| {width=5000} | Inverse spinning disk microscope (Nikon Ti2E double-deck stand) for fast pseudo-confocal imaging, preferably for long-term live applications. Fast high-resolution sCMOS or sensitive EM-CCD camera, multi-positioning and full environmental control. Pulsed 355 nm laser for nanodissection, all lasers available for photomanipulation (FRAP, photoactivation). Widefield illumination: 8-color SPECTRA-III LED, white LED for transmitted light (room 1.318). |
| {width=5000} | Inverse spinning disk microscope (Nikon Ti2E double-deck stand) for fast pseudo-confocal imaging, preferably for long-term live applications. Fast high-resolution sCMOS or sensitive EM-CCD camera, multi-positioning and full environmental control. Pulsed 355 nm laser for nanodissection, all lasers available for photomanipulation (FRAP, photoactivation). Widefield illumination: 8-color SPECTRA-III LED, white LED for transmitted light (room 1.318). |
- For the EM-CCD (low resolution, high sensitivity), pull the handle on the spinning disk unit up and put the small plastic box underneath to fix it (see image right).
- For the EM-CCD (low resolution, high sensitivity), pull the handle on the spinning disk unit up and put the small plastic box underneath to fix it (see image right).
- For the sCMOS (fast, high resolution), push it down. Hold it with your hand while pushing (see image left).
- For the sCMOS (fast, high resolution), push it down. Hold it with your hand while pushing (see image left).
- Windows login: pick the right user account (Imaging, FRAP or Nanodissection). Nanodissection is password protected, the others are not.
- Windows login: pick the right user account (Imaging, FRAP or Nanodissection). Nanodissection is password protected, the others are not.
- Launch VisiView. If you have not turned on all lasers, an info message will list inactive lasers being emulated (Obis, Obis-2, and/or Toptica for the 405, 488, and/or 640 nm, respectively). Acknowledge by clicking OK. Likewise, acknowledge Hard drive not found warnings (the startup script will reset the path for saving files to the standard hard drive D:).
- Launch VisiView. If you have not turned on all lasers, an info message will list inactive lasers being emulated (Obis, Obis-2, and/or Toptica for the 405, 488, and/or 640 nm, respectively). Acknowledge by clicking OK. Likewise, acknowledge Hard drive not found warnings (the startup script will reset the path for saving files to the standard hard drive D:).
- Startup dialog:
- Startup dialog:
- Camera & Spinning Disk: enter the selected camera & SD (W1 or X1, sCMOS or EM-CCD)
- Camera & Spinning Disk: enter the selected camera & SD (W1 or X1, sCMOS or EM-CCD)
- Objective: choose the one you wish to use from the drop-down list
- Objective: choose the one you wish to use from the drop-down list
- Yes (recommended) moves the objective to the lowest (safe) position
- Yes (recommended) moves the objective to the lowest (safe) position
- No (are you sure?) should only be chosen if you had to restart VisiView during a session
- No (are you sure?) should only be chosen if you had to restart VisiView during a session
- Keep settings/positions:
- Keep settings/positions:
- No (recommended) clears any previous stage positions and illumination settings
- No (recommended) clears any previous stage positions and illumination settings
- Yes should only be chosen if you had to restart VisiView during a session
- Yes should only be chosen if you had to restart VisiView during a session
- After running the startup dialog, the objective should be in the lowest (safe) position.
- After running the startup dialog, the objective should be in the lowest (safe) position.
- If the ESC (Escape) button on the microscope lights green, the focus is locked. This happens if the previous user has lowered the objective turret at the end of the session using the ESC button. Press it and keep it pressed until the green light turns off.
- If the ESC (Escape) button on the microscope lights green, the focus is locked. This happens if the previous user has lowered the objective turret at the end of the session using the ESC button. Press it and keep it pressed until the green light turns off.
- Launch the webcam in the box with the camera icon on the taskbar.
- Launch the webcam in the box with the camera icon on the taskbar.
- Turn on the light in the chamber.
- Turn on the light in the chamber.
- At 37°C, rotate the collar so that the mark on the objective body coincides with the 0.15-mm scale mark on the collar. On the 40x silicone objective, adjust to the mark halfway between 0.13 and 0.17, on the 60x water to the mark labeled "15".
- At 37°C, rotate the collar so that the mark on the objective body coincides with the 0.15-mm scale mark on the collar. On the 40x silicone objective, adjust to the mark halfway between 0.13 and 0.17, on the 60x water to the mark labeled "15".
- If the CO2 insert is not used, it should be stored inside the box (to keep it on temperature) at the front left corner (caution: avoid blocking the travel range of the XY stage). The click-in plates for the CO2 insert should be stored on one of the shelves inside the box.
- If the CO2 insert is not used, it should be stored inside the box (to keep it on temperature) at the front left corner (caution: avoid blocking the travel range of the XY stage). The click-in plates for the CO2 insert should be stored on one of the shelves inside the box.
- Other inserts (standard slide, multiwell) should be kept in the small drawer next to the door. In the drawer, there is also a click-in plate for two 35-mm dishes.
- Other inserts (standard slide, multiwell) should be kept in the small drawer next to the door. In the drawer, there is also a click-in plate for two 35-mm dishes.
**Important:** when using the click-in plate for two 35-mm dishes, always move the objective down using the ESC button before moving from one dish to the other! Do not program stage positions to move the stage from one dish to the other in a VisiView sequence. There is a high risk of damaging the objective! Lower the objective to the load position and move manually from one dish to the other using the joy stick.
**Important:** when using the click-in plate for two 35-mm dishes, always move the objective down using the ESC button before moving from one dish to the other! Do not program stage positions to move the stage from one dish to the other in a VisiView sequence. There is a high risk of damaging the objective! Lower the objective to the load position and move manually from one dish to the other using the joy stick.
- The Nikon focus turns in the opposite direction as compared with Zeiss microscopes. An arrow (UP) on the focus drives and on the Nikon joystick pad indicates the direction.
- The Nikon focus turns in the opposite direction as compared with Zeiss microscopes. An arrow (UP) on the focus drives and on the Nikon joystick pad indicates the direction.
- The Z^ button toggles between coarse (fast) and fine (slow) mode. In coarse mode, the focus indicator on the frame lights green. The current mode is also indicated on the LED of the joystick.
- The Z^ button toggles between coarse (fast) and fine (slow) mode. In coarse mode, the focus indicator on the frame lights green. The current mode is also indicated on the LED of the joystick.
- The PFS buttons on the focus drives and joystick control the Perfect Focus hardware autofocus.
- The PFS buttons on the focus drives and joystick control the Perfect Focus hardware autofocus.
- Pressing the button briefly turns it on or off. The PFS indicator on the frame lighting green indicates an active PFS. It blinks if it is off, but detects a surface within its travel range. It remains dark if there is no surface in reach.
- Pressing the button briefly turns it on or off. The PFS indicator on the frame lighting green indicates an active PFS. It blinks if it is off, but detects a surface within its travel range. It remains dark if there is no surface in reach.
- Pressing it and holding it for >1 sec turns the PFS off and removes the dichroic from the light path. The indicator on the frame turns dark. The LED on the joystick pad displays "PFs:OUT".
- Pressing it and holding it for >1 sec turns the PFS off and removes the dichroic from the light path. The indicator on the frame turns dark. The LED on the joystick pad displays "PFs:OUT".
- The Nikon XY joystick is inactive. To move the stage in XY, use the ASI controller next to the microscope. Pressing the button on the top of its joystick toggles between slow and fast mode.
- The Nikon XY joystick is inactive. To move the stage in XY, use the ASI controller next to the microscope. Pressing the button on the top of its joystick toggles between slow and fast mode.
- To ease finding the focus at startup, click on the Focus Up button in the VisiView toolbar. In the dialog popping up, select the name of the installed stage insert.
- To ease finding the focus at startup, click on the Focus Up button in the VisiView toolbar. In the dialog popping up, select the name of the installed stage insert.
- Center the objective below the opening of the stage insert using the ASI XY joystick and click OK. The objective will move up, close to the sample. It will not be exactly in focus, though, and you will have to focus a bit manually. Clicking the Cancel button in the dialog will exit without action.
- Center the objective below the opening of the stage insert using the ASI XY joystick and click OK. The objective will move up, close to the sample. It will not be exactly in focus, though, and you will have to focus a bit manually. Clicking the Cancel button in the dialog will exit without action.
- To replace the sample once focused, press the ESC button on the microscope frame. The objective turret will move down. All focus controls will be locked and the ESC indicator will light up green. Briefly pressing the ESC button again will move the objective back to last focus position and enable the focus controls (wait until the green light is off). When keeping the ESC button pressed until the green light goes off, the focus lock will be released, but the objective turret will remain in the lowest (safe) position.
- To replace the sample once focused, press the ESC button on the microscope frame. The objective turret will move down. All focus controls will be locked and the ESC indicator will light up green. Briefly pressing the ESC button again will move the objective back to last focus position and enable the focus controls (wait until the green light is off). When keeping the ESC button pressed until the green light goes off, the focus lock will be released, but the objective turret will remain in the lowest (safe) position.
### 9. CO2 & temperature control (optional)
### 9. CO2 & temperature control (optional)
- By default, the system should be running at 37°C. Do not switch off the controller after your session. If you need lower temperatures, please contact the person signed in after you far in advance and ask for their permission, as the temperature will need some time to increase again after your experiment.
- By default, the system should be running at 37°C. Do not switch off the controller after your session. If you need lower temperatures, please contact the person signed in after you far in advance and ask for their permission, as the temperature will need some time to increase again after your experiment.
- Open the main valve of the CO2 tank by turning it counter clockwise for 1 round (no need to rotate it up to the stop). Close it clockwise once you are done. Don't touch the other valves!
- Open the main valve of the CO2 tank by turning it counter clockwise for 1 round (no need to rotate it up to the stop). Close it clockwise once you are done. Don't touch the other valves!
- Power on the CO2 controller (power switch on its back side).
- Power on the CO2 controller (power switch on its back side).
- Place the CO2 cover on the CO2 insert and make sure that the humidifier bottle is filled with enough distilled water (>50 mL; the bottle can be disconnected with two luer connectors). To replenish water (deionized, autoclaved water only), contact the facility staff!
- Place the CO2 cover on the CO2 insert and make sure that the humidifier bottle is filled with enough distilled water (>50 mL; the bottle can be disconnected with two luer connectors). To replenish water (deionized, autoclaved water only), contact the facility staff!
- File format: in the top menu, select Configure → System. In the dialog popping up, select the Acquisition tab. Under Save File, select file format:
- File format: in the top menu, select Configure → System. In the dialog popping up, select the Acquisition tab. Under Save File, select file format:
Metamorph STK is recommended for all kinds of multidimensional acquisitions.
Metamorph STK is recommended for all kinds of multidimensional acquisitions.
OME Tiff is recommended single-channel FRAP experiments. It stores FRAP regions in the metadata. Check the auto checkbox. VisiView will automatically select the proper OME Tiff subtype (32 bit as *.ome.tif, 64 bit version as *.ome.btf).
OME Tiff is recommended single-channel FRAP experiments. It stores FRAP regions in the metadata. Check the auto checkbox. VisiView will automatically select the proper OME Tiff subtype (32 bit as *.ome.tif, 64 bit version as *.ome.btf).
Also, there are many useful videos from the company: https://www.youtube.com/channel/UCY8kMsXVHCt5rI57j9tRt7w
Also, there are many useful videos from the company: https://www.youtube.com/channel/UCY8kMsXVHCt5rI57j9tRt7w
### 1. Choosing the right illumination
### 1. Choosing the right illumination
In the 'illumination' dropdown menu, you can choose the illumination that matches your hardware settings - meaning the selected SD & camera (W1 or X1, sCMOS or EM-CCD). Choose the laser line you need to excite your fluorophore (DAPI, GFP, mCherry & Cy5 - meaning 405, 488, 561 and 647 excitation lasers). The 'Quadband' illuminations have a filter that works for all wavelengths (so your recordings will be faster in case of multicolor imaging), however you will potentially experience more crosstalk, which you can check under https://www.fpbase.org/spectra/. If you are not sure, ask the facility for their help in figuring out whether you can use the Quadband. For each illumination (in case of multi-color imaging) go to 'Show live' adapt the following parameters to achieve the desired image quality (in that order):
In the 'illumination' dropdown menu, you can choose the illumination that matches your hardware settings - meaning the selected SD & camera (W1 or X1, sCMOS or EM-CCD). Choose the laser line you need to excite your fluorophore (DAPI, GFP, mCherry & Cy5 - meaning 405, 488, 561 and 647 excitation lasers). The 'Quadband' illuminations have a filter that works for all wavelengths (so your recordings will be faster in case of multicolor imaging), however you will potentially experience more crosstalk, which you can check under https://www.fpbase.org/spectra/. If you are not sure, ask the facility for their help in figuring out whether you can use the Quadband. For each illumination (in case of multi-color imaging) go to 'Show live' adapt the following parameters to achieve the desired image quality (in that order):
- the EMCCD Gain (only available for the EMCCD, a good starting value is 300), which only increases your signal on the detection side (no increase in photobleaching)
- the EMCCD Gain (only available for the EMCCD, a good starting value is 300), which only increases your signal on the detection side (no increase in photobleaching)
- the Exposure (if you have a mobile sample, you might save time by reducing the illumination time)
- the Exposure (if you have a mobile sample, you might save time by reducing the illumination time)
- the Laser power (be aware that higher laser powers and illumination times can result in irreversible photobleaching, which impairs e.g. brightness analysis). Do not use more than 10% of the laser power! If you are getting close to 10%, let the facility know and they will realign the lasers
- the Laser power (be aware that higher laser powers and illumination times can result in irreversible photobleaching, which impairs e.g. brightness analysis). Do not use more than 10% of the laser power! If you are getting close to 10%, let the facility know and they will realign the lasers
When acquiring the brightfield channel, you only have to adapt the intensity of the Dialamp.
When acquiring the brightfield channel, you only have to adapt the intensity of the Dialamp.
Always tick 'Save Sequence to Disk' and set a path under 'Directory' (ideally save it directly to the Hive or Fileshare). Only tick 'Separate Files' if you want to save each recording individually (meaning each color channel etc.). **Important:** Sometimes the box next to 'Save Sequence to Disk' or 'Separate Files' disappears (which is a bug). Solution: untick 'Time-lapse', 'Wavelength', 'Z-series' and 'Stage positions', then you should see it again. Tick the box next to 'Save Sequence to Disk' or 'Separate Files' and then tick again 'Time-lapse', 'Wavelength', 'Z-series' or 'Stage positions'. Even if the box disappears again now, it will remain active.
Always tick 'Save Sequence to Disk' and set a path under 'Directory' (ideally save it directly to the Hive or Fileshare). Only tick 'Separate Files' if you want to save each recording individually (meaning each color channel etc.). **Important:** Sometimes the box next to 'Save Sequence to Disk' or 'Separate Files' disappears (which is a bug). Solution: untick 'Time-lapse', 'Wavelength', 'Z-series' and 'Stage positions', then you should see it again. Tick the box next to 'Save Sequence to Disk' or 'Separate Files' and then tick again 'Time-lapse', 'Wavelength', 'Z-series' or 'Stage positions'. Even if the box disappears again now, it will remain active.
Tick Time-lapse and choose your number of time points, your time interval or your duration. You only have to choose two of them and the third one will adapt automatically. If you are only doing a time-lapse (no other wavelengths, z-series or stage positions), you can also tick 'streaming' for a faster recording. The time interval is only the waiting time between two time points, after which an image is recorded and needs to be read out from the camera chip (so there will always be a couple of 10-100ms added to your time interval, which you can read out from your metadata). If you set a time interval of 0 the recording will be done as fast as possible (meaning the time interval is then only the Exposure time plus the readout time of the camera - multiplied by the number of wavelengths, z-planes and positions, depending on your experiment: also switching in between wavelengths or moving to different z-planes or positions takes time!). If you set a time interval >0 then it will automatically be increased, in case you have chosen it too short for your number of wavelengths, z-planes and/or positions. If you still want to further decrease your time interval, you have to reduce your number of wavelengths, z-planes and/or positions per time point.
Tick Time-lapse and choose your number of time points, your time interval or your duration. You only have to choose two of them and the third one will adapt automatically. If you are only doing a time-lapse (no other wavelengths, z-series or stage positions), you can also tick 'streaming' for a faster recording. The time interval is only the waiting time between two time points, after which an image is recorded and needs to be read out from the camera chip (so there will always be a couple of 10-100ms added to your time interval, which you can read out from your metadata). If you set a time interval of 0 the recording will be done as fast as possible (meaning the time interval is then only the Exposure time plus the readout time of the camera - multiplied by the number of wavelengths, z-planes and positions, depending on your experiment: also switching in between wavelengths or moving to different z-planes or positions takes time!). If you set a time interval >0 then it will automatically be increased, in case you have chosen it too short for your number of wavelengths, z-planes and/or positions. If you still want to further decrease your time interval, you have to reduce your number of wavelengths, z-planes and/or positions per time point.
In the Time-lapse tab you can also tick Autofocus and FRAP (for a detailed explanation see further below).
In the Time-lapse tab you can also tick Autofocus and FRAP (for a detailed explanation see further below).
Tick Wavelength and choose your number of fluorophores (brightfield counts as an extra wavelength). For each slot in the 'Current' dropdown menu pick an illumination in the 'Illumination' dropdown menu (in the order in which you want to record them). Ticking 'same exposure/gain for all wavelengths' increases the speed of your recording, it it however not recommended, as different fluorophores usually exhibit different brightnesses, which you then have to compensate with the laser power only.
Tick Wavelength and choose your number of fluorophores (brightfield counts as an extra wavelength). For each slot in the 'Current' dropdown menu pick an illumination in the 'Illumination' dropdown menu (in the order in which you want to record them). Ticking 'same exposure/gain for all wavelengths' increases the speed of your recording, it it however not recommended, as different fluorophores usually exhibit different brightnesses, which you then have to compensate with the laser power only.
If you have also ticket the 'z-Series' tab, you will have the option in the 'Wavelength' tab to tick 'Z-Plane Series' for each illumination **individually** (meaning you could e.g. record a brighfield image without a z-Series and than 4 colors with a z-Series each). You can also apply a flatfield correction, in case you are doing quantitative brightness analysis (for a detailed explanation go to the Visiview helpcenter and search for flatfield correction)
If you have also ticket the 'z-Series' tab, you will have the option in the 'Wavelength' tab to tick 'Z-Plane Series' for each illumination **individually** (meaning you could e.g. record a brighfield image without a z-Series and than 4 colors with a z-Series each). You can also apply a flatfield correction, in case you are doing quantitative brightness analysis (for a detailed explanation go to the Visiview helpcenter and search for flatfield correction)
Tick Z-Series. Go to 'show live', focus on your sample, then tick 'Centered Range'. Do not change to focus after that. You can now either stay with the ticked 'Centered Range' and choose a value for the 'Centered Range' (=size of your z-stack) or you can untick it and go to 'View top offset' and 'View bottom offset' and live-change the value next to it to change the top and the bottom of your z-Series. Be aware that the z-Series tab does not recognize a change in the focus (as e.g. the Zeiss Zen software does), so you can **only** adapt your range by changing the values for 'View top offset' and 'View bottom offset'.
Tick Z-Series. Go to 'show live', focus on your sample, then tick 'Centered Range'. Do not change to focus after that. You can now either stay with the ticked 'Centered Range' and choose a value for the 'Centered Range' (=size of your z-stack) or you can untick it and go to 'View top offset' and 'View bottom offset' and live-change the value next to it to change the top and the bottom of your z-Series. Be aware that the z-Series tab does not recognize a change in the focus (as e.g. the Zeiss Zen software does), so you can **only** adapt your range by changing the values for 'View top offset' and 'View bottom offset'.
You can now adapt the number of steps or the size of the steps. For optimal resolution in z, click the macro 'set opt z' in the right sidebar. If you experience a lot of photobleaching, you might consider trading resolution (larger steps) for less photobleaching.
You can now adapt the number of steps or the size of the steps. For optimal resolution in z, click the macro 'set opt z' in the right sidebar. If you experience a lot of photobleaching, you might consider trading resolution (larger steps) for less photobleaching.
#### Stage
#### Stage
##### Different stage positions
##### Different stage positions
Tick 'Stage positions'. Go to your desired positions using the joystick, **focus properly(!)**, change the name of your position (e.g. 1,2,3,...) and click 'Add'. Importantly, also the z-value is saved with each position. If you want to update your positions (e.g. just the z-focus right before starting your experiment or after adding a substance to your probe), click on each of your positions, click 'Go to', change the focus, click 'Add' and confirm that the current position will be overwritten. You can also pick a 'Settle time' (meaning a waiting time after moving to a new position), in case your sample exhibits some motion after being moved.
Tick 'Stage positions'. Go to your desired positions using the joystick, **focus properly(!)**, change the name of your position (e.g. 1,2,3,...) and click 'Add'. Importantly, also the z-value is saved with each position. If you want to update your positions (e.g. just the z-focus right before starting your experiment or after adding a substance to your probe), click on each of your positions, click 'Go to', change the focus, click 'Add' and confirm that the current position will be overwritten. You can also pick a 'Settle time' (meaning a waiting time after moving to a new position), in case your sample exhibits some motion after being moved.
a. Tick 'Stage positions' and then choose 'Scan slide' in the dropdown menu.
a. Tick 'Stage positions' and then choose 'Scan slide' in the dropdown menu.
b. Choose the Fill Order 'Vertical' and click on the square symbol right below (it only shows when you hover over it with your mouse) - the Scan Slide Detail View should open
b. Choose the Fill Order 'Vertical' and click on the square symbol right below (it only shows when you hover over it with your mouse) - the Scan Slide Detail View should open
c. Click on 'Setup' in the right upper corner, choose the right objective and overlap, then close just the Scan Slide Detail View
c. Click on 'Setup' in the right upper corner, choose the right objective and overlap, then close just the Scan Slide Detail View
d. Put on a sample, go to 'Show Live' and find a central, significant, solitary object. Choose an illumination time <100ms, put your joystick's/controller's speed to S (slow) and draw a ROI (not larger than 100x100 pixels) around your object.
d. Put on a sample, go to 'Show Live' and find a central, significant, solitary object. Choose an illumination time <100ms, put your joystick's/controller's speed to S (slow) and draw a ROI (not larger than 100x100 pixels) around your object.
f. If the calibration is sucessful confirm by clicking 'Yes'. If it has failed repeat with a different object, different laserpower or a different ROI. If it does not work after a couple of tries, you have to restart the software/PC and potentially the microscopes hardware and try again (unfortunately it is sometimes buggy).
f. If the calibration is sucessful confirm by clicking 'Yes'. If it has failed repeat with a different object, different laserpower or a different ROI. If it does not work after a couple of tries, you have to restart the software/PC and potentially the microscopes hardware and try again (unfortunately it is sometimes buggy).
h. Click 'clear'. Then go to 'Show Live' and use the joystick/controller to move around your sample and define the area you want to scan. If you have moved to far, go to the side that you want to reduce (corners do not work), click inside the Scan Slide Detail View window & press Control + Shift while moving the joystick to reduce the area.
h. Click 'clear'. Then go to 'Show Live' and use the joystick/controller to move around your sample and define the area you want to scan. If you have moved to far, go to the side that you want to reduce (corners do not work), click inside the Scan Slide Detail View window & press Control + Shift while moving the joystick to reduce the area.
i. Choose a desired overlap between tiles
i. Choose a desired overlap between tiles
j. Record an overview image
j. Record an overview image
k. Drag a rectangular ROI around the structure you want to record in your overview image
k. Drag a rectangular ROI around the structure you want to record in your overview image
l. You can set focus points by going to 'show live', double clicking in your overview image and changing the focus
l. You can set focus points by going to 'show live', double clicking in your overview image and changing the focus
m. Record your sequence and process it in VisiView our Imaris
m. Record your sequence and process it in VisiView our Imaris
The hardware autofocus permits focus stabilization under non-bleaching conditions. It uses backreflection of grid pattern generated with a weak LED (850 nm) to measure the distance between the objective and the cover glass and to keep it constant. An aqueous medium (with a refractive index close to that of water) is required. It cannot be used with glycerol-based or hardening mounting media.
The hardware autofocus permits focus stabilization under non-bleaching conditions. It uses backreflection of grid pattern generated with a weak LED (850 nm) to measure the distance between the objective and the cover glass and to keep it constant. An aqueous medium (with a refractive index close to that of water) is required. It cannot be used with glycerol-based or hardening mounting media.
Objects above the cover glass/medium interface are not monitored. If objects of interest can change their focus position (e.g., cells rounding up during mitosis), the software autofocus should be used.
Objects above the cover glass/medium interface are not monitored. If objects of interest can change their focus position (e.g., cells rounding up during mitosis), the software autofocus should be used.
#### A. Continuous mode (constant offset)
#### A. Continuous mode (constant offset)
This mode is convenient to keep the focus at a contant distance above the cover glass while moving the XY stage across large areas of the sample.
This mode is convenient to keep the focus at a contant distance above the cover glass while moving the XY stage across large areas of the sample.
1. Click the button "HW-AF Dialog" in the toolbar on the right side of the application. Alternatively, the dialog can be opened with the menu command Configure → Autofocus.
1. Click the button "HW-AF Dialog" in the toolbar on the right side of the application. Alternatively, the dialog can be opened with the menu command Configure → Autofocus.
2. Click on the "Find Focus" button. The device will try to find the z-position of the cover glass/medium interface
2. Click on the "Find Focus" button. The device will try to find the z-position of the cover glass/medium interface
3.Focus on the desired height above the cover glass and click on the "On" radio button Once the offset is determined, the "Find Focus" button is disabled.
3.Focus on the desired height above the cover glass and click on the "On" radio button Once the offset is determined, the "Find Focus" button is disabled.
To use a different offset, turn off the autofocus, focus manually on a different position and turn it on again. For convenience, the toolbar also contains "HW-AF ON" and "OFF" buttons. The button "AutoFocus NOW" has no function (it works only with devices from other manufacturers).
To use a different offset, turn off the autofocus, focus manually on a different position and turn it on again. For convenience, the toolbar also contains "HW-AF ON" and "OFF" buttons. The button "AutoFocus NOW" has no function (it works only with devices from other manufacturers).
#### B. Stage positions with multiple offsets
#### B. Stage positions with multiple offsets
The following procedure describes the setup of a multiposition time series with multiple offsets (i.e., positions may have different z-coordinates above the cover glass).
The following procedure describes the setup of a multiposition time series with multiple offsets (i.e., positions may have different z-coordinates above the cover glass).
1. In the Time lapse tab, check "Time-lapse series" and the "Autofocus every x time points" option ("every 1 time points" is recommended for multiple stage positions. **Important:** Sometimes the box 'every x time points' disappears (which is a bug). Solution: untick 'Time-lapse', 'Wavelength', 'Z-series' and 'Stage positions', then you should see it again. Change the value in 'every x time points' to 1 and then tick again 'Time-lapse', 'Wavelength', 'Z-series' or 'Stage positions'. Even if the box disappears again now, it will remain active.
1. In the Time lapse tab, check "Time-lapse series" and the "Autofocus every x time points" option ("every 1 time points" is recommended for multiple stage positions. **Important:** Sometimes the box 'every x time points' disappears (which is a bug). Solution: untick 'Time-lapse', 'Wavelength', 'Z-series' and 'Stage positions', then you should see it again. Change the value in 'every x time points' to 1 and then tick again 'Time-lapse', 'Wavelength', 'Z-series' or 'Stage positions'. Even if the box disappears again now, it will remain active.
Click the "Config" button on the right of the "Autofocus" option
Click the "Config" button on the right of the "Autofocus" option
2. In the "Configure AutoFocus" dialog, check "Hardware Autofocus" and "use Continuous Focus". For very distant stage positions, additionally check "during stage move". Close the dialog ("x" on the right upper corner)
2. In the "Configure AutoFocus" dialog, check "Hardware Autofocus" and "use Continuous Focus". For very distant stage positions, additionally check "during stage move". Close the dialog ("x" on the right upper corner)
3. In the Z-series tab, check "Stage Postions" and "AF Offset". For very distant positions, a settle time of 200-500 msec is recommended (to let the immersion oil film keep up with the XY stage movement)
3. In the Z-series tab, check "Stage Postions" and "AF Offset". For very distant positions, a settle time of 200-500 msec is recommended (to let the immersion oil film keep up with the XY stage movement)
4. Turn on hardware autofocus in continuous mode: in the toolbar (right side of the GUI), click "HW-AF Dialog" and then select "Continuous Auto Focus Parameter" = "On"
4. Turn on hardware autofocus in continuous mode: in the toolbar (right side of the GUI), click "HW-AF Dialog" and then select "Continuous Auto Focus Parameter" = "On"
5. Move to the desired stage positions. In the "Stage" tab, add them by clicking the "Add" button. Coordinates added successfully with offset should should display the suffix "a=Set". Start the sequence as usual.
5. Move to the desired stage positions. In the "Stage" tab, add them by clicking the "Add" button. Coordinates added successfully with offset should should display the suffix "a=Set". Start the sequence as usual.
