- 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:).
- 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".
- Place a drop of the appropriate immersion fluid on the objective.
- Place a drop of the appropriate immersion fluid on the objective.
- Plan Apo 20x Air: None
- Plan Apo 20x Air: None
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- Mount your sample in the stage insert.
- Mount your sample in the stage insert.
#### 8. Focusing
### 8. Focusing
- 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.
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- 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!
- On the microscope, press the ESC button to bring the objective in a safe position.
- On the microscope, press the ESC button to bring the objective in a safe position.
- If using CO2 control:
- If using CO2 control:
- Turn off the CO2 flow on the CO2 controller.
- Turn off the CO2 flow on the CO2 controller.
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@@ -156,9 +156,48 @@ OME Tiff is recommended single-channel FRAP experiments. It stores FRAP regions
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@@ -156,9 +156,48 @@ OME Tiff is recommended single-channel FRAP experiments. It stores FRAP regions
- Swith off all laser keys (4).
- Swith off all laser keys (4).
- Power off the power strips in reverse order (3, 2, 1).
- Power off the power strips in reverse order (3, 2, 1).
## Technical Specifications
## Hardware Autofocus
### Objectives
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.
### 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.
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
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).
### 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).
1. In the Time lapse tab, check "Time-lapse series" and the "Autofocus" option. Click the "Config" button 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)
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"
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.
