Table of Contents
Summary of Contents for ASI CRISP
- Page 1 CRISP Autofocus Instruction Manual pplied cientific nstrumentation, Inc. 29391 West Enid Road Eugene, OR 97402-9533 USA Phone: (800) 706-2284 (541) 461-8181 Fax: (541) 461-4018 Web: www.ASIimaging.com E-mail: Support@ASIimaging.com...
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Page 2: Table Of Contents
Adjusting Position of the LED Light Source ............... 20 Adjusting the Primary Mirror ....................21 Advanced Techniques ......................22 Troubleshooting Steps ......................25 Computer Control of the CRISP System .................. 26 TTL Control of the CRISP focus lock ................. 29... - Page 3 Figure 1: CRISP with DCMS photo-port splitter................4 Figure 2: Schematic diagram of CRISP optical system ..............5 Figure 3: Semrock Dichroic FF408/504/581/667/762-Di01.............. 7 Figure 4: Semrock LF405/488/561/635-A-000 ................. 8 Figure 5: C-mount Splitter (DCMS) contains dichroic mirror and blocking filter......11 Figure 6: Photo detector difference signal for a scan through a microscope slide.
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Page 4: Crisp Continuous Autofocus System
The unit is a C-mount device, that can be placed at the C-mount port. Usually it is used in conjunction with the a dual C-mount Splitter (DCMS) so both the CRISP unit and a data recording camera can share the same microscope photoport. -
Page 5: Fluorescent Filter Considerations
As shown in Figure 2, a dichroic beam splitter that reflects light from the IR LED and passes visible light to the camera is used to couple the CRISP unit to the system at the C-mount photo- port. Figure 2: Schematic diagram of CRISP optical system Fluorescent Filter Considerations The CRISP system commonly utilizes an 850nm LED that is projected onto the sample. -
Page 6: Led Characteristics And Filters
It may be possible to place the CRISP in the excitation path or to find an alternative location between the objective and the microscope’s filter cube to insert the CRISP coupling beam splitter. Although these solutions are perhaps better optically, they probably require customization for the particular case. - Page 7 Pinkle set. The upper transmission band of the dichroic is perfect for the standard 780nm CRISP IR LED. Used in this way, this filter set can be installed in the microscope’s filter cube in the usual manner. A 750nm IR block is place in the DCMS splitter camera C-mount to block the upper band from the camera.
- Page 8 IR. If not, consider alternatives that have such a pass band. Listed below are several filter sets from major filter manufacturers that will work with CRISP. Some of them require special non-standard LED color.
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Page 9: Led Power And Eye Safety
Uses a multiband emission filter that can be placed in the camera’s DCMS C-mount This set will also work for CRISP in the microscope’s filter cube if the Cy5 channel is used for CRISP – Specify 700nm LED for CRISP for this application, and place 650nm SP block in front of camera. - Page 10 100µW (typically about 70 µW) with the LED set to 100% intensity and the internal aperture stop open fully. The CRISP LED mask appears to be about 1.0 mm × 6.8 mm in the image plane. The brightest part of the LED emitter depends slightly on the LED used and the exact focus, but is about 1.0 mm square at the image plane.
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Page 11: Installation
Mount the CRISP unit on the reflected port of the DCMS. Mount the camera on the “straight-through” port of the DCMS. Connect the DB9 cable from the CRISP unit to the labeled connector on the back of the MS2000 control unit. -
Page 12: Photodiode Displacement Signal
position. Often the reflected light comes from small refractive index discontinuities at sample surfaces. The amount of light reflected at a dielectric interface is given by – n R = (n / (n where n and n are the refractive indexes of the adjoining dielectric materials. The table below shows the refractive index of several optical materials and the magnitude of the reflection expected at various interfaces between materials. - Page 13 Figure 6: Photo detector difference signal for a scan through a microscope slide. The figure above shows the difference signal from the photodiode pair as the focus is scanned through a standard microscope slide. You will notice two green shaded zones corresponding to the front and back surface of the slide.
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Page 14: Control Of The Crisp System
UP position. The MS-2000 controller provides an easy means to turn on and off the CRISP LED as well as to initiate the focus lock. The LCD display shows the status of the system. The figure below shows the typical display. -
Page 15: Crisp System States
LK F=<decimal number> (You can use the serial command as shown in column two on the table below to directly force a CRISP system state. For example, to set the CRISP state to Balance LK F=66. issue the serial command Use with care, as out-of-sequence events are not necessarily handled smoothly.) -
Page 16: Asi Console Support For Crisp
ASI Console support for CRISP The ASI Console program has built-in support for the CRISP unit that makes it easy to setup and calibrate the CRISP unit. Using the ASI Console program eliminates the need to learn all of the special button presses to accomplish the calibration steps. -
Page 17: Crisp Operations
CRISP Operations The following guide assumes that the default CRISP parameter settings are adequate and will provide an adequate focus lock with many objectives and sample types. Focus on your sample. Quick Start Instructions Using ASI_Console 1) Download and install ASI_Console from the ASI website: http://www.asiimaging.com/support/downloads/asi-console/... -
Page 18: Engaging The Lock For Normal Operation
Engaging the LOCK for Normal Operation In addition to the quick start instructions above… If you have calibrated the system, but then perhaps changed samples or significantly disturbed the system, you may find that the focus-error shown on the LCD is nowhere near zero when in the state prior to locking. - Page 19 Ready Parameters used with the CRISP system The serial commands give the user access to several parameters used with the CRISP system. Advanced users may find that they have a need to change particular settings from the default values for specific purposes.
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Page 20: Optical Adjustment
Optical Adjustment The CRISP unit is pre-adjusted at the factory, and should not need major adjustments. However, this guide will allow anyone to test and check the proper adjustment. The recording camera is helpful for adjusting the primary mirror position. In order to see the illumination light, any blocking filter in front of the camera needs to be removed. -
Page 21: Adjusting The Primary Mirror
Adjusting the position of the LED slightly off center or with a small tilt can reduce this glare and significantly improve the performance of the CRISP unit. Adjusting the Primary Mirror... -
Page 22: Advanced Techniques
Advanced Techniques A common problem is that the CRISP system will be able to hold focus best at a location that is not in the center of the best focus range for the sample. This can typically happen because the reference cover-slip is slightly in front of the region where the sample is best in focus. - Page 23 Using the Iris LED Beam Stop The internal iris in the CRISP unit can be used to improve the returned beam quality by reducing the amount of stray LED light that cannot be accepted by the objective aperture. The system is shipped with the iris all the way open so as to be able to accommodate high brightness objectives.
- Page 24 Cut and paste into Excel to plot the numbers. You can see the strong slope near the surface (Z=0) that provides the focus feedback. For a focus at the interface, the capture range includes any Z-position that has the correct polarity of focus error signal to bring the stage back to the lock point.
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Page 25: Troubleshooting Steps
IR block from the optical path. Usually there is sufficient light that leaks through the dichroic to be able to see the CRISP LED light on the camera when there is no other illumination. Verify that the CRISP LED light is present at the sample an go... -
Page 26: Computer Control Of The Crisp System
Computer Control of the CRISP System The focus controller responds to several commands dedicated to controlling the feedback system. Please see the MS-2000 Programming Manual for further information about using serial commands. Command: LOCK Shortcut: Format: LK [X] [Y] [Z=lock_offset] [F=code]... - Page 27 If the controller can handle more than one Z-axis focus device, you can specify the focus_index to select which one is active for the CRISP system. Save the parameter change (SS Z) and reset the controller. “:A” is returned upon receipt of the command.
- Page 28 Command: KADC Shortcut: Format: KA [X=n] [Y=n] [Z=n] [F=n] Function: Adjusts a gain multiplier in the CRISP servo loop where n is a signed integer. “:A” is returned upon receipt of the command. Reply: KA Z? returns the current value. Query:...
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Page 29: Ttl Control Of The Crisp Focus Lock
TTL Control of the CRISP focus lock In some instances it may be desirable to be able to turn on/off the CRISP lock using a TTL signal. Imaging software that does not support CRISP explicitly may support controller TTL I/O which can provide a simple method to synchronize the autofocus function with other imaging functions.
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