crisp_manual
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+ | ====== CRISP : Continuous 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. | ||
+ | |||
+ | |||
+ | |||
+ | ===== Software ===== | ||
+ | There are 3 main programs to interact with the CRISP device through a GUI. CRISP can also be operated by sending serial commands and even completely stand-alone using buttons on the controller. | ||
+ | |||
+ | [[crisp_mm_plugin|ASI CRISP Control]] - Micro-Manager Plugin\\ | ||
+ | [[crisp_ninja|CRISP Ninja]] - Standalone Windows Application\\ | ||
+ | [[https:// | ||
+ | |||
+ | |||
+ | ===== System Overview ===== | ||
+ | |||
+ | The CRISP system consists of optical, electronic, and mechanical components. | ||
+ | |||
+ | ==== Optical placement ==== | ||
+ | |||
+ | The CRISP unit is a C-mount device that is placed optically conjugate to the camera (and sample). A dichroic beam splitter used to couple the CRISP unit to the microscope system. | ||
+ | |||
+ | **__Dichroic in focus space:__** A shortpass dichroic is placed between the tube lens and camera, most often using ASI's DCMS (dual C-mount splitter) so that CRISP shares the microscope photoport with a camera. | ||
+ | |||
+ | **__Dichroic in collimated space:__** A dichroic is placed between the objective and tube lens. **Placement in collimated space is preferred when it is possible, e.g. with ASI's modular infinity microscopes.** | ||
+ | |||
+ | [{{ crisp_2_.jpg? | ||
+ | |||
+ | [{{ crisp_1_.png? | ||
+ | |||
+ | ===== CRISP LED Options and Filters ===== | ||
+ | |||
+ | CRISP uses an internal LED light source, and many different LED wavelengths are available. | ||
+ | |||
+ | The table below shows the LEDs that can be supplied, along with the suggested dichroic beam splitter and blocking filters. | ||
+ | < | ||
+ | |||
+ | ^LED Part Number ^LED Color (nm) ^Typ. LED Power @50mA (mW) ^FW HM (nm) ^FW to 2% wings ^Short Pass Dichroic Beam Splitter ^^Short Pass Camera Block Filter ^^Band Pass LED Cleanup Filter ^^ | ||
+ | ^::: | ||
+ | ^VLCS5830 |625 |10 |18 |580-660 |600 |69216 |600 |84710 |628/ | ||
+ | ^L660-06 |660 |3 |20 |615-700 |600 |69216 |600 |84710 |650/ | ||
+ | ^L700-06 |700 |13 |30 |650-740 |650 |69217 |650 |84712 |700/ | ||
+ | ^L720-06 |720 |13 |30 |670-760 |700 |69218 |650 |84712 |725/ | ||
+ | ^L735-06 |735 |18 |30 |680-780 |700 |69218 |700 |84714 |725/ | ||
+ | ^L740-06 |740 |18 |30 |685-785 |700 |69218 |700 |84714 |750/ | ||
+ | ^L780-06 |780 |20 |30 |710-830 |750 |69219 |750 |64332 |775/ | ||
+ | ^TSHG8200 |830 |25 |40 |750-900 |750 |69219 |750 |64332 |825/ | ||
+ | ^TSHG5210 |850 |27 |40 |790-930 |800 |69220 |800 |64333 |850/ | ||
+ | ^TSFF5210 |870 |23 |40 |810-950 |800 |69220 |800 |64333 |875/ | ||
+ | ^TSHF5210 |890 |23 |40 |830-970 |850 |69221 |850 |64334 |900/ | ||
+ | ^L940-06 |940 |17 |50 |840-1040 |900 |69222 |900 |64335 |950/ | ||
+ | ^L970-06 |970 |5.5 |50 |910-1070 |900 |69222 |900 |64335 |975/ | ||
+ | ^L1050-06 |1050 |2.5 |50 |950-1130 |1000 |86695 |1000 |64337 |1050/ | ||
+ | |||
+ | </ | ||
+ | |||
+ | ===== Fluorescent Filter Considerations ===== | ||
+ | |||
+ | The CRISP system utilizes a (near) IR LED that is projected onto the sample, most commonly 780nm or 850nm. | ||
+ | |||
+ | 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. | ||
+ | |||
+ | Some configurations provide an easier solution to the filter problem. | ||
+ | |||
+ | It may be possible to place the CRISP in the excitation path or to find an alternative location in focus space (the preferred approach with ASI's RAMM/MIM systems). | ||
+ | |||
+ | |||
+ | ==== Filter Sets Suitable for CRISP ==== | ||
+ | |||
+ | It is important to make sure the filters in your microscope are compatible with CRISP. | ||
+ | |||
+ | When retrofitting an existing microscope, CRISP is usually added near the camera using a DCMS. The DCMS has an internal dichroic mirror and two C-mounts, one for the camera and the other for CRISP. | ||
+ | |||
+ | - If both the dichroic and emission filter transmit the CRISP wavelength, then the filter set is completely compatible with CRISP and the complete filter set can be used inside the body of the microscope. | ||
+ | - If the dichroic transmits the CRISP wavelength but the emission filter does not, then the filter set will work with CRISP as long as the emission filter is placed just before the camera instead of inside the microscope. | ||
+ | - If neither the dichroic nor emission filter transmit the CRISP wavelength, then the filter set is not usable with CRISP. | ||
+ | |||
+ | The first category is obviously the best. Most often these filter sets are are marketed as simple longpass filters, not bandpass filters. | ||
+ | |||
+ | The second category is the most common. | ||
+ | |||
+ | The third category needs no further explaining. | ||
+ | |||
+ | |||
+ | Contact ASI with your filter specifications for further guidance. | ||
+ | |||
+ | === Using multi-band filter sets with CRISP === | ||
+ | |||
+ | Frequently the dichroic beam splitter on multi-band filter sets has limited transmission outside the data-channel color bands. Nevertheless, | ||
+ | |||
+ | [{{ crisp_2_.png? | ||
+ | |||
+ | 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. | ||
+ | |||
+ | Another Semrock multi band set, LF405/ | ||
+ | |||
+ | [{{ crisp_3_.png? | ||
+ | |||
+ | To determine the correct filter set for your application, | ||
+ | |||
+ | == Semrock multi-band filter sets that will work with CRISP == | ||
+ | |||
+ | * DA/ | ||
+ | * Uses FF408/ | ||
+ | * 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. | ||
+ | |||
+ | * LF405/ | ||
+ | * Uses Di01-R405/ | ||
+ | * Uses a multiband emitter that can be placed in the camera’s DCMS C-mount | ||
+ | |||
+ | * LF405/ | ||
+ | * Uses Di01-R405/ | ||
+ | |||
+ | * LF442/ | ||
+ | * Uses Di01-R442/ | ||
+ | * Uses a multiband emission filter that can be placed in the camera’s DCMS C-mount | ||
+ | |||
+ | * LF488/ | ||
+ | * LF488/ | ||
+ | * Uses Di01-R488/ | ||
+ | * Uses a multiband emission filter that can be placed in the camera’s DCMS C-mount | ||
+ | |||
+ | * FRET - GFP/RFP –C-000 | ||
+ | * Uses FF 495-Di03 dichroic which passes 780 to 850 IR LED | ||
+ | * Requires switched emission filter before camera for two channels | ||
+ | |||
+ | * FRET-CFP/ | ||
+ | * 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/ | ||
+ | * 69300 DAPI/ | ||
+ | * 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/ | ||
+ | * 69308 ECFP/ | ||
+ | * 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/ | ||
+ | * 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, | ||
+ | |||
+ | 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. | ||
+ | |||
+ | 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. | ||
+ | |||
+ | For example, a 60× objective will expose some parts of the sample to a maximum of about 0.36W/mm2 of IR radiation. | ||
+ | |||
+ | \begin{equation} | ||
+ | \frac{Total Power}{Area} | ||
+ | \end{equation} | ||
+ | |||
+ | |||
+ | You can reduce the radiative power at the sample by using a lower LED intensity and/or reducing the internal aperture stop. | ||
+ | |||
+ | |||
+ | ===== Installation ===== | ||
+ | |||
+ | Install the Z‑axis drive or PZ‑2000 stage as described in its manual. | ||
+ | |||
+ | The CRISP device is designed to be used at a camera C-Mount location. | ||
+ | |||
+ | [{{crisp_7_.jpg? | ||
+ | |||
+ | There should also be a blocking filter on the camera C-mount to keep LED light out of the camera. | ||
+ | |||
+ | Mount the CRISP unit on the reflected port of the DCMS. | ||
+ | |||
+ | Mount the camera on the “straight-through” port of the DCMS. | ||
+ | |||
+ | ==== CRISP Cable Connection ==== | ||
+ | |||
+ | Connect the DB9 cable from the CRISP unit to the labeled connector on the back of the MS2000 control unit. | ||
+ | |||
+ | Pin-outs for this cable are shown below. | ||
+ | |||
+ | ^ CRISP DB-9 Connector | ||
+ | ^ PIN ^ SIGNAL | ||
+ | | 1 | PD_CH1 | ||
+ | | 2 | N.C. | Not Connected | ||
+ | | 3 | GND | Ground | ||
+ | | 4 | LED | LED control voltage, 0 to 5v DC input | | ||
+ | | 5 | N.C. | Not Connected | ||
+ | | 6 | PD_CH2 | ||
+ | | 7 | +5V | +5V power | | ||
+ | | 8 | N.C. | Not Connected | ||
+ | | 9 | N.C. | Not Connected | ||
+ | |||
+ | |||
+ | ===== Theory of Operation ===== | ||
+ | |||
+ | The CRISP autofocus device uses a pupil obscuration method for determining focal position. | ||
+ | |||
+ | During operation of the CRISP unit -- in the " | ||
+ | |||
+ | At a user-specified update rate, the target position of the focus motor is changed by an amount proportional to the averaged '' | ||
+ | |||
+ | When the '' | ||
+ | |||
+ | Larger '' | ||
+ | |||
+ | Increasing averaging will reduce the influence of perturbations in the reflected signal but also make the feedback less responsive. | ||
+ | |||
+ | CRISP updates the target position of the focus motor continuously. | ||
+ | |||
+ | ASI's firmware has reasonable default settings for '' | ||
+ | ==== Sample Considerations ==== | ||
+ | |||
+ | |||
+ | There are several classes of samples that are common in microscopy and present very different challenges for focus systems. | ||
+ | |||
+ | \begin{equation} | ||
+ | R = \frac{(n_1 – n_2)^2}{(n_1 + n_2)^2} | ||
+ | \end{equation} | ||
+ | |||
+ | where $n_1$ and $n_2$ are the refractive indexes of the adjoining dielectric materials. | ||
+ | |||
+ | <wrap em> | ||
+ | |||
+ | A good test sample for oil objectives is just a fingerprint on a cover-slip-bottom dish with water in it. | ||
+ | |||
+ | |||
+ | |||
+ | == Table 1: Reflection Intensity from a Dielectric Interface == | ||
+ | |||
+ | ^Material ^Refractive index @ 800nm ^Reflectance at interface (%) ^^^ | ||
+ | ^::: ^::: | ||
+ | ^Air |1.000 | — |2.0 |4.3 | | ||
+ | ^Water |1.329 |2.0 | — |0.46 | | ||
+ | ^Immersion Oil |1.518 |4.2 |0.45 | ||
+ | ^Glycerol |1.473 | | ||
+ | ^Glass (typical) |1.523 |4.3 |0.46 | — | | ||
+ | ^Plastic (Polystyrene) |1.575 |5.0 |0.72 |0.03| | ||
+ | ^Plastic (PMMA acrylic) |1.483 |3.8 |0.30 |0.02| | ||
+ | ^PDMS |1.410 |2.9 |0.09 |0.43| | ||
+ | ^Fused Silica |1.453 |3.4 |0.20 |0.05| | ||
+ | |||
+ | It can sometimes be difficult to discriminate between light coming from two closely spaced interfaces, for example, the two sides of a coverslip, or the variable spacing between a cover slip and a slide. | ||
+ | |||
+ | ==== Photodiode Displacement Signal ==== | ||
+ | |||
+ | |||
+ | The heart of the focus system is the split photodiode displacement sensor. | ||
+ | |||
+ | |||
+ | {{ crisp_4_.png? | ||
+ | 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. | ||
+ | |||
+ | The brown shaded regions have opposite slope compared to the regions near the surface. | ||
+ | |||
+ | |||
+ | {{ crisp_3_.gif? | ||
+ | Figure 7: Reflections from a glass bottomed Petri dish. | ||
+ | |||
+ | |||
+ | |||
+ | A common typical sample is a glass-bottomed Petri dish with a water sample. | ||
+ | |||
+ | ===== Control of the CRISP system ===== | ||
+ | |||
+ | |||
+ | To use the LCD display, ensure that the display-mode DIP switches 1 and 2 located on the back of the controller are in the UP position. | ||
+ | |||
+ | ==== LCD Display ==== | ||
+ | |||
+ | On the MS2000 controller, the bottom line of the LCD display shows information about the photo-detector signals and CRISP system state. | ||
+ | |||
+ | {{ crisp_lcd.jpg? | ||
+ | | ||
+ | |||
+ | The meaning of the quantitative information on the display changes depending up on the system state. | ||
+ | |||
+ | For a Tiger controller, the text that would be shown on bottom line of the LCD display is accessible by sending the serial command '' | ||
+ | |||
+ | As of Tiger '' | ||
+ | * State: '' | ||
+ | * Sum: '' | ||
+ | * Error: '' | ||
+ | |||
+ | There are 3 states meant to read sensor values and display them on the LCD: | ||
+ | |||
+ | ^ State Character ^ Sum Changed ^ Error Changed ^ | ||
+ | | A - Signal | Yes | Yes | | ||
+ | | B - Balance | Yes | Yes | | ||
+ | | M - Background Difference | No | Yes | | ||
+ | |||
+ | This table shows which states change the Sum and Error result on the LCD display. This also applies when using the '' | ||
+ | |||
+ | ==== Button Actions ==== | ||
+ | |||
+ | |||
+ | The @ button is used to manually control the CRISP system. | ||
+ | |||
+ | |||
+ | |||
+ | ^Function ^Button ^ | ||
+ | |Advance to next focus state |Press @ briefly and release | | ||
+ | |Back to Previous state or Advance to Calibration state |Press @ >3 sec. and release | | ||
+ | |Set Focus Offset to zero from READY state |Press @ >10 sec. and release | | ||
+ | |||
+ | ==== CRISP System States ==== | ||
+ | |||
+ | Activating and calibrating the CRISP system is done by moving to the next CRISP state using the @ button on the controller and pressing it for various amounts of time as shown in the “Next State” and “Previous State” columns in the table below. It is recommended that you use [[crisp_manual# | ||
+ | |||
+ | You can use the serial command '' | ||
+ | |||
+ | The state character in column 1 of Table 2 shows up as a character on the LCD display on MS2000. | ||
+ | |||
+ | Next State - @ button short press \\ | ||
+ | Previous State - @ button long press | ||
+ | |||
+ | === Table 2: CRISP System States === | ||
+ | ^ State ^ Code ^State Name ^Next State | ||
+ | |I |79 (O) |Idle |R |G |LED is tuned off going from Ready to Idle | | ||
+ | |R |85 (U) |Ready |K (D) |I |LED on - @ button locks | | ||
+ | |D | |Dim |(R) |I |Low returned light signal (prevents Ready state) | ||
+ | |K |83 (S) |Lock |R(k) |R |Active but not within focus tolerance; @ button unlocks | ||
+ | |F | |In Focus |R |R |Active and within focus tolerance; @ button unlocks | ||
+ | |N | |Inhibit |R |I |Low returned signal (unlocks system) | ||
+ | |E | |Error | |R |Usually Out-of-Range Error | | ||
+ | |G |72 (H) |loG_cal |R |1 |Initiate basic Log-Amp Calibration | ||
+ | | |67 (C) |gain_Cal |(2, | ||
+ | |f (g, | ||
+ | | †c |97 (a) |Curve |(R) | |Generate focus curve data | | ||
+ | | †B |66 (B) |Balance |R | |Display shows signal from each half of detector. | ||
+ | | †o |111 (o) |Set Offset |(R) | |Resets focus offset to zero for present focal position. | ||
+ | |||
+ | †States can only be initiated with the '' | ||
+ | ===== ASI Console support for CRISP ===== | ||
+ | |||
+ | {{crisp_6_.png? | ||
+ | |||
+ | |||
+ | 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. | ||
+ | |||
+ | <WRAP center round download 40%> | ||
+ | [[http:// | ||
+ | </ | ||
+ | |||
+ | |||
+ | In operation, the CRISP control is found on the MORE tab. Clicking on the CRISP button will bring up the main CRISP control panel. | ||
+ | |||
+ | The main initialization steps are presented with three buttons. | ||
+ | |||
+ | After you have calibrated the system with the three steps indicated, you may wish to obtain a plot of the focus curve. | ||
+ | |||
+ | Once the system is basically working, the Loop Gain slider is the easiest way to optimize the performance. | ||
+ | |||
+ | |||
+ | ===== 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. | ||
+ | |||
+ | ==== Quick Start Instructions Using ASI Console ==== | ||
+ | |||
+ | - Download and install [[http:// | ||
+ | - Using ASI Console, connect to the '' | ||
+ | - Follow the three step initialization and calibration procedure in the CRISP control window. Use the Lateral adjustment thumb screw to maximize the ERR signal for Step 2 dither. | ||
+ | |||
+ | ==== Quick Start Instructions Using Controller Only ==== | ||
+ | |||
+ | * Press @ button for 3 seconds to achieve reflectivity calibration. | ||
+ | * Press @ button for 3 seconds to initiate the Z-axis focus dither. | ||
+ | < | ||
+ | * Adjust the detector lateral adjustment screw on the CRISP unit for maximum absolute value of the focus error change. | ||
+ | * Press @ button briefly to advance to the READY state. | ||
+ | * Press @ button briefly to advance to the Lock state. | ||
+ | * Press @ button briefly to unlock and return to the READY state. | ||
+ | |||
+ | For optimum performance, | ||
+ | |||
+ | ==== 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 Ready state prior to locking. | ||
+ | |||
+ | Once the Lock is engaged, the Z‑axis control knob on the controller can be used to manually adjust the reference lock value. | ||
+ | |||
+ | To unlock the system, again, a short-press of the '' | ||
+ | |||
+ | When the Lock is engaged, any commanded move to the focus axis will fail and will generate a '' | ||
+ | |||
+ | ==== Saving Calibration and Offsets ==== | ||
+ | |||
+ | Once you are satisfied with the focus performance and adjustments, | ||
+ | |||
+ | Now, as long as you stay with the same sample preps and objective lens, you should not need to go through the first three steps above. | ||
+ | |||
+ | [[commands: | ||
+ | |||
+ | If you are trying to save the calibration state manually by querying serial commands, you will also need the **cal_gain** ('' | ||
+ | ==== Calibration Details ==== | ||
+ | |||
+ | Different samples and objective lenses can result in dramatically different levels of signal of returned light and different sensitivity of the detector to focus error. | ||
+ | |||
+ | === Log-Amp Calibration === | ||
+ | |||
+ | Before calibration, | ||
+ | |||
+ | This calibration step is initiated from the Idle state by a long press (3 sec.) of the '' | ||
+ | |||
+ | During this step the controller automatically adjusts the range of the internal log amplifier so that the light level on the photodiode corresponds to ~75% of full scale. | ||
+ | |||
+ | === Focus Sensitivity Calibration and Detector Lateral Adjustment === | ||
+ | |||
+ | |||
+ | Before this step, first focus on the sample and perform the Log-Amp Calibration described above. | ||
+ | |||
+ | === Focus Dither for Optical Adjustments === | ||
+ | |||
+ | In the Dither state the focus is changed by up and down by the cal_range amount. | ||
+ | |||
+ | Slowly adjust the detector lateral adjustment screw for a maximum absolute value of the '' | ||
+ | |||
+ | When satisfied that the focus slope is the best possible, a short press of the @ button will cause the controller to return the stage to the initial position, check and set the error offset to zero, and leave the system in the Ready state. | ||
+ | |||
+ | |||
+ | === Parameters used with the CRISP system === | ||
+ | |||
+ | |||
+ | The serial commands give the user access to several parameters used with the CRISP system. | ||
+ | |||
+ | ^ cal_range | ||
+ | ^ lock_range | ||
+ | ^ loop_gain | ||
+ | ^ cal_gain | ||
+ | ^ lock_offset | ||
+ | ^ LED_Intensity | ||
+ | ^ LogAmp_AGC | ||
+ | ^ in_focus_mm | ||
+ | |||
+ | The values of some of the parameters that are set during calibration will be sent to the serial port if the verbose mode VB X=16 is set. | ||
+ | |||
+ | ==== Optical Adjustment ==== | ||
+ | |||
+ | The CRISP unit is pre-adjusted at the factory, and should not need major adjustments. | ||
+ | |||
+ | === Adjusting the Relay Lens position === | ||
+ | |||
+ | |||
+ | The relay lens should be set to the center of its range. | ||
+ | |||
+ | === Adjusting Position of the LED Light Source === | ||
+ | |||
+ | |||
+ | Focus on a glass slide with a 10X or 20X objective so that a typical glass/air reflected beam is obtained. | ||
+ | |||
+ | a){{crisp_13_.jpg? | ||
+ | |||
+ | 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. | ||
+ | |||
+ | Sometimes you will notice an unfocused glare from the LED in the camera. | ||
+ | |||
+ | === Adjusting the Primary Mirror === | ||
+ | |||
+ | |||
+ | {{ crisp_19_.jpg? | ||
+ | Figure 9: Mirror Adjusting Screw | ||
+ | |||
+ | The most critical alignment in the CRISP device is the primary mirror that injects the LED light into half of the optical aperture and allows light from the other half of the optical aperture to reach the photo-detector. | ||
+ | |||
+ | One approach for adjusting the mirror position is to maximize the error signal received when in Dither mode. First center the photo-detector board so it is in the middle of its travel range. | ||
+ | |||
+ | You should also find that when the detector and LED are aligned so that you are getting a reasonable dither ERR, you can adjust the mirror position to maximize the Sum signal. However, a high dither Err number is most important for proper operation, so be sure you can return to a good Err number is you use the Sum as a guide to the mirror adjustment. | ||
+ | |||
+ | With the mirror properly adjusted, you should observe a strong lateral movement of the LED mask image in the camera as the focus is changed back and forth. | ||
+ | |||
+ | ===== Advanced Techniques ===== | ||
+ | |||
+ | |||
+ | ==== Offset the locking plane from the imaging plane ==== | ||
+ | |||
+ | A common issue 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. | ||
+ | |||
+ | \begin{equation} | ||
+ | D = \frac{δ}{n} M^2 | ||
+ | \end{equation} | ||
+ | |||
+ | For high magnification objectives, small focal plane changes can require substantial extension at the C-mount. | ||
+ | |||
+ | [{{ crisp_21_.jpg? | ||
+ | |||
+ | The C-mount extension lengths recommended for various objectives to optimize the focus range of the system within the sample are included in the table below. | ||
+ | |||
+ | === Table 3: Focus Properties for Typical Objectives === | ||
+ | |||
+ | ^ Microscope Objective | ||
+ | | 100X NA 1.25 | 2.4 | 4.5 | 0.43 | 14-29 | +/-3 | > +/- 10 | | ||
+ | | 60X NA 1.4 | ||
+ | | 40X NA 1.3 | ||
+ | | 40X NA 0.7 | ||
+ | | 20X NA 0.75 | ||
+ | | 20X NA 0.4 | ||
+ | | 10XNA 0.25 | 0.39 | 9.0 | 10.8 | 0 | +/-50 † | > +/- 100 | | ||
+ | |||
+ | †Glass/ | ||
+ | |||
+ | The focus range of the system is largely determined by the numerical aperture of the objective used. Once the reflective interface is well outside the depth of focus (DOF) of the objective, little useful light is returned to the detector and it is difficult to capture focus. | ||
+ | |||
+ | The amount of light available for CRISP depends not only on the type of reflective interface, but also on the light gathering ability of the objective lens. CRISP both illuminates and collects through the objective, so the relative brightness goes as NA< | ||
+ | |||
+ | With low power, low numerical aperture objectives, the light reflected from the air/glass interface will begin to significantly contribute to the focus signal. | ||
+ | |||
+ | ==== 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. | ||
+ | |||
+ | ==== Using Focus Curve Generation for Optimizing Adjustments ==== | ||
+ | |||
+ | |||
+ | Sometimes it's helpful to plot the focus error response so you can determine the usable lock range and capture range of the system. There are two programs that can generate and display the focus curve data: | ||
+ | |||
+ | * **Tiger:** [[crisp_mm_plugin|ASI CRISP Control]] (Micro-Manager 2.0 plugin) | ||
+ | * **MS2000:** [[https:// | ||
+ | |||
+ | The '' | ||
+ | |||
+ | **Note: Obtaining the Tiger focus curve requires version 2.5.0 of the CRISP plugin.** | ||
+ | |||
+ | To obtain the focus curve, first lock focus and adjust the lock optimally. | ||
+ | |||
+ | Prior to Whizkid firmware v9.51, if you change the [[commands: | ||
+ | |||
+ | <asi> $ LK F=97 | ||
+ | :A a | ||
+ | T: | ||
+ | T: 50 -10.4 -1 | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: | ||
+ | T: 1000 | ||
+ | T: 1050 | ||
+ | T: 1100 0.2 2 # Focus | ||
+ | T: 1150 0.7 -6 | ||
+ | T: 1200 1.2 -16 | ||
+ | T: 1250 1.7 -25 | ||
+ | T: 1300 2.2 -30 | ||
+ | T: 1350 2.7 -32 | ||
+ | T: 1400 3.2 -33 | ||
+ | T: 1450 3.7 -31 | ||
+ | T: 1500 4.2 -34 # -Peak | ||
+ | T: 1550 4.7 -30 | ||
+ | T: 1600 5.2 -30 | ||
+ | T: 1650 5.7 -27 | ||
+ | T: 1700 6.2 -27 | ||
+ | T: 1750 6.7 -25 | ||
+ | T: 1800 7.2 -24 | ||
+ | T: 1850 7.7 -22 | ||
+ | T: 1900 8.2 -22 | ||
+ | T: 1950 8.7 -19 | ||
+ | T: 2000 9.2 -19 | ||
+ | T: 2050 9.7 -19 | ||
+ | T: 2100 | ||
+ | end | ||
+ | </ | ||
+ | |||
+ | {{ crisp_1_.gif? | ||
+ | |||
+ | |||
+ | **Example graph:** You can see the strong slope near the surface (Z=0) that provides the focus feedback. | ||
+ | |||
+ | ==== Focus Variation Reduction by Averaging ==== | ||
+ | |||
+ | |||
+ | In many instances, the focus lock mechanism is used to hold the subject in focus for long periods of time. Dynamic performance is secondary, and stable focus on a weak interface may be more important. | ||
+ | |||
+ | ==== Dynamic Performance Optimization ==== | ||
+ | |||
+ | |||
+ | For application of automated image acquisition, | ||
+ | |||
+ | ===== Troubleshooting Steps ===== | ||
+ | |||
+ | If you cannot get sufficient Err difference in the Dither state, check these troubleshooting steps. | ||
+ | |||
+ | * Verify that the electronics are working. | ||
+ | * Set LED Intensity to 0%. Apply. | ||
+ | * Click Step 1 Log Amp Calibration. | ||
+ | * Set LED Intensity to ~70%. Apply. Save. Be sure you can get AGC value >35. | ||
+ | * Verify that you have an appropriate sample that is in focus. | ||
+ | * Verify that the IR light can reach the objective. | ||
+ | * Flat glass windows and prism surfaces can reflect more IR light than is coming from the desired interface. | ||
+ | * If problems persist, achieve and image of the CRISP LED on the system camera by removing the 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. | ||
+ | |||
+ | ===== Electromagnetic Interference ===== | ||
+ | |||
+ | Interference from certain cellular phones, microwave ovens, and other devices that emit radiation in the 0.1-10 GHz range has been known to interfere with normal CRISP operation when operated in close proximity and simultaneously. | ||
+ | |||
+ | ===== TTL Control of the CRISP focus lock ===== | ||
+ | |||
+ | === TTL input function | ||
+ | |||
+ | |||
+ | In some instances it may be desirable to be able to turn on/off the CRISP lock using a TTL signal. | ||
+ | |||
+ | The MS2000 TTL Input can be programmed for several functions. | ||
+ | |||
+ | Use ASI_Console, | ||
+ | |||
+ | === TTL output function (TTL Y=11, 12) === | ||
+ | |||
+ | You can also use TTL control to determine when the system is in focus. | ||
+ | |||
+ | The TTL output can be used to directly monitor if CRISP is in the ‘F’ state or not using TTL Y=12. TTL OUT0 is high when CRISP is ‘F’ state, low otherwise. | ||
+ | |||
+ | ===== Serial polling of the CRISP focus lock ===== | ||
+ | |||
+ | The TTL control discussed above can eliminate polling delays, but sometimes it is easier to implement serial polling. | ||
+ | |||
+ | For any other TTL Y=n state (n|=11) the global status command will only respond to the XY stage busy status. | ||
+ | |||
+ | ===== Computer Control of the CRISP System ===== | ||
+ | |||
+ | The focus controller responds to several commands dedicated to controlling the feedback system. | ||
+ | |||
+ | {{topic> | ||
+ | |||
+ | ==== Serial Command Cheatsheet ==== | ||
+ | |||
+ | The only deprecated command is '' | ||
+ | |||
+ | '' | ||
+ | |||
+ | On TG-1000 don't forget to prefix the card address to the command. For instance, if the '' | ||
+ | |||
+ | ^ CRISP Serial Commands |||| | ||
+ | ^ Property ^ Set ^ Get ^ Notes ^ | ||
+ | | CRISP State | '' | ||
+ | | Idle State | '' | ||
+ | | Log Cal State | '' | ||
+ | | Dither State | '' | ||
+ | | Set Gain State | '' | ||
+ | | Set Offset State | '' | ||
+ | | Get Focus Curve | '' | ||
+ | | Lock State | '' | ||
+ | | Unlock State | '' | ||
+ | | LED Intensity | '' | ||
+ | | Objective NA | '' | ||
+ | | Loop Gain | '' | ||
+ | | Number of Averages | '' | ||
+ | | Update Rate (ms) | '' | ||
+ | | Lock Range (mm) | '' | ||
+ | | In Focus Range (um) | '' | ||
+ | | SNR | - | '' | ||
+ | | Sum | - | '' | ||
+ | | Error Number | - | '' | ||
+ | | Lock Offset | '' | ||
+ | | Focus Axis | '' | ||
+ | | Knob Speed | '' | ||
+ | | LogAmp_AGC | '' | ||
+ | | Calibration Range | '' | ||
+ | | Calibration Gain | '' | ||
+ | |||
+ | ==== Accessing Diagnotistics over Serial ==== | ||
+ | |||
+ | The text normally shown on bottom line of the LCD display is accessible by sending the serial command '' | ||
+ | |||
+ | {{tag> | ||
+ |
Address: 29391 W Enid Rd. Eugene, OR 97402, USA | Phone: +1 (541) 461-8181