ASI CRISP User manual

CRISP Autofocus
Instruction Manual
Applied Scientific Instrumentation, Inc.
29391 West Enid Road
Eugene, OR 97402-9533 USA
Phone: [phone]
[phone]
Fax: [phone]
Web: www.ASIimaging.com
E-mail: [e-mail]

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Table of Contents
CRISP Continuous Autofocus System....................................................................................... 4
System Overview...................................................................................................................... 4
Fluorescent Filter Considerations.......................................................................................... 5
LED Characteristics and Filters ............................................................................................. 6
Commercial Filter Sets Suitable for CRISP........................................................................... 6
LED Power and Eye Safety..................................................................................................... 9
Installation.............................................................................................................................. 11
Theory of Operation.............................................................................................................. 11
Sample Considerations......................................................................................................... 11
Photodiode Displacement Signal.......................................................................................... 12
Control of the CRISP system ............................................................................................... 14
Button Actions...................................................................................................................... 14
CRISP System States............................................................................................................ 15
ASI Console support for CRISP ........................................................................................ 16
CRISP Operations................................................................................................................. 17
Quick Start Instructions Using ASI_Console....................................................................... 17
Quick Start Instructions Using Controller Only................................................................... 17
Engaging the LOCK for Normal Operation......................................................................... 18
Calibration Details................................................................................................................. 18
Optical Adjustment................................................................................................................ 20
Adjusting the Relay Lens position ....................................................................................... 20
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

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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. ......................... 13
Figure 7: Reflections from a glass bottomed Petri dish................................................................... 13
Figure 8: Reflection from glass slide of a) LED exit slit and b) focused deeper, the LED emitter,
when the LED holder is properly aligned by moving c) LED holder...................................... 21
Figure 9: Mirror Adjusting Screw.................................................................................................... 21
Figure 10: C-mount extension. ........................................................................................................ 22

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CRISPContinuous Autofocus System
The Continuous Reflection Interface Sampling and Positioning (CRISP) system provides for a
very high level of focus stability, allowing a specimen to remain accurately focused for hours at a
time with drift <0.1 μm. The system compensates for focus changes caused by temperature
variations as well as mechanical drifts of the microscope mechanisms. The CRISP system
promises to be a solution to focus drifts that plague time-lapse experiments at high magnification.
The CRISP system uses a pupil obscuration method to determine focus from reflective surfaces.
The control system allows adjustment of the focal lock position, relative to a nearby surface, once
the system is locked. 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.
System Overview
The CRISP system consists of optical, electronic, and mechanical components. The optical
system injects IRLED light into the microscope, captures the beam reflected from the specimen
slide or cover slip, and routes the reflected beam onto a position-sensitive detector (PSD). The
signal from the PSD is conditioned by an amplifier circuit in the MS2000 controller and used as
the feedback signal for Z-axis control. The MS-2000 Z-axis controller changes the focal position
of the microscope either with a servomotor or with a PZ-2000 piezo Z-axis stage.
Figure 1: CRISP with DCMS photo-port splitter.

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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. Proper
arrangement of the light filters in the microscope is necessary for the system to function properly.
A dichroic beam splitter that reflects the IR light is used in the dual C-mount splitter (DCMS). No
other filters can be in the path to the objective that block the IR light. An emission filter that
blocks the IR LED should be placed in front of the camera and can be located in the C-mount
fitting of the DCMS for the camera. Fluorescence dichorics need to have a “window” in the IR to
pass the CRISP LED. See the list of commercial filter sets that work with CRISP below.
The long C-mount adapter on the Olympus IX-71 or BX scopes permits the use of both a filter
wheel and the CRISP unit in the provided space. This allows use of specific emission filters in
conjunction with either a multi-band dichrioc with an IR pass band, or with a single excitation
wavelength and a long pass dichroic in the scope.
Some configurations provide an easier solution to the filter problem. If a spinning disk confocal
unit attached to the C-mount port is used for fluorescent microscopy, the filter cube is located in
the confocal head and not in the microscope. In this case the CRISP mounted on the DCMS will
work fine and not be impeded by any fluorescence filters in the microscope.

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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. Contact ASI for details
LED Characteristics and Filters
Several LED wavelengths are available that will provide good performance with the CRISP
system. Usually the unit is supplied with an IR LED with 780nm peak wavelength. The table
below shows other LEDs that can be supplied, along with the suggested dichroic beam splitter and
blocking filters. With sufficient spectral distance between the LED wavelength and the dichroic
and camera block cut-off wavelength, a cleanup filter for the LED may not be required. The
detector in the CRISP unit begins to lose sensitivity after about 1000nm limiting the maximum
useable wavelength to about 1050nm.
LED Part
Number
LED
Color
(nm)
Typ.
LED
Power
@50
mA
(mW)
FWHM
(nm)
FW to
2% wings
Short Pass
Dichroic Beam
Splitter
Short Pass
Camera Block
Filter
Band Pass
LED Cleanup
Filter
cutoff
(nm)
Part
Number
cutoff
(nm)
Part
Number
Cutoff
(nm)
Part
Number
VLCS5830
625
10
18
580-660
600
69216
600
84710
628/32
84087
L660-06
660
3
20
615-700
600
69216
600
84710
650/50
84774
L700-06
700
13
30
650-740
650
69217
650
84712
700/50
84775
L720-06
720
13
30
670-760
700
69218
700
84712
700/50
84775
L735-06
735
18
30
680-780
700
69218
700
84714
750/50
84776
L740-06
740
18
30
685-785
700
69218
700
84714
750/50
84776
L780-06
780
20
30
710-830
750
69219
750
64332
800/50
84777
TSHG8200
830
25
40
750-900
750
69219
750
64332
TSHG5210
850
27
40
790-930
800
69220
800
64333
850/50
84778
TSFF5210
870
23
40
810-950
800
69220
800
64333
TSHF5210
890
23
40
830-970
850
69221
850
64334
900/50
84779
L940-06
940
17
50
840-1040
900
69222
900
64335
950/50
84780
L970-06
970
5.5
50
910-1070
900
69222
900
64335
L1050-06
1050
2.0
50
950-1130
1064/80
NFD01-
1064
1000
64337
1050/50
85881
Commercial Filter Sets Suitable for CRISP
For fluorescent applications, choosing the correct filters is important. Matching the best CRISP
LED color with the filter set will give the best results.
Single band sets with single edge long pass dichroic beam splitters
There are many filter sets from several manufacturers that have a single edge, long pass
dichroic. Usually the emission filter provides the data pass-band in the region just above the
dichroic edge. Frequently the dichroic will continue to pass light well above the emission filter

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pass-band. Wavelengths above the emission filter pass-band, where the dichroic is still
transmitting, provide the ideal wavelength region for CRISP. Specify the CRISP LED color to be
as far in the red/IR as possible away from the emission band, yet still where the dichroic has good
transmission (and the emission filter has good blocking for the CRISP LED). Often the
fluorescence emission filter can be placed directly in front of the camera in the DCMS C-mount
splitter where it will serve to block the CRISP IR LED from the camera.
Contact ASI with your filter specifications for further guidance.
Multi-band filter sets that will work with CRISP
Frequently the dichroic beam splitter on multi-band filter sets has limited transmission outside
the data-channel color bands. Nevertheless, there are several multi-band commercial filter sets that
can be used with CRISP. One interesting filter set is the Semrock five-band with the dichroic filter
characteristics below.
Figure 3: Semrock Dichroic FF408/504/581/667/762-Di01
This dichroic is used with either individual emitters and exciters for each band, or with
individual exciters only as a 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.
Another Semrock multi band set, LF405/488/561/635-A-000, has an extended region for the red
band that would pass IR light. The pass band above 700nm is open, allowing easy operation with
a 780nm IR LED for CRISP. In this case, the emission filter would be installed in the DCMS
splitter C-mount in front of the camera and would act as the IR block for the CRISP LED.

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Figure 4: Semrock LF405/488/561/635-A-000
To determine the correct filter set for your application, first check the filters you have to see if
there is a pass band in the 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.
Semrock multi-band filter sets that will work with CRISP
LF405/488/594-A-000
Uses Di01-R405/488/594 dichroic which passes 780 to 800 IR LED
Uses a multiband emitter that can be placed in the camera’s DCMS C-mount
LF405/488/532/635-4x-A-000
Uses Di01-R405/488/532/635 dichroic which passes 780 to 820 IR LED
LF442/514/561-3X-A-000
Uses Di01-R442/514/561 dichroic which passes 780 to 830 IR LED
Uses a multiband emission filter that can be placed in the camera’s DCMS C-mount
Uses a multiband emitter that can be placed in the camera’s DCMS C-mount
LF488/561-2x-B-000
LF488/561-A-000
Uses Di01-R488/561 dichroic which passes 780 to 830 IR LED
Uses a multiband emission filter that can be placed in the camera’s DCMS C-mount
DA/FI/TR/Cy5/Cy7-5x-A-000
Uses FF408/504/581/667/762-Di01 dichroic with passes 780 to 850 IR LED
This five band set has the top band situated perfectly for CRISP
Uses a multiband emission filter with pass band in IR so can be used in microscope filter cube.
FRET - GFP/RFP –C-000

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Uses FF 495-Di03 dichroic which passes 780 to 850 IR LED
Requires switched emission filter before camera for two channels
FRET-CFP/YFP-C-000
Uses FF458-Di02 dichroic which passes 780 to 850 IR LED
Requires switched emission filter before camera for two channels
Chroma multiband filter sets that will work with CRISP
59004 FITC/TRITC –ET
59204 FITC/TRITC
Uses 59004bs dichroic with available pass band at 740nm –specify 740nm LED for CRISP.
Uses a multiband emission filter that can be placed in the camera’s DCMS C-mount
59017 ECFP/EYFP –ET
59217 ECFP/EYFP
Uses 59017bs dichroic with available pass band at 650nm –specify 660nm LED for CRISP.
Uses a multiband emission filter that can be placed in the camera’s DCMS C-mount
69000 DAPI/FITC/TRITC
69300 DAPI/FITC/TRITC
Uses 69000bs dichroic with available pass band at 700nm –specify 700nm LED for CRISP.
Uses a multiband emission filter that can be placed in the camera’s DCMS C-mount
69008 ECFP/EYFP/mCherry
69308 ECFP/EYFP/mCherry
Uses 69008bs dichroic with available pass band at 735nm –specify 735nm LED for CRISP
Uses a multiband emission filter that can be placed in the camera’s DCMS C-mount
88000v2 DAPI/FITC/TEXAS RED/Cy5
Uses 88100bs dichroic with available pass band at 830nm –specify 830nm LED for CRISP
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.
Contact ASI or your filter supplier if you have further questions.
LED Power and Eye Safety
The CRISP system uses an IR LED to illuminate the sample and provide a reflected beam that
is used to determine focus. Although relatively bright IR LEDs are used in the CRISP unit, the
distributed nature of the LED source, masking of the LED, reduction in aperture and reduce duty
cycle combine to make the CRISP light source eye-safe. Never-the-less, please do not stare into

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the CRISP C-mount when the unit is powered up. IR LED sources do not generate visible
radiation, so prolonged exposure is possible and should be avoided.
The maximum measured average power for a typical CRISP unit at the C-mount is less than
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. Based upon the objective used, you can use these
numbers to calculate typical maximum intensity of IR illumination at the sample. However, be
aware that many objectives will not pass the full aperture at the CRISP aperture stop, so the
number you get this way will be a maximum.
For example, a 60×objective will expose some parts of the sample to a maximum of about
0.36W/mm2of IR radiation.
Total Power / Area = 100µW / (1/60 mm × 1/60 mm) = 0.36 W/mm2
You can reduce the radiative power at the sample by using a lower LED intensity and/or
reducing the internal aperture stop.