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crisp_manual [2019/10/30 16:57]
jon fixed table sort order
crisp_manual [2019/10/30 17:23]
jon [System Overview]
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 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. 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.
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 ===== System Overview ===== ===== 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 PZ‑2000 ​piezo Z‑axis stage.+The CRISP system consists of optical, electronic, and mechanical components. ​ The optical system injects ​IR LED 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 ASI controller changes the focal position of the microscope either with a motorized Z device ​or a piezo Z‑axis stage. 
 [{{ crisp_2_.jpg?​200 |Figure 1: CRISP with DCMS photo-port splitter.}}] [{{ crisp_2_.jpg?​200 |Figure 1: CRISP with DCMS photo-port splitter.}}]
 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. 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.
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 [{{ crisp_1_.png?​300 |Figure 2: Schematic diagram of CRISP optical system}}] [{{ crisp_1_.png?​300 |Figure 2: Schematic diagram of CRISP optical system}}]
  
 ===== Fluorescent Filter Considerations ===== ===== 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 CRISP system utilizes ​a (near) IR LED that is projected onto the sample, most commonly 780nm or 850nm.  Proper arrangement of the light filters in the microscope is necessary for the system to function properly, and requires some thought especially when the CRISP is placed at the C-mount.  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. ​ 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. ​
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 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. 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.
  
-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+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.  This is the preferred solution with our RAMM/MIM systems.  Although these solutions are better optically, they may require customization for the particular case.  Contact ASI for details.
  
 ==== LED Characteristics and Filters ==== ==== 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. ​+Several LED wavelengths are available that will provide good performance with the CRISP system.  ​For CRISP systems at the C-mount a common wavelength ​is 780nm because that can often make it through multi-band filter sets.  On CRISP systems on ASI's MIM2 the CRISP path diverges right under the objective, making it trivial to choose an appropriate filter and the default ​wavelength ​is 850nm.  The table below shows the 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 ​usable ​wavelength to about 1050nm. ​
 <​datatables paging="​false"​ ordering="​false">​ <​datatables paging="​false"​ ordering="​false">​
  
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 Contact ASI with your filter specifications for further guidance. Contact ASI with your filter specifications for further guidance.
  
-Multi-band filter sets that will work with CRISP+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. 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.
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crisp_manual.txt · Last modified: 2019/10/30 17:23 by jon