Imaging CryoMicrotome BS-8 - Biomedical...

BBarlow SScientific, IInc.

Imaging CryoMicrotome™ BS-8 Version 2010.UCD TECHNICAL MANUAL

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BARLOW SCIENTIFIC, INC.

6307 Tamoshan Drive Olympia, WA 98502 USA

Phone: (360) 867-6053 Fax: (603) 462-9083

Imaging CryoMicrotome™ Manual

Model BS-8 Version 2010.UCD

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The information in this manual has been reviewed and is believed to be reliable, however, no responsibility is assumed for inaccuracies. The material in this manual is for informational purposes only, and may be changed without notice. The equipment that this manual describes is subject to design change and product improvement. Warning

Barlow Scientific, Inc., assumes no liability to damages consequent to the use of this product. This product is not designed with components of a level of reliability suitable for use in life support or critical applications.

Trademarks Any tradenames or trademarks not belonging to Barlow Scientific, Inc., used in this document are used for reference purposes only. Barlow Scientific, Inc., disclaims any interest in the tradenames or trademarks other than its own. Alta is a trademark of Apogee Imaging Systems. LabVIEW is a trademark of National Instruments Corp. Luxtel is a trademark of LuxteL LLC. Windows is a trademark of Microsoft Corp.

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Table of Contents 1. System Description 1.1 Physical Components 1.2 Software 1.3 Component Interconnections 2. Hardware Setup 2.1 Installing the Microtome Knife 2.2 Refrigeration 2.3 Sample Preparation

2.3.1 Freezing Sample 2.3.2 Embedding and Mounting Sample 2.3.3 Tissue Embedding Media

2.4 Sample Plate Mounting 2.5 Filter Wheel Mounting

2.5.1 Excitation Filter Wheel 2.5.2 Emission Filter Wheel

2.6 Changing Optical Filters 2.7 Camera Mounting 2.8 Lamp Operation 3. System Start-Up/Shut-Down 3.1 Power Up Order 3.2 Power Down Order 4. Software Operation 4.1 Starting the Program 4.2 Program Description 4.3 Panel Descriptions

4.3.1 Main Panel 4.3.2 Image Acquisition Panel 4.3.3 Subsystem Setup Panel 4.3.4 Camera Settings 4.3.5 Image File Settings 4.3.6 Filter Wheel Settings 4.3.7 Imaging Settings

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4.3.8 Sample Settings 4.4 NI Vision Tools Palette 4.5 ICM 2010.UCD Initialization File 5. Image Processing 5.1 Measuring Image Scale 5.2 Subtracting Dark Current Noise 5.3 Correcting Nonuniform Illumination 5.4 Replacing Defective Images 6. Operational Review 6.1 Freezer Startup 6.2 Sample Preparation 6.3 Lamp Startup 6.4 ICM Status 6.5 Program Startup 6.6 Instrument Configuration 6.7 Image Acquisition 6.8 System Shut Down 6.9 Image Processing 7. System Maintenance 7.1 Maintenance Schedule 7.2 ICM Cleaning 7.3 Knife Maintenance 7.4 Lamp Maintenance 7.5 Optical Filter Care 8. Technical Information 8.1 Contact Information 8.2 H2W Crossed Axis Roller Gantry 8.3 Apogee Alta U32 Camera 8.4 Optical Filter Spectra 8.5 CeraLux CL300BF Xenon Lamp 8.6 Cold Mirror Chimney 8.7 Other Optical Characteristics 9. Conditions of Acceptance

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1. System Description

WARNING! Read and understand all of the instructions and safety information in this manual before operating this instrument. Failure to observe this warning can result in bodily injury.

Thank you for purchasing the Barlow Scientific, Inc., Imaging CryoMicrotome™ (ICM). Before starting to use your new ICM, we recommend reading these instructions carefully to obtain optimum performance and a longer service life from the instrument. Be sure to retain this manual for future reference. The Barlow Scientific, Inc., Imaging CryoMicrotome™ is an automated image acquisition and analysis system consisting of hardware and software designed to vascular, pulmonary, and respiratory flow from organs filled with fluorescent microspheres. A linear-motor-driven microtome serially sections frozen tissue while filtered light excites fluorescence from microspheres in the exposed surface of the remaining tissue block. After each slice, a CCD camera records fluorescence images of the block and saves the digital images to storage media. Images are later analyzed to provide quantitative results for luminescent features in the organ. Images created by the ICM have a variety of applications in biomedical research.

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1.1 Physical Components 1.1.1 BSI Imaging CryoMicrotome Model# BS-8 S/N BS8001 1.1.2 Computer and Parts 1. Dell Computer T3500

Service Tag 70T8JQ1 Product Key 384MK-HJDVK-HVWTC-BJ9MQ-RXPQY

2. Dell P2210 monitor S/N CN-0U828K-74445-14R-JJWS

3. Dell Keyboard and Mouse 1.1.3 ScienTemp Custom Freezer Model# BS40-12A

S/N S8004606 1.1.4 H2W XY Crossed Axis Roller Positioning Gantry 1. Model# XRS-09-19-XY-001 2. Linear Motors (Model# BLDD-2B-NC-WD3S-13)

X-Axis, 19", S/N 110824R0193 Y-Axis, 9", S/N 110824R0192

3. Technosoft IDM3000-ER Servo Drivers (Model# MCSA-IDM3000-10-2) X-Axis S/N GH1519 Y-Axis S/N GH1520

4. Advanced Motion Controls Power Supply (340 VDC, 30A, Model# PS30AB) S/N 58352-1003

5. Meanwell Power Supply (24 VDC, 1.5 A, Model# RS-35-24) S/N EB04347306

1.1.5 DDK Custom Microtome Knife o Custom 2-piece Knife

6" Tungsten Carbide edge 7.5 " Steel back

o 49 degrees Total Angle 34 degrees on front edge 15 degrees on back edge

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1.1.6 Luxtel Xenon Arc Lamp with Housing and Power Supply 1. CeraLux 300 W Xenon Lamp (P/N CL300BF)

S/N 2. CeralLux Lamp Module (P/N CL1338)

S/N 3185738 3. Power Supply (P/N CL1470) 1.1.7 Apogee Alta U-32 Digital Camera

Model# D01F-VS25D-U03200-MM2A o D01F body (C-Mount back-focal distance) o Vincent Shutter o Thermoelectric Cooling o Grade 2 Kodak KAF-3200ME CCD Sensor o USB 2.0 Interface

1.1.8 FLI Seven-position Excitation and Emission Filter Wheels 1. Excitation (Model# CFW2-7)

S/N FW0181011 2. Emission (Model# CFW2-7)

S/N FW0251071 1.1.9 Optical Filters Excitation Filter Wheel Positions: 0. Open (no filter) 1. Automated Entertainment UV PASS Blacklight (360/40) 2. Chroma D440/20x (Lot 239681) 3. Chroma D510/20x (Lot 239629) 4. Chroma D560/20m (Lot 239682) 5. Chroma D650/30x (Lot 250085) 6. Chroma D750/30x (Lot 249951) Excitation Filter Wheel Positions: 0. Open (no filter) 1. Chroma D480/30m (Lot 230831) 2. Chroma D505/30m (Lot 231051) 3. Chroma D555/30m (Lot 231233) 4. Chroma D635/30m (Lot 249783) 5. Chroma D700/30m (Lot 249046) 6. Chroma D800/30m (Lot 249899)

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1.1.10 OCLI Cold Mirrors 1. 45 Degree UV Cold Mirror, Borofloat glass 2. 45 Degree Cold Mirror, Borofloat glass 1.1.11 AC Power Connections 1. Computer power (120V AC) 2. Monitor power (120V AC) 3. Freezer power (120V AC) 4. Lamp power supply (120V AC) 5. Lamp Housing power supply (120V AC) 6. Instrument power supply (120V AC) 7. Instrument power supply (208V, 1Ph AC) 8. Camera power (120V AC) 9. Viewport fan (120V AC) 1.1.12 Tools o SAE Ball-Point Hex Key Set o Large Crescent Wrench (not included) o Phillips Head Screwdriver (not included) o Flat Head Screwdriver (not included) 1.2 Software 1. Windows 7 Professional 32-bit Operating System

Product ID 00371-OEM-8992671-00524 2. BSI ICM Control Software Ver. 2010.UCD 3. National Instruments LabVIEW 2009 SP1 Run-Time License 4. NI Vision 2010 Run-Time License

User Clyde Barlow, Barlow Scientific, Inc. S/N M73X55199

5. NI-DAQmx Device Driver 9.0.2 6. NI-IMAQ Vision Acquisition Driver 4.5 7. Technosoft TML_LIB for LabVIEW 2.0 8. Easy Motion Studio, Build 2012022202 9. Apogee SDK Release 3.12.28.2150 10. FLI Library SDK Release 1.71

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1.3 Component Interconnections

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2. Hardware Setup 2.1 Installing the Microtome Knife When changing a knife, be extremely careful. Please do not touch the edge of the microtome knife. Using a knife cover at all times is advised. When the knife is not installed in the ICM, it should be stored in its plastic case. Barlow Scientific, Inc. does not assume any responsibility for injuries (see section 8, Conditions of Acceptance). The knife holder is aluminum and located inside of the freezer towards the front wall. Bring the freezer up to room temperature before attempting to remove the knife holder.

Shield the knife edge with the cover. Take notice of the position of the cap screw in the arced recess. To remove the knife holder from the instrument, unthread the two 3/8" cap screws with a hex key. Carefully lift out the knife assembly.

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Remove the knife by carefully unthreading the four 1/4" cap screws attaching it to the holder. Place the old blade in its plastic case. Carefully position the new or resharpened knife against the holder; thread the four 1/4" caps screws with washers until tight.

Reinstall the knife assembly in the instrument by reversing the above removal instructions. Rotate the knife holder to the previously noted blade angle. Tighten both cap screws well. 2.2 Refrigeration Plug in the freezer and turn on the control switch to begin cooling the sample chamber. All openings to the chamber should be covered. It takes 6 to 8 hours to cool to the currently set temperature of -20 °C. Refer to the freezer manual for instructions on modifying the target freezer temperature. Note: To avoid freezer burn when working inside the cold Imaging CryoMicrotome™, a long sleeved shirt and gloves are recommended during setup. 2.3 Sample Preparation After the organs have been experimentally prepared, the sample should be frozen and mounted in a black embedding medium. The sample is then attached to a sample holder by freezing. Once set, the sample holder is placed in the Imaging CryoMicrotome™. 2.3.1 Freezing Sample Freeze the sample in a free-standing/hanging position to retain the organ’s anatomic structure. Internal chambers may be infused with black embedding medium before freezing. 2.3.2 Embedding and Mounting Sample Prepare the sample by trimming any excess tissue. Fat tissue may fluoresce under certain filter combinations, so be careful to remove it all.

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Select a sample holder and an appropriately sized sample mold. The sample mold should fully enclose the sample without touching the sides and fit within the machined grooves on the sample holder. After taping the sample mold together and placing it on the sample holder, freeze the assembly until cold.

Once the sample mold and holder have been frozen, fill the mold with a 1/2"-1" layer of black embedding medium. Freeze until hard; remove from freezer and melt the top of the frozen block with warmed metal. Attach tissue to softened surface with a dollop of black embedding medium. After a few seconds, the tissue will be firmly frozen to the surface. When all tissue samples have been treated in this manner, fill the mold with enough black embedding medium to cover all tissue samples. Make a note of the tissue sample positions within the block for later reference. Freeze until solid, about 4 to 8 hours, depending on volume. Take block from freezer and remove the sample forms. Wash the forms and set aside to reuse later. If the cross-sectional area of the sample block is 1 in² or less, add bulk by surrounding it with white embedding medium using the above described method. Soften the four block sides with warmed metal. Surround the black block with a larger form. Fill new form with white embedding medium until it just covered the frozen block. Return to the freezer until solid.

Sample may stay in freezer as long as needed. Leave the sample block in its forms and cover the exposed surface with plastic wrap. This slows the dehydration of the embedding medium.

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After the sample is securely frozen onto the plate, mount the sample plate on the tool post in the cryomicrotome.

2.3.3 Tissue Embedding Media 2.3.3.1 8% Polyvinyl Alcohol (PVA) Solution 1 Liter 8 Liters PVA 540, g 80 640 DI Water, g 920 7360 Total, g 1000 8000 Heat water in double boiler until hot. Add PVA while stirring vigorously. Elevate solution temperature to 93 °C or higher and hold for 60 min. Heat solution until lumps are dissolved. Turn off heat and leave covered until solution is cool. Store PVA solution in refrigerator to retard bacterial growth. 2.3.3.2 4% Carbon Black PVA Solution 100 mL 800 mL Carbon Black, g 4 32 8% PVA Soln, g 96 768 Total, g 100 800 Add carbon black to PVA solution in a bottle with mixing stones. Place the bottle on a roller until well mixed. Avoid shaking the solution and introducing air bubbles. Store black PVA solution in refrigerator to retard bacterial growth. 2.3.3.3 Chemical Supplies o Celvol 540 Polyvinyl Alcohol (Hydrolysis 87-89%; Viscosity, %4 aqueous, 45-55

cP), Sekisui Specialty Chemicals America, LLC, Dallas, TX (www.sekisui-sc.com/sekisui/home.htm or search www.ebay.com)

o Lamp Black, Daniel Smith Dry Pigment, Part No. 284 030 040, Daniel Smith Inc.,

Seattle, WA (www.danielsmith.com)

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2.4 Sample Plate Mounting The sample plate attaches to the Imaging CryoMicrotome™ by a tool post bolted on the linear motor carriage. Clamp a tool holder onto the back of the sample plate. Slide the sample plate assembly over the tool post's tapered gib. Use the knurled nut to adjust the sample's vertical position. Rotate the post handle clockwise to lock the sample plate in place.

2.5 Filter Wheel Mounting To replace filters, the filter wheels must be removed. The Changing Filters instructions are located in section 2.6. 2.5.1 Excitation Filter Wheel The excitation filter wheel is located in front of the Luxtel lamp and is perpendicular to the front plate. To mount, slide the filter wheel over the ring attached to the cold mirror chimney and tighten the thumbscrew and the cap screw. When clamping the filter wheel ensure it is flush to the surface of the cold mirror chimney. To dismount, loosen the same two screws. When working with the excitation filter wheel, be careful not to touch the front-surface mirror used to position the light from the lamp. 2.5.2 Emission Filter Wheel The emission filter wheel is located in front of the viewing window, behind the camera, and parallel to the front plate. It is attached to the front plate by two thumb screws. 2.6 Changing Optical Filters Detach the filter wheel from the Imaging CryoMicrotome™. Remove the filter wheel cover by unthreading the 10 Philips-head screws.

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Optical filters are held in place with threaded retaining rings. Remove the rings by carefully inserting a flat screw driver or tweezers into the notches and unscrewing. Take great care and do not scratch the surface of the optical filter. Use a piece of tissue paper to push upward on the edge of the filter you would like to remove. Handle the filter by the metal rim, being very careful not to touch the glass surface with bare fingers.

Optical glass filters have a specific orientation, which is necessary to minimize autofluorescence and maximize performance. There is a caret (or arrow) marked on the edge of each filter to aid orientation. Filters should be positioned with the arrow pointing towards the sample (for an excitation filter, the arrow points away from the light source; for an emission filter the arrow points away from the detector). Gently place the filter into an empty pocket. Record the position number for the filter. Be careful not to touch the glass surfaces of the filter.

Carefully thread the retaining ring to secure the filter. Gently snug the ring. IMPORTANT: Over tightening the ring may damage the filter. Do not cross-thread the retaining ring. It should rotate freely within the threads. If the ring binds, rotate the ring in the opposite direction to correctly start threading.

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Screw the cover back on and reattach the wheel to the ICM. 2.7 Camera Mounting The Apogee camera is mounted on a moveable bracket. Slide the bracket to change the camera's focal distance. Secure the camera bracket to the Imaging CryoMicrotome™ with the 1/4" thumb screw. 2.8 Lamp Operation Before igniting the xenon arc lamp for the first time, review the Luxtel Safety and Operating Hazards for CeraLux Lamps. Following the instructions outlined in the manual for the Luxtel CL 1470 Power Supply, ignite the lamp. Use the front-surface mirror to position the beam on the sample. The high intensity radiation of the lamp can damage eyes and burn skin. Observe all of the safety recommendations outlined in the manuals. Precautions: o Never look directly into the output beam of the housing when operating the lamp. o Never look at a specular (mirror) reflection of the beam, even for short periods of

time. o Never put any part of your body in the path of the output beam. o Use the interlock system to prevent access to a working lamp. Ignition of the arc lamp requires high voltage high frequency pulses followed by a high current dump. Both of these are sources of electromagnetic interference, radiated and conducted. Recommendations: o Start the arc lamp before powering nearby computer systems. o Keep the computer at least 6 feet away from the ignitor/power supply. o Use a different outlet and line for the computer and ignitor/power supply. Minimize the amount of time that the lamp shines through a single optical filter in the excitation filter wheel by employing the shutter. Prolonged exposure to the intensity of the lamp may damage the filter by scorching or breaking.

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3. System Start-Up/Shut-Down 3.1 Power Up Order 1. Ignite Arc Lamp. 2. Turn on ICM Motor Control Box. 3. Start Computer. 4. Turn on Camera. 5. Turn on Filter Wheels. 6. Start ICM Program. 3.2 Power Down Order 1. Turn off ICM Motor Control Box 2. Turn off Arc Lamp. 3. Exit ICM Program. 4. Turn off Filter Wheels. 5. Turn off Camera. 6. Turn off Computer.

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4. Software Operation 4.1 Starting the Program Double-click the BSI ICM 2010.UCD icon to start the program. When BSI Imaging CryoMicrotome 2010.UCD starts, the software reads default information from the initialization file, then checks for the presence of the excitation and emission filter wheels, the linear motor and the camera. 4.2 Program Description Imaging CryoMicrotome 2010.UCD has three primary panels for system control and five secondary panels for subsystem configuration.

Main: The initial panel that opens when the application starts. Displays the camera temperatures and cooler status. Acquire: Controls the automated image acquisition. Setup: The gateway to instrumental subsystems configuration:

Camera: Modify the camera configurations for each color setting. File: Modify the image file destinations and configurations for each color

setting. Filter Wheel: Assign and test the filter wheels. Imaging: Modify the imaging configurations for each color setting. Sample: Position and plane the sample and set the slice width.

A hierarchy of the program panels follows:

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4.3 Panel Descriptions 4.3.1 Main Panel BSI Imaging CryoMicrotome 2010 [ICM2010UCD.vi] is the top-level instrument panel for the Imaging CryoMicrotome™ program. It also includes a display of current camera temperatures. This program controls the Imaging CryoMicrotome, which is an automated digital image acquisitioning and serial cryostat-sectioning instrument. Written with LabVIEW 2009 SP1, NI Vision 10.0, NI-DAQmx 9.0.2, NI-IMAQ 4.5, MS Windows 7 Professional SP1 April, 2012

Control Descriptions CCD o The current temperature of camera sensor in degrees Celsius. Cooler Status o The current status of the cooler.

Off: No drive is applied to the cooler. Ramping: The cooler is ramping to the Set Point temperature. At Set Point: The cooler is at the Set Point temperature. Revision: The controller has generated a new Set Point.

Heatsink o The current temperature the camera heatsink in degrees Celsius. Top Level Control o Top level control buttons.

Main: Top-level instrument panel with camera temperature display. Acquire: Automated image acquisition panel. Setup: The gateway panel to subsystem configurations with current settings

display. Camera: Modify the camera configurations for each color setting. File: Modify the image file destinations and configurations for each color


Exit: Exit the program.

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4.3.2 Image Acquisition Panel ICM Image Acquisition [ICM.UCD Acquire.vi] is the automated image acquisition panel for ICM experiments.

Control Descriptions Colors o A list of the color settings for the

experiment. Current Scan No. o The number of the current scan. Display Palette o Select a psuedo-color palette for

the image window display. The palette is for display purposes only. Images will be saved without a palette, as grayscale. Gray: Linear Black - White Gradation Binary: Repeating 16 Color Pattern

Useful for visualizing individual graylevels. Gradient: Gradation from Red - Lt. Blue - White Rainbow: Gradation from Blue - Green - Red Temperature: Gradation from Black - Dk. Red - White Pastels: Repeating Pastel Pattern with Blk - Wht

Useful for visualizing dim images. Hot Spot: Gradation from Black - White with Red Max

Useful for locating saturated pixels. Image Acquisition o Image acquistion control buttons.

Start: Start slicing the sample and collecting images for each color setting in the experiment.

Pause: Pause the slicing and the imaging after the current scan is completed. Stop: Stop the slicing and the imaging after the current scan is completed. Note: The current scan number will be reset to the starting scan number when

scanning is restarted. Progress (mm) o Displays the amount of sample block removed (mm). Save Dark-Current o Save a dark-current image for each color setting in the experiment to a file on

disk. The dark-current image is an image taken with the shutter closed. Starting Scan No. o The number of the starting scan. The scan number will be used to incrementally

name image files.

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Top Level Control o Top level control buttons.




Exit: Exit the program. Total Scans o Total number of scans to be collected. One scan is an image collected for each

color setting. Width (µm) o Displays the width of a single microtome slice. (microns)

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4.3.3 Subsystem Setup Panel ICM Subsystems Setup [ICM.UCD Setup.vi] is the gateway panel to the ICM subsystem configurations. It includes a display of current instrument settings.

Control Descriptions Camera o Camera: Modify the camera

configurations for each color setting in the experiment. Also display and acquire live images.

Colors o A list of the current color

settings for the experiment. Select a color to display its imaging and file configurations.

File Configuration o The current image file

configuration for the selected color setting.

File o File: Modify the image file

destinations and configurations for each color setting in the experiment. Also add new or remove existing color settings.

Filter Wheel o Filter Wheel: Assign and test the filter wheels Image Bit Depth o The current number of bits to output per image pixel.

16-bit: Image intensity range is 0 - 65535 graylevels. Digitization speed is 1 MHz.


Image File Type o The current image file type.

PNG: Portable Network Graphics (*.png) JPEG2000: Joint Photographic Experts Group 2000 (*.jp2) TIFF: Tagged Image File Format (*.tif)

Images are a nonstandard extension of the TIFF standard. Note: All formats use lossless image compression.

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Imaging Configuration o The current camera configuration for the selected color setting. Sample o Imaging: Modify the imaging configurations for each color setting in the

experiment. Sample o Sample: Position and plane the sample block and set the microtome slice width. Slice Width (µm) o The current width of a single microtome slice in microns. Top Level Control o Top level control buttons.





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4.3.4 Camera Settings Panel ICM Camera Settings [Setup ICM.UCD Camera.vi] displays and allows the user to modify camera digitization resolution and camera cooler settings.

Control Descriptions Backoff Point o Specify the Backoff temperature of the

cooler in degrees Celcius. o Note: If the cooler is unable to reach

the Set Point, a new Set Point is determined by the Backoff Point number of degrees up from the lowest temperature reached.

o The Backoff Point is used to prevent the cooler from being constantly driven at maximum power to an unreached temperature.

CCD o The current temperature of the CCD

sensor in degrees Celcius. Camera Model o The model name of the Apogee

camera. Drive Level o The drive level applied to the temperature controller. (0% - 100%) Fan Speed o Select the fan speed. (Off, Low, Medium or High) Heatsink o The current temperature of the camera's heatsink in degrees Celcius. Image Bit Depth o Select the number of bits to output per image pixel.



Mode o Control cooler operation. Sensor Model o The model name of the sensor installed in the Apogee camera.

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Set Point o Specify the cooler temperature in degrees Celsius. o Note: If the cooler cannot reach the Set Point, a new Set Point is determined by

the Backoff Point number of degrees up from the lowest temperature reached. Status o The current status of the cooler.

Off: No drive is applied to the cooler. Ramping: The cooler is ramping to the Set Point temperature. At Set Point: The cooler is at the Set Point temperature. Revision: The controller has generated a new Set Point.






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4.3.5 Image File Settings Panel ICM Image File Settings [Setup ICM.UCD Files.vi] modifies the image file destinations and configurations for each color setting in the experiment. The user may also add or remove color settings to/from the experiment.

Control Descriptions Add New o Add a new color setting to

the experimental setup. Colors o A list of the color settings

for the experiment. Select a color to review and/or edit its image file configuration.

Delete Selected o Delete the selected color setting from the experimental setup. Image File Configuration o Image file configuration for the selected color setting. Review and/or edit the

configuration. Color Label: Type a unique brief description of this color (fluorochrome) setting. Image Directory: Enter the directory path for stored image files. Filename Root: Type the root text for incrementally named image files.

Image File Type o The current image file type.

PNG: Portable Network Graphics (*.png) JPEG2000: Joint Photographic Experts Group 2000 (*.jp2) TIFF: Tagged Image File Format (*.tif)

Images are a nonstandard extension of the TIFF standard. Note: All formats use lossless image compression.




setting. Filter Wheel: Assign and test the filter wheels.

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Imaging: Modify the imaging configurations for each color setting. Sample: Position and plane the sample and set the slice width.


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4.3.6 Filter Wheel Settings Panel ICM Filter Wheel Settings [Setup ICM.UCD Filter Wheels.vi] assigns the filter wheels as either the excitation or the emission wheel.

Control Descriptions Select Filter Wheel: o Select a filter wheel for moving to a

filter position or changing device filename.

Swap o Exchange the excitation and emission

filter wheels assignment. Emission Filter Wheel Model o Model number of emission filter wheel. Excitation Filter Wheel Model o Model number of excitation filter

wheel. Filename o Unique filename of FLI filter wheel. Filter Position o Move the selected filter wheels to a position. Top Level Control o Top level control buttons.





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4.3.7 Imaging Settings Panel ICM Imaging Settings [Setup ICM.UCD Imaging.vi] modifies the imaging configurations for each color setting in the experiment. The user may also view and acquire live images.

Control Descriptions Colors o A list of the current color

settings for the experiment. Select a color to display its imaging and file configurations.

Display Palette o Select a psuedo-color palette

for the image window display. The palette is for display purposes only. Images will be saved without a palette, as grayscale. Gray: Linear Black - White

Gradation Binary: Repeating 16 Color

Pattern Useful for visualizing individual graylevels.

Gradient: Gradation from Red - Lt. Blue - White Rainbow: Gradation from Blue - Green - Red Temperature: Gradation from Black - Dk. Red - White Pastels: Repeating Pastel Pattern with Blk - Wht


Useful for locating saturated pixels. Exposure (ms) o Enter the camera exposure time in milliseconds. The valid value range is 1.0 to

100,000.0 ms. Gain (dB) o Enter the camera gain (dB). The valid value range is 0.0 to 40.3 dB. o Note: The gain setting only applies to 12-bit images. It has no effect on 16-bit

images. Imaging Configuration o The current camera configuration for the selected color setting.

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Save Live Image o Save the currently displayed image to a file on disk. Top Level Control o Top level control buttons.




Exit: Exit the program. Update Settings

o Update the imaging configuration for the selected color setting by copying the live display settings to the acquisition settings.

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4.3.8 Sample Settings Panel ICM Sample Settings [Setup ICM.UCD Sample.vi] positions and planes the sample block and sets the microtome slice width.

Control Descriptions Display o Select the image display mode. o Valid modes are:

Snap: An image will be acquired after each jog or slice.

Live: Images will be continually acquired until the display mode is changed. Positioning is not active during live display.

Display Palette o Select a psuedo-color palette for

the image window display. The palette is for display purposes only. Images will be saved without a palette, as grayscale. Gray: Linear Black - White

Gradation Binary: Repeating 16 Color

Pattern Useful for visualizing individual graylevels.

Gradient: Gradation from Red - Lt. Blue - White Rainbow: Gradation from Blue - Green - Red Temperature: Gradation from Black - Dk. Red - White Pastels: Repeating Pastel Pattern with Blk - Wht


Useful for locating saturated pixels. Exposure (ms) o Enter the camera exposure time in milliseconds. The valid value range is 1.0 to

100,000.0 ms. Focal Point X-axis o The X-axis coordinate of the focal point setting. The focal point is the position

the sample will be imaged. o Note: If the coordinates are (0,0), then the focal point is unset.

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Focal Point Y-axis o The Y-axis coordinate of the focal point setting. The focal point is the position

the sample will be imaged. o Note: If the coordinates are (0,0), then the focal point is unset. Gain (dB) o Enter the camera gain (dB). The valid value range is 0.0 to 40.3 dB. o Note: The gain setting only applies to 12-bit images. It has no effect on 16-bit

images. Jog Mode o Select the jog increment. o Valid modes are:

Slow: 5.0 µm Medium: 500.0 µm = 0.5 mm Fast: 2000.0 µm = 2.0 mm

Lighted Indicator o Indicates whether the sample is being sliced or not. Position Arrows o Position the sample using either the arrow buttons on the front panel or the arrow

keys on the keyboard. o Sample Movement:

Up Arrow: Moves back, towards the rear of the freezer (Positive Y-axis). Down Arrow: Moves forward, towards the front of the freezer (Negative Y-axis). Left Arrow: Moves left (Negative X-axis). Down Arrow: Moves right (Positive X-axis).

Position X-axis o Displays the position of the sample along the X-axis (Left - Right). The position

is reported in units of 0.1 µm. Position Y-axis o Displays the position of the sample along the Y-axis (Front - Back). The position

is reported in units of 0.1 µm. Set Focal Point o Set the focal point to the current X-axis and Y-axis coordinates. Set Slicing Plane o Set the slicing plane to the current Y-axis coordinate. Slice Sample o Continually remove slices from the sample at the specified width and speed. Slice Speed (mm/s) o Set the speed that the sample passed the blade. (mm/s) Slice Width (µm) o Set the width of a single microtome slice. (µm) Slicing Plane o The Y-axis coordinate where the sample intersects with the microtome knife. o Note: If the coordinate is 0, then the slicing plane is unset.

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4.4 NI Vision Tools Palette The NI Vision Tools palette is a floating palette of the available region tools for the image display window.

Information Descriptions 1. Pixel Intensity 2. Image Type Indicator (8-bit, 16-bit, Float,

Complex, 32-bit RGB, HSL, 64-bit RGB) 3. Coordinates of the mouse on the active

image window 4. Anchoring coordinates of an ROI 5. Size of an active ROI 6. Length and horizontal angle of a line

region

Tool Descriptions

Icon Tool Name Function

Selection Tool

Select an ROI in the image and adjust the position of its control points and contours. Action: Click ROI or control points.

Point Select a pixel in the image.

Action: Click the position of the pixel.

Line Draw a line in the image.

Action: Click the initial position and click again at the final position.

Rectangle Draw a rectangle or square in the image.

Action: Click one corner and drag to the opposite corner.

Oval Draw an oval or circle in the image.

Action: Click the center position and drag to the required size.

Polygon Draw a polygon in the image.

Action: Click to place a new vertex and double-click to complete the ROI element.

Freehand Region

Draw a freehand region in the image. Action: Click the initial position, drag to the required shape and release the mouse button to complete the shape.

Annulus Draw an annulus in the image. Action: Click the center position and drag to the required size. Adjust the inner and outer radii, and adjust the start and end angle.

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Icon Tool Name Function

Zoom Zoom-in or zoom-out in an image. Action: Click the image to zoom in. Hold down the <Shift> key and click to zoom out.

Pan Pan around an image. Action: Click an initial position, drag to the required position and release the mouse button to complete the pan.

Broken Line Draw a broken line in the image.

Action: Click to place a new vertex and double-click to complete the ROI element.

Freehand Line

Draw a freehand line in the image. Action: Click the initial position, drag to the required shape, and release the mouse button to complete the shape.

Rotated Rectangle

Draw a rotated rectangle in the image. Action: Click one corner and drag to the opposite corner to create the rectangle. Then click the lines inside the rectangle and drag to adjust the rotation angle.

You can alter the functionality of region tools by using a tool while pressing certain keyboard keys, as follows: o <Shift> while drawing constrains X and Y dimensions of an ROI to be the same.

This forces rectangles into squares, ellipses into circles, and line segments into horizontal or vertical segments. You can also use it to constrain the rotation of the rotated rectangles and annuluses to multiples of 45 degrees.

o <Ctrl> <Click> adds an ROI without erasing the previous ROI elements. The previous elements are erased if you do not use <Control> when starting a new element.

o <Click> <Click> (double-click) while drawing produces the last point of a polygon or broken line.

You can erase an ROI in an image window by selecting it and pressing <Delete>. Use the selection tool to select any currently existing ROI by clicking its border. You can then manipulate the ROI in the following ways: o To resize a rectangle or ellipse, click in a grab handle and drag it to a new

location. o To reposition a vertex in a broken line, polygon, or line, click in a grab handle and

move it to a new location. o To reposition a rectangle or ellipse, click in the interior and drag it to a new

location. o To reposition a point, click on it and drag it to a new location. o To reposition lines, broken lines, and polygons, click on any segment and drag it

to a new location. o To reposition freehand lines and freehand regions, click anywhere on the line

and drag it to a new location.

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4.5 ICM 2010.UCD Initialization File ; Barlow Scientific, Inc. ; Imaging CryoMicrotome(TM), Version 2010.UCDavis ; Initialization File ; ; IMPORTANT: This INI file must remain in the same directory as ; the ICM program. Spelling and capitalization of Section ; and Key names must match those in the comments. ; ; ----------------------------------------------------------------- ; KEY DEFINITIONS ; ----------------------------------------------------------------- ; Common ; Total Colors: Total number of color settings in entire experiment ; Slice Width: Width of a single microtome slice, microns ; Image Type: Image file type (0=PNG, 1=JPEG2000, 2=TIFF) ; Notes: JPEG2000 format is lossless. TIFF format is non-standard. ; Color<i> ; Label: Unique brief (1 or 2 words) description of fluorochrome ; Dir Path: Directory path for storing image files ; File Root: Filename root of incremently named images ; Exposure: Camera exposure setting, ms (0.003-100000.000 ms) ; Gain: Camera gain setting, dB (0.0-40.3 dB) ; Gain is applicable only to 12-bit images. ; Ex Position: Position of excitation filter in wheel (0-6) ; Em Position: Position of emission filter in wheel (0-6) ; Increment: Number of slices to increment between image acquisitions ; Notes: If increment = 1, then every slice will be imaged. ; If increment = 10, then every 10th slice will be imaged. ; Camera ; Bit Depth: Number of bits output per image pixel (0=16-bit, 1=12-bit) ; Cooler Enable: Cooler operation (F=Off, T=On) ; Set Point: Desired cooler temperature, degrees C ; Backoff Point: Number of degrees C to raise the lowest temperature ; reached if the cooler cannot reach the set point temperature ; Fan Mode: Fan speed (0=Off, 1=Low, 2=Medium, 3=High) ; Filter Wheels ; Ex Fname: Unique filename of FLI excitation filterwheel (flifil0) ; Em Fname: Unique filename of FLI emission filterwheel (flifil1) ; Linear Motor ; COM Port: Serial RS232 communication port (COM1, COM2, COM3, ...) ; Setup Dir: Directory path where configuration setups are saved ; Xaxis Setup: Configuration setup for the X-axis (*.t.zip) ; Yaxis Setup: Configuration setup for the Y-axis (*.t.zip) ; Cut Speed = Sample cutting speed, mm/seconds (default=1500 mm/s) ; ----------------------------------------------------------------- [Common] Total Colors = 6 Slice Width = 25.0 Image Type = 2 [Color1] Label = Blue Dir Path = C:\data\test\bl

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File Root = bl Exposure = 4000.0 Gain = 12.0 Ex Position = 1 Em Position = 1 Increment = 1 [Color2] Label = Green Dir Path = C:\data\test\gr File Root = gr Exposure = 2100.0 Gain = 8.0 Ex Position = 2 Em Position = 2 Increment = 1 [Color3] Label = Yellow Dir Path = C:\data\test\yl File Root = yl Exposure = 2000.0 Gain = 6.0 Ex Position = 3 Em Position = 3 Increment = 1 [Color4] Label = Red Dir Path = C:\data\test\rd File Root = rd Exposure = 1000.0 Gain = 0.0 Ex Position = 4 Em Position = 4 Increment = 1 [Color5] Label = NIRed Dir Path = C:\data\test\nr File Root = nr Exposure = 500.0 Gain = 0.0 Ex Position = 5 Em Position = 5 Increment = 1 [Color6] Label = NIR Dir Path = C:\data\test\nir File Root = nir Exposure = 500.0 Gain = 0.0 Ex Position = 6 Em Position = 6 Increment = 1

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[Camera] Bit Depth = 1 Cooler Enable = T Set Point = -20.0 Backoff Point = 2.0 Fan Mode = 1 [Filter Wheels] Ex Fname = flifil0 Em Fname = flifil1 [Linear Motor] COM Port = COM1 Setup Dir = C:\Program Files\Technosoft\ESM\Archives Xaxis setup = X Axis (19in).t.zip Yaxis Setup = Y Axis (9in).t.zip Cut Speed = 1500

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5. Image Processing 5.1 Measuring Image Scale o Acquire an image of grid paper. o Count the number of pixels within a known distance in both the X- and Y-axis to

get the number of microns per pixel in the horizontal and vertical directions. 5.2 Subtracting Dark Current Noise o Covering the lens and shuttering the lamp collect a dark-current image for each

different camera setting used in the experiment. o To correct experimental images for dark current noise, subtract the appropriate

dark-current image from the data. 5.3 Correcting Nonuniform Illumination o Acquire a bright, but not saturating, white-field image of a uniformly fluorescent

and flat object. Two, an amber and a red, fluorescent acrylic sheets are provided for this purpose.

o Acquire a dark-current image at the same camera settings as the white-field image and subtract it from the white-field image.

o Smooth the resulting difference image with a boxcar filter. o To correct experimental images for nonuniform illumination, divide the data

images by the smoothed white-field image. 5.4 Replacing Defective Images o Replace a single defective image with a renamed copy of the previous image in

the data sequence. o If there are more than a couple defective images in series, instead of copy

previous and following images use a creeping median filter of 3 images.

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6. Operational Review 6.1 Freezer Startup 1. Power up freezer and viewport ventilation. 6.2 Sample Preparation 2. Select sample tissue. 3. Embed sample on plate. 4. Mount plate in ICM when freezer is at temperature. 6.3 Lamp Startup 5. Insert lamp shutter. 6. Turn on xenon lamp power. 6.4 ICM Status 7. Verify microtome knife condition. 8. Check for correct optical filters installed in filter wheels. 9. Confirm the viewport window is clear. 10. Remove camera lens caps. 6.5 Program Startup 11. Power up camera, filter wheels and computer. 12. Launch ICM Program. 6.6 Instrument Configuration 13. Verify filter wheels have correct assignment. 14. Create/confirm data folders and labels are set for each fluorochrome (color) in

the experiment. 15. Slice the sample until there is a flat surface across the sample. 16. Set the Slicing Plane. 17. Attach a small piece of grid paper to flat sample surface. 18. Position sample in field of view and set focus, by moving camera and sample. 19. Set Focal Point. 20. Position light beam to illuminate sample surface. 21. Determine and set imaging parameters for each color in the experiment. 22. Double check camera focus at each color setting. 23. Save an image of the grid paper. 24. Slice sample until fluorescent markers begin to show.

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25. Set slice thickness. 26. Double-check the imaging exposure settings. 27. Triple-check the camera focus. 6.7 Image Acquisition 28. Start acquiring images and slicing sample. 29. Grab dark-current images after finished acquiring. 30. Save a white-field image of fluorescent acrylic sheet. 31. Save a dark-current image for the white-field image -- turn off lamp and cover

camera lens. 6.8 System Shut Down 32. Turn off ICM Motor Contol Box. 33. Remove fluorescent acrylic from ICM. 34. Power down ICM. 35. Clean and dry ICM. 6.9 Image Processing 36. Process the images.

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7. System Maintenance 7.1 Maintenance Schedule o Clean and dry ICM after each use. o Vacuum refrigerator coils monthly. 7.2 ICM Cleaning Tissue shavings need to be cleaned out periodically, because they can build up to the point where they block the view of the camera. Leaving the freezer running for a day will lyophilize the shavings and make removal easier. The shavings will melt if removed earlier, although it may be unavoidable. After the freezer is turned off and the shavings have been removed, use a fan to quickly dry the interior of the ICM, minimizing corrosion. 7.3 Knife Maintenance Knives should be sharpened after 15,000 slices or when dull. BSI recommends choosing a professional and experienced sharpening company. The tungsten carbide knife furnished with the instrument was custom manufactured by Delaware Diamond Knives. They also provide a knife sharpening service.

Delaware Diamond Knives 3825 Lancaster Pike, Wilmington, DE 19805 USA 800-222-5143

7.4 Lamp Maintenance The xenon arc lamp for the ICM has a limited lifetime. To replace the lamp follow the instructions outlined in the Ceralux Lamp Replacement manual. Observe all of the safety recommendations. After ignition, the lamp must remain lit for at least 3 minutes to allow for warm-up. Shutting it off before this warm-up time will greatly reduce lamp lifetime.

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7.5 Optical Filter Care Remove loose particles from excitation and emission filters with a bulb puffer or filtered, pressurized air cleaner. If necessary, gently wipe surface using anhydrous alcohol and lint-free lab towels. Use a new surface of towel for each wipe. Avoid touching filter surface with bare fingers.

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8. Technical Information 8.1 Contact Information In case of difficulty or to obtain warranty or non-warranty service, please contact:

Clyde Barlow, President [email protected] Dawn Rorvik, CTO [email protected] Barlow Scientific, Inc. 6307 Tamoshan Drive NW Olympia, WA 98502 USA Tel: 360-867-6053 FAX: 603-462-9083 [email protected]

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8.2 H2W Crossed Axis Roller Gantry Connections 8.2.1 Servo Drive Arrangement

8.2.2 IDM3000 Drive Connector Layout

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8.2.3 Feedback Connector, J2A Connect the hall and encoder 26-pin high-density connector from the linear motor to the J2A Feedback Connector.

8.2.4 Motor Supply Connector, J2B Connect the +291 VDC Advanced Motion Controls power supply to the J2B Motor Supply Connector on the drive. Wire Color Connector White +VMOT (Pin 1) Black GNDM (Pin 2)

8.2.5 Linear Motor Connector, J3 Connect the linear motor leads to the J3 Motor Connector on the drive. Wire Color Connector Red A Orange B Brown C Green Ground lug

8.2.6 Logic Supply Connector, J6 Connect the +24 VDC Meanwell power supply to the J6 Logic Supply Connector on the drive. Wire Color Connector Red +VLOG (Pin 1) Black GND (Pin 2)

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8.2.7 Serial Connector, J7 Connect the RS232 communication cable from the computer to the J7 Serial Connector on the X-Axis Drive.

8.2.8 DIP-Switch Settings, SW1 Set the DIP-switch array on each axis drive to its axis address and activate AUTORUN mode.

X-Axis Linear Motor (19") Axis ID = 1 (Pin 7 = ON) AUTORUN = ON (Pin 8 = ON)

Y-Axis Linear Motor (9") Axis ID = 2 (Pin 6 = ON) AUTORUN = ON (Pin 8 = ON)

8.2.9 Meanwell +24 VDC Logic Power Supply Connect 115 V AC power to the +24 VDC Meanwell power supply AC In Wire Color Connector Black L White N Green Ground DC Out Wire Color Connector Red -V Black +V

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8.2.10 Advanced Motion Controls +291 VDC Motor Power Supply Connect 208 V 1PH AC power to the +291 VDC AMC power supply AC In Wire Color Connector Black 1 AC 1 White 2 AC 2 Green 4 CASE GND DC Out Wire Color Connector White 1 High Voltage Black 2 Power GND

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8.3 Apogee Alta U32 Camera 8.3.1 CCD Specifications Category Specification Sensor Model Kodak KAF-3200ME Sensor Type Full Frame Progressive Scan CCD Array Size (pixels) 2184 x 1472 Pixel Size 6.8 x 6.8 microns Imaging Area 14.8 x 10.0 mm (148.7 mm2) Imaging Diagonal 17.9 mm Linear Full Well (typical) 55K electrons Dynamic Range 77 dB QE at 400 nm 53% Peak QE (610 nm) 86% 8.3.2 CCD Sensitivity

8.3.3 System Performance Category Specification Digital Resolution 16 bits at 1 MHz and 12 bits at 7 MHz System Noise (typical) 8 e- RMS at 1 MHz and 2 counts at 7 MHz Frame Sizes Full frame Cooling (typical) Thermoelectric cooler with forced air.

Maximum cooling 50°C below ambient temperature

Dark Current (typical) 0.05 e-/pixel/sec (-25°C) Temperature Stability ± 0.1°C Operating Environment -22° to 27°C. Relative humidity: 10 to 90%

non-condensing.

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8.4 Optical Filter Spectra 8.4.1 Set 1

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UV PASS BlackliteD480/30m

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8.4.3 Set 3

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8.4.5 Set 5

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8.4.6 Set 6

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8.5 CeraLux CL300BF Xenon Lamp 8.5.1 Operational Specifications Category Specification Power (Watts) Nominal 300 Range 180 - 330 Current (Amps DC) Nominal 21 Range 10 - 22 Voltage (volts DC) Nominal 14 Range 13 - 16 Minimum Ignition Voltage (kV) 23 Maximum Temperature 150 °C 8.5.2 Spectral Output

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8.6 Cold Mirror Chimney 8.6.1 Cross-Sectional Diagram

8.6.2 OCLI 45° Cold Mirror Spectra

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Transmission Average: 750 - 1200 nm, 93.37% Reflection Average: 420 - 630 nm, 95.02% 8.6.3 OCLI 45° UV Cold Mirror Spectra

Transmission Average: 600 - 1200 nm, 88.54% Reflection Average: 325 - 475 nm, 95.87%

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8.7 Other Optical Characteristics 8.7.1 Glass View Port Spectrum

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9. Conditions of Acceptance In the following agreement, Barlow Scientific, Inc., 6307 Tamoshan Drive NW, Olympia, WA 98502, shall be referred to hereinafter as “BSI” and the customer/client, shall be referred to as “Buyer”. Buyer agrees, upon acceptance of Model BS-8 of the Barlow Scientific, Inc., Imaging CryoMicrotome™, to the following provisions connected to its use: Article 1. Protection of Humans

A. Buyer agrees that the BS-8 Imaging CryoMicrotome™ is for experimental use only and is not for use on human subjects. B. Buyer shall bear full responsibility for the proper and safe performance of all work using the BS-8. No provision of this agreement shall be deemed to constitute Buyer as an agency or employee of BSI. Buyer agrees that in its use of this instrument he will discharge its obligations, duties and undertakings and the work pursuant thereto whether requiring professional judgment or otherwise as an independent institution and without liability on the part of BSI.

Article 2. Indemnification

Buyer agrees to indemnify and hold BSI and its employees harmless from any and all claims and costs made against BSI arising from the use of the BS-8 Imaging CryoMicrotome™.

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