16 17 added a functionality that would bookmark certain X Y Z locations for quick return or reference In addition I added fields so that X Y Z coord
52. mage and using the mouse scroll wheel to focus The user uses CTRL click on the control image to define the first corner and then the user repeats this process to define the second corner These are listed at Point A and Point B in the indicator above the control image To finish defining the imaging pattern the Slice Depth control must be set to the desired distance between z slices Once the imaging pattern is defined the optical settings above the control image should be defined followed by the light intensity and exposure settings to the right and beneath the control image respectively This process should be repeated for each fluorescence channel the user would like to image Then the desired imaging channels should be selected from the channel list above the recording controls Finally the Interleave control should be enabled to reduce mechanical movement delay through the image space and the Switch Flour control should be set to at the end of sequence to prevent time consuming filter wheel changes between frames 49 Once these pattern settings have been entered the user presses Snap to begin acquisition As always the user may press the Stop recording button to discontinue the capture and save what has been acquired so far 4 2 2 Multiple Timestamped Image Sets The workflow for 3 D movie capture is exactly the same as for capturing single image sets Section 4 2 1 until the user presses
53. mmediately after the recording is complete Also an option to stop the recording at any time during capture was implemented to abort excessively long or mistimed captures 2 4 0 New Development Environment At this stage in the development hardware conflicts with the legacy imaging system prompted a change in development procedure Specifically the new camera drivers installed for use with the new control application were not compliant with the legacy ImagePro imaging system This caused the legacy system to fail repeatedly and repair of the legacy system caused data loss and set back development of the new application Therefore a new hardware configuration was designed and implemented using separate hard disks and operating systems to boot the legacy and new imaging systems 20 Once installed an imaging user could choose to use the legacy or the new microscopy application with a simple selector switch Regardless of which system was used to image recorded data was still available in both operating systems ensuring a seamless transition between applications while the new system was still in development This hardware configuration remained the standard for the remainder of the development process 2 5 0 Fluorescent Lamp In order to incorporate the Lambda DG 4 into the imaging system control information was obtained from the National Instruments support department from a previous joint venture between National Instrument and
54. n as well as the image toolbox that correspond to the new emission filters Cyan and Yellow These new channels added dimensionality to an ever expanding settings and preferences array and this introduced memory based instability into the system Therefore anew memory manager function was added to read and write these settings to disk as opposed to leaving them loaded in program memory A new section was added to the communication module and the RS232 connection to the Sutter instrument was given highest priority 29 55 Runnells Brent Interview with Nathan Shepard National Instruments Nov 29 2007 Media Cybernetics Inc Image pro Plus Start up Guide for Windows Silver Spring MD 2004 57 Runnells Brent Interview with Nathan Shepard National Instruments Nov 29 2007 38 Yu Diana Interview with Nathan Shepard Silva Research Group Jan 26 2007 32 Yu Diana Interview with Nathan Shepard Silva Research Group Jan 26 2007 6 Buibas Marius Interview with Nathan Shepard Silva Research Group Jan 26 2007 Sutter Instrument Company News and Downloads url http www sutter com news software_downloads html 2003 George Nicolas Olympus RS232 Commands Personal communication via e mail Jan 8 2008 Olympus International 1X2 Host I F Specifications N Olympus Development Document April 4 2005 Davidson Marie Interview with Nathan Shepard Silva Research Group Jan 26 2007 6 Olympus Internat
55. n calcium binding changes the conformation of the dye molecule and changes the emission wavelength By calculating the ratio of higher frequency emission unbound dye to lower frequency emission calcium bound dye the normalized concentration of intracellular calcium can be determined To accomplish this mitotic primary spinal cord astrocytes from neonatal rat pups are transfected with genes to express the FRET marker in vitro via a viral agent After a 2 4 hour transfection period and 2 4 hours exposed to a rat specific promoter the cultures are washed and incubated overnight Currently a FRET based dye called Premo Cameleon is being evaluated by the Silva lab as a ratiometric calcium sensor The transfection process leads to successful Preno Cameleon expression in about 50 of spinal cord astrocytes but much fewer in cortical neuronal cells This dye excites at 420nm and emits 485nm cyan when unbound and 570nm yellow when bound to calcium Mechanical or ATP stimulation is used to excite the astrocyte networks and the cell to cell activation time is between 300 800ms Because two separate imaging filters must be used simultaneously to calculate a ratio these filters must be quickly switched between frames To accomplish this the Lambda 10 3 Sutter Instrument was implemented to switch between CFP cyan fluorescent protein and YFP yellow fluorescent protein emission filters at frequencies as high as 13Hz 1
56. nnel meaning that there is no 35 communication from the DG 4 back to the control PC This requires constant maintenance of the DG 4 to make sure that its state is correct Figure 10 fa 25 DG 4 Filters Value Change Image Format Objective IX Condenser heck filter Light Intensity Es DG 4 Filter DG4 ON 121 0 Confocal Disk Edd Flourescence o Shul EEES eo IDG 4 U8 Commands 00 OF Filt Cng Imm 10 1F Filt Cng strobe 144 Open Shutter True_ Y Bright Field Pval San volts ye tH Flourescence Pial Sanl leo IEE pter time val Soni Gain Yal Sari Brightness Oov e ELE En Palan Recall settings Figure 10 LabVIEW User Interface Module featuring control algorithm for DG 4 Optical Filter This section of the user interface module responds when the position of the DG 4 filter is changed by the user Using the previous value of the filter in combination with eight other settings Image Format Objective IX condenser DSU Cube Prism Fluorescence shutter state Confocal Disk position and Emission filter this routine saves the previous settings for six exposure and light variables bright field intensity fluorescence intensity exposure time camera gain camera brightness and camera gamma and loads the previously saved settings for the new DG 4 filter if the user has enabled the Reca
57. ocations channels array into the recording mode so that recorded imaging can begin from the top left corner of the image space Also the Snap record control is disabled in this routine 3 5 0 Image Toolbox In order to take advantage of the image processing capabilities of the software resources must be allocated that are not available during imaging Thus when the image toolbox is opened the imaging module is put on standby until the toolbox is closed The toolbox is responsible for providing methods of opening and manipulating the acquired images and saving them to disk as smaller more intuitive color image files The ability to quickly tile and step through the z axes of 3 D images is also provided Histogram shaping and analysis is provided for both monochrome and color images Channels can be selected or deselected or selected only above a certain intensity value Channel color representations may be changed A customizable burn in scale feature is also available Figure 13 Gain and brightness may also be modified as a post 40 processing feature and several measurements including original normalized brightness may be easily calculated for each channel Once post processing operations are completed the final image may be saved as a common bitmap type file that can be easily read by any imaging application or simply incorporated into any publication Figure 14 Open Image Sets Qdots indicate the presence of the tar
58. of the hardware exactly as it was at startup upon shutdown Based on these and other requirements of experiments underway the imaging system was designed to coordinate image capture with subject position changes and optical setting modifications 1 3 0 Available Hardware Components The imaging hardware revolves around an IX 81 inverted confocal microscope complete with periphery joystick and button panel controls The servos that control the included objective turret wavelength filters light prism condenser bright field light shutters and confocal disk are coordinated by a separate Olympus hardware control the IX2 UCB The IX2 UCB is interfaced via a RS232 serial connection Figure 5 In addition to the scope itself the Hamamatsu ORCA ER IEEE 1394 Camera is placed in the image space opposite the eyepiece This camera is interfaced via an IEEE 1394 fire wire protocol through a separate ORCA ER camera controller Excitation light for the fluorophores is provided by a Lambda DG 4 ultra high speed wavelength switcher and routed through fiber optic cable onto the image subject The DG 4 is interfaced via a RS232 serial connection The horizontal position of the subject is controlled by a Prior Stage Controller interfaced via a RS232 serial connection The high speed emission filter switch is controlled by the Sutter 10 3 instrument interfaced via a RS232 serial connection through a serial to USB Universal Serial Bus c
59. onverter into an emulated RS232 port on the host PC Figure 5 je Control and coordination of the system is executed on a Dell dual core PC Personal Computer running the Windows XP operating system which interfaces directly with each hardware component or its control hardware The imaging control software is run on this PC and the user interface is managed via a pair of flat screen high resolution monitors as well as a mouse and keyboard adjacent to the confocal microscope Figure 5 Hamamatsu Camera Firewire Olympus IX 81 Scope Controller Scion Stage Lambda DG 4 Apa Sutter 10 3 RS232 ScopePC LabVIEW Controls devices Records Images Sends images and scope data position magnification info to 4 corePC in real time 10 4 core PC Receives images and image position data Reconstructs 3D structure Displays 4D structure 3D structure plus time of network and Ca waves Allows rotation and visualization with network structure overlay Image from www swindale ecc ubc ca gallery html Figure 5 Ultimate hardware layout including post processing 4 core Linux machine diagram courtesy of Marius Buibas Silva Research Group 1 4 0 LabVIEW Development Software Early in the design process it became necessary to choose a development environment that would meet a number of important criteria Control of the confocal microscope requires careful timing and monitoring at
60. or cells grafted in the hippocampus of GFAP Vim mice Stem Cells 2007 Jul 12 4 Nakazawa T Takeda M Lewis GP Cho KS Jiao J Wilhelmsson U Fisher SK Pekny M Chen DF Miller JW Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin Invest Ophthalmol Vis Sci 2007 Jun 48 6 2760 8 Lin J Cai W Effect of Vimentin on reactive gliosis in vitro and in vivo analysis J Neurotrauma 2004 Nov 21 11 1671 82 Martinez Contreras A Huerta M Lopez Perez S Garcia Estrada J Luquin S Beas Zarate C Astrocytic and microglia cells reactivity induced by neonatal administration of glutamate in cerebral cortex of the adult rats J Neurosci Res 2002 Jan 15 67 2 200 10 7 Damodaran TV Bilska MA Rahman AA Abou Doni MB Sarin causes early differential alteration and persistent overexpression in mRNAs coding for glial fibrillary acidic protein GFAP and vimentin genes in the central nervous system of rats Neurochem Res 2002 May 27 5 407 15 8 Nakazawa T Takeda M Lewis GP Cho KS Jiao J Wilhelmsson U Fisher SK Pekny M Chen DF Miller JW Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin Invest Ophthalmol Vis Sci 2007 Jun 48 6 2760 8 Stringer JL Repeated seizures increase GFAP and vimentin in the hippocampus Brain Res 1996 Apr 22 717 1
61. pus IX 81 Inverted Microscope sssesssessesssesesesessseesseessresseresseesseeesseee 33 iv 3 23 Lambda DORA is 34 3 2 4 Sutter 10 3 High speed Emission Filter SWitCh ooooocccnnncccnoncccnonaccnonncnonnnnnos 35 33 0 Us r Interface Mod le nin eei a E E T AE 36 3 4 0 R cording A a E E EAEE 37 3 5 0 Image Toolbox eiiean n u e ga aar S 39 4 0 0 Final Worktlo Weir taa 43 ALO SM de 43 4 1 Smele Praise 43 4 1 2 Multiple Timestamped Frames 0 eecccesscecessececesececeeeeeceeeeeceeeeesseeeeesteeeesaes 46 4 20 3 D IMASE tiet r a i e a ede aeta 47 42 SUITS IE SU as os sy ankle cdg EE E IN 48 4 2 2 Multiple Timestamped Image Sets 000 0 ceeecceeseecesnceceeececeeneeeceeeecseeeeseeeeesaes 49 5 0 0 DISCUSSION o wrth caers eat cates nahi dora t ie A E E ee ld E eet ee TAE 51 5 1 0 Real time 3 D Rendering ii ie aaa cited 51 5 1 1 Communication with External Rendering Hard War8 ooonooccccnncccnonccononccinnncnnns 51 5 1 2 Graphical User Interface for Image ManipulatiON coococnnncccnnnncccnoncncnnncnnnnnnno 51 5 2 0 Real time Network Nodal Analysis oooooonnccccnoncccnoncccnnncconnnnnononcnononcnononcncnnncnnnnnos 52 5 3 0 Confocal A n a e e cach AE E A E a S 52 5 4 0 Integration with a Two Photon Microscope oococococcconcnnonnononcnononannnnnn conan ccoo ncconennnos 32 A AN 54 APpendix Version History o E NOE eeano nad EEES ESS ianea 55 Referentes hainesi sta ed A eed a 63 List of Figures Figure 1 As
62. re hasn t been any user input in over 500 milliseconds ms however this brief maintenance algorithm does not put any notable strain on the processor 37 Figure 11 LabVIEW User Interface Module featuring user click on image to re center This section of the user interface module responds when the user left clicks on a portion of the control image If the program has current information on the position of the stage then the program measures the distance between the location where the user clicked and the center of the image converts this vector to units of um and then moves the stage accordingly in order to center the image on the location where the user clicked This routine then logs this position into PosA or PosB depending on which one is being requested and calculates the mean squared difference between PosA and PosB and reports this distance to the display on the user interface 3 4 0 Recording Module The recording module is responsible for coordinating all functions relating to recording image data sets Itis given the lowest processor priority because its functions are not time dependant and its routines are relatively simple The recording module is responsible for calculating the series of physical coordinates that will be sampled in the subject as determined from user inputs on the front panel Also this module is responsible for initiating the recording process when stimulated by the user to do so Figure
63. relatively high frequency 150Hz which led to an event driven and interrupt based program architecture From a user interface standpoint the resulting application must provide an intuitive workflow and a simple layout of features and controls Also the new program must operate within the Microsoft Windows XP Professional operating system on a 2 99GHz Pentium 4 Datel Systems PC with 2 00GB of RAM Native image processing and display capabilities were necessary as well as an ability to perform these operations quickly with a minimum of required processing and memory resources 11 In light of these criteria the LabVIEW development suite presented the best overall production environment option The event structures inherent to LabVIEW programming architecture natively support event driven programming necessary for scenarios involving broad user input possibilities such as the confocal microscope LabVIEW was built from the ground up for signal processing applications operating in less than dedicated environments like Microsoft Windows XP LabVIEW also allows for processor specific routines so that certain high priority or time critical processes can be pre allocated specific processing resources The LabVIEW development suite also provides a simple reliable and pleasing user interface free of redundant work multiple control windows and hardware communication failures 1 5 0 Theory of Implementation Imaging is
64. s already been developed with the rendering algorithm a smart signal tracking algorithm could be designed to actively track the progression of network signaling A simple decision algorithm could decide where the most interesting network information may be imaged and move the image plane to that location 5 3 0 Confocal Disk Currently the confocal disk must be manually moved into and out of the image path As soon as the command set for this module is delivered by the Olympus corporation the command should be incorporated into the software control of the confocal disk 5 4 0 Integration with a Two Photon Microscope The Silva Research Group has been interested in implementing two photon microscopy in the near future which was one reason why the application was designed to 101 be flexible across hardware platforms This design lends itself towards easy integration with another microscopy setup however an additional and separate hardware 53 control PC will be necessary in order to leverage the appropriate processing resources required for another control load Buibas Marius Buibas Marius 2 Buibas Marius Buibas Marius 2 Buibas Marius Interview with Nathan Shepard Interview with Nathan Shepard Interview with Nathan Shepard Interview with Nathan Shepard Interview with Nathan Shepard Silva Research Group May 23 2007 Silva Research Group May 23 2007 Silva Research Group J
65. s of the glial network These molecules excite at 350nm and emit at 605nm In order to map the structural filaments to the cellular structure the glial nuclei are marked using DAPI 4 6 diamidina 2 phenylindole a fluorescent stain that binds strongly with DNA This dye excites at 350nm and emits around 470nm Figure 2 gly y Vian 7 Figure 1 Astrocyte cells with nuclei stained with DAPI blue and GFAP stained with FITC green Control experiment no primary antibody Figure 2 Astrocyte cells with nuclei stained with DAPI blue and GFAP stained with FITC green Once a successful drug has been found to reduce gliosis using the relatively simple ICC technique western blot analysis is used to confirm those findings Once successful results can be reliable repeated further study in human retinal cells is anticipated 1 1 2 Current project Flour 4 Calcium wave Imaging A sudden intracellular increase in calcium is the primary mechanism of astrocyte activation In astrocyte networks cell to cell activation can be tracked by monitoring intracellular calcium concentration and calcium waves of multiple networked astrocyte activation are observed using this method Working towards a functional understanding of extra cellular coordination of these networks it is desirable to quickly and accurately f 18 monitor the calcium concentrations across an astrocyte network Figure 3
66. stable neutral density filter is installed in the camera light path as a grey filter that diminishes intensity equally across all wavelengths A simple routine was written to govern the position of this filter usually set in the open or 100 position Similar to the DSU cube and IX condenser exposure settings and light intensity settings for each neutral density filter are recorded and reinstated as the filter grey level is changed 2 6 7 Bright Field The bright field lamp and shutter are internal to the scope and they are controlled using the same command set as the objective turret In order to conserve the life of the bright field lamp the new algorithm cuts the power to the lamp when the shutter is closed unlike the legacy system This modification does not impede performance as the bright field lamp lights more quickly than the shutter opens 2 6 8 Joystick Jog Wheel The joystick and jog wheel controls can be set to operate internally without supervision from the control application However in order to implement motion restrictions and axis inversions the joystick and jog wheel have been modified to interact 24 with the control application and receive permission before performing any modifications to the stage of turret positions This does not cause any noticeable lag in performance 2 6 9 Confocal Disk Although the confocal disk is internal to the scope the confocal disk controls are not part of t
67. terface Module e Feature Standard Format 7 image format change without user cycling camera power e Feature Calibration data saved in profile e Feature Stop Reset sets fail safe condition shutters closed objective turret down etc e Feature ESC button same as pressing Stop Reset e Feature Automatic error handler enabled e Feature Non essential controls disabled during recording e Control Print profile e Indicator X axis ruler e Indicator Y axis ruler Recording Module e Feature Profile exposure and light intensity settings saved with each frame e Feature Image data backup to RAM is disk writing error 3 2 1 02 16 2007 User Interface Module e Feature Mouse wheel duplicates jog wheel action e Feature Shutter switch defaults and operation modified Recording Module e Control Snap image button single image recording 322 02 19 2007 User Interface Module e Feature Redesign of control indicator layout on front panel e Feature Robustness testing debugging several small issues 3 2 3 02 24 2007 User Interface Module e Feature Settings remembered for combinations of DSU Cube DG 4 filter IX Condenser Prism and DG 4 On 3 2 4 02 25 2007 Hamamatsu ORCA ER Camera e Feature Shutter time error tracked and corrected in real time User Interface Module e Feature Redesign of control placement on front 59 panel 3 2 5 03 03 2007 IX 81 Scope e Feature Escape button toggles objecti
68. th the bright field and fluorescence shutters are temporarily closed to prevent photo bleaching 2 8 0 Z stack and Tiling Two critical features that the legacy imaging system boasted were z stack acquisition and tiling capability In order to implement these features in the new software the recording module was completely rewritten in April 2007 Using the same algorithm that measured distance in the stage control module two X Y Z coordinates can be selected as absolute limits of imaging defining a 3 D box which the program would image and stitch together into a large tiled 3 D image Several iterations of this image may also be recorded with timestamps to create a 3 D movie Also multi channel recording was also added as a feature to allow for multiple emission spectra to be images simultaneously and overlaid in the Image Toolbox 2 9 0 Image Toolbox In order to incorporate image processing techniques without compromising valuable system resource during recording a separate associated application was built to process the captured images These applications are linked and work well together however they utilize the same resources so the imaging application goes into standby mode while the Image Toolbox is open While in standby the imaging application is designed to close the bright field and fluorescence shutters to minimize photo bleaching The Image Toolbox was designed around the multi channel recording feature inst
69. trocyte cells with nuclei stained with DAPI blue and GFAP stained with FITC green Control experiment no primary antibody ooooocnnocccnoncccnonccnnnonaninnnnnos 3 Figure 2 Astrocyte cells with nuclei stained with DAPI blue and GFAP stained with FITC Freen A A A PRE ae eat ee 3 Figure 3 Astrocyte network exhibiting calcium wave via Fluor 4 AM calcium indicator after mechanical stimulation de caes 4 Figure 4 Functional diagram of Fluor 4 AM calcium marker image courtesy of Diana VANE a 5 Figure 5 Ultimate hardware layout including post processing 4 core Linux machine diagram courtesy of Marius Buibas Silva Research Group ooooococccocccnocaninncnoncnanoss 10 Figure 6 Timing diagram for 3 D image acquisition oooccoccconononnnononcnoncnononnnnnnnnncnnn corn ncnns 12 Figure 7 LabVIEW Camera Module featuring Image Format change oooonocccnnnccccnnncc n 32 Figure 8 LabVIEW Communication Module featuring PRIOR stage current X Y POSO acquisition o io tasa 33 Figure 9 LabVIEW Communication Module featuring Scope Port ooonnoccnnncccccoccccnnnnnss 34 Figure 10 LabVIEW User Interface Module featuring control algorithm for DG 4 A A A 35 Figure 11 LabVIEW User Interface Module featuring user click on image to re center 37 Figure 12 LabVIEW Recording Module featuring Capture SetUP coooocnoncccnonnccnnnccnnnnnnos 39 Figure 13 Image Toolbox user interface 2 iii ia ta Boe 40 Figure 14 Image Toolbox display of a saved image
70. turret would return to its former position but it would be grossly out of focus requiring a manual refocus each time Because each objective has a different focal length it was beneficial to design an algorithm to remember these focal lengths and automatically move each objective into focus when it was selected This algorithm also protects the larger oil objectives from punching into the slides by moving the turret down switching to the oil objective at the lowest setting and then waiting until the Escape button is pressed before returning to the memorized focus level This allows time for the user to apply oil adjust the imaging subject and ensure a proper scope configuration before moving the objective close to the subject 2 6 3 DSU Cube The mirror unit on the scope is used as a wavelength filter to distinguish emission wavelengths There are six total filter wheel positions however one is permanently reserved for viewing the subject through the eyepiece The other five can be changed at 22 any time but currently are five of six possibilities a polarized filter a DAPI filter excitation 350nm emission 470nm a FITC filter excitation 495nm emission 520nm a TRITC filter excitation 557nm emission 576nm a Q dot filter excitation 350nm emission 605nm and a dichroic mirror for use with emission filters in the Sutter 10 3 A simple algorithm was designed to control the filter wheel itself but the state of the
71. ule e Feature Open sample of recorded images for review during save process e Feature Close shutters during image save process to prevent photo bleaching e Feature Redesign of control indicator layout on front panel e Feature Robustness testing debugging several small issues Recording Module e Complete capture information added to file recorded with each image file 4 2 1 03 24 2007 User Interface Module e Feature Button permissions updated Recording Module e Feature Automatic handling of multi channel imaging 4 2 2 03 28 2007 User Interface Module e Control Open Image Toolbox Image Toolbox e Control Indicator Open image sets list select Control Open new image set Control Close image set Control Indicator Indicator Saved images list Control Save image Control Rename image Control Selected channels Control Channel colors Control X axis Control Y axis Control Z axis Control Time axis Control Original histogram Control Auto mark histogram cutoffs Control Histogram shaping select Control Burnt in scale placement Control Burnt in scale color Control Burnt in scale length um Control Close Image Toolbox Indicator Resulting IEEE 1394 image display Indicator Image description 61 e Indicator Available channels e Indicator Final histogram e Indicator Processor busy light 4 2 3 04 09 2007 Recording Module e Feature Optimization of 3 D imaging workflow e Contro
72. ve turret up down User Interface Module e Feature Don t allow user to close minimize window e Feature Dialogue for save profile e Feature Dialogue for new features at startup e Control Real time full scale contrast stretch of control image e Control Load saved image Recording Module e Feature Multi channel recording e Control Flour color e Control Edit colors 3 2 6 03 07 2007 Hamamatsu ORCA ER Camera e Control Auto set gain brightness and gamma Recording Module e Feature Advanced real time image analysis e Indicator Image histogram 3 2 7 03 08 2007 Recording Module e Indicator Low Saturation warning light e Indicator High Saturation warning light e Indicator Low Distribution warning light e Indicator High Distribution warning light e Indicator Advice text on how to improve current image acquisition based on histogram analysis 4 0 0 03 11 2007 IX 81 Scope e Control Confocal disk in out User Interface Module e Feature Automatic conversion of profiles from previous profiles Recording Module e Feature Automatic mapping of 3 D imaging given two X Y Z corner coordinates and slice depth nm e Control Image mode 2 D or 3 D e Control Interleave 4 1 0 03 17 2007 Recording Module e Feature Multi channel color recording for single image and movie modes e Control Selected channels 60 e Control Channel colors e Indicator Available channel list 4 2 0 03 18 2007 User Interface Mod

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