Imaging on the CT-ASLM-V1 and CT-ASLM-V2
This is a case study in using the software to image with a CT-ASLM-V1 and CT-ASLM-V2 microscopes.
Setting up the chamber
Make sure the chamber is clean and dry. If in doubt, fill the chamber with deionized water to see if there is any residue. To clean the chamber, rinse it with deionized water and ethanol, then gently clean the chamber with Q-tips. Repeat the process a few times and end the process with a rinse of 100% ethanol. Gently clean the objectives with lens paper and 100% ethanol. Finally, let the chamber air dry. To speed up the drying process, one can gently blow some air into the chamber.
Once the chamber is completely dry, fill the chamber with imaging media.
Sample loading and finding the samples
It’s recommended to start the software before loading the sample on the stage.
Mount the sample on custom cut glass slide with silicon or super glue.
Mount the glass slide onto the sample holder.
Mount the sample holder onto the stage with the sample facing the illumination and detection objective so the glass slide is 45 degrees with both objectives.
Decrease the numerical aperture of the illumination beam such that it covers the entire field of view. Typically this is achieved with a magnetic mounted slit aperture that can be readily adjusted.
Go to Camera Settings. Select Normal under Sensor Mode.
Go to the . A popup named Waveform Parameter Settings will appear.
Set the wavelength’s Amplitude and Offset to
0.0.Select the channel with a proper laser under the Channels tab and set the laser power to an appropriate value.
Select “Continuous Scan” from the dropdown next to the Acquire button. Press Acquire. This will launch a live acquisition mode.
Scroll around with the stage either via joystick or using the controls in the Stage Control tab until the sample comes into view.
Focus on the sample in the center of the beam. Zoom in by placing the mouse over the image and scrolling the mouse wheel. Slowly adjusting the focus by scrolling the piezo controller to move the detection objective along the z axis. Lower the laser power if the image is saturated.
Imaging a Z-Stack with Stelzer mode
Stelzer mode is the normal non-ASLM light sheet mode, it gives more signal while offering around 1040 nm (CT-ASLM-V1) and 500 nm (CT-ASLM-V2) axial resolution.
Go to the . A popup named Waveform Parameter Settings will appear.
Set the wavelength’s Amplitude and Offset to
0.0.Go to Camera Settings, select Normal under Sensor Mode.
Put a slit into the setup.
Select the channel with a proper laser under the Channels tab and set the laser power to an appropriate value.
Select “Continuous Scan” from the dropdown next to the Acquire button. Press Acquire. This will launch a live acquisition mode.
If needed, slowly adjust the slit opening until the image sharpness looks uniform across the whole field of view. Uncheck Autoscale in Camera View under LUT and adjust the Min Counts and Max Counts if needed.
Go to Stage Control, set the Z position in Stage Positions to be
0.Find the region of interest by using the joystick or using the controls in the Stage Control tab.
Move along the Z axis with the joystick or the “Focus” in the Stage Control tab to one end of the region of interest. Under the Channels tab, in Stack Acquisition Settings (um), press Set Start Pos/Foc.
Go to Stage Control, change the Z position in Stage Positions to set the scan range. Be aware the range for z-piezo is 0 - 200. Going outside of the range will cause the stage to have issues.
Go back to Channels tab, in Stack Acquisition Settings (um), press Set End Pos/Foc.
Setup Step Size under the Channels, recommend 3.0 (CT-ASLM-V1) and 1.0 (CT-ASLM-V2).
Under the Channels, make sure Enable is unchecked under Multi-Position Acquisition.
Under the Channels, make sure Save Data is checked under Timepoint Settings.
Select “Z-Stack” from the dropdown next to the Acquire button. Press Acquire. A popup named File Saving Dialog will appear.
Fill out the fields and press Acquire Data.
Imaging a Z-Stack with ASLM mode
ASLM mode is the high-resolution light sheet mode, it gives leas signal but offering around 950 nm (CT-ASLM-V1) and 480 nm (CT-ASLM-V2) isotropic resolution.
Switch the slit out of the setup.
Go to Camera Settings, select “Light-Sheet” under Sensor Mode.
Select the channel with a proper laser under the Channels tab and set the laser power to an appropriate value.
Select “Continuous Scan” from the dropdown next to the Acquire button. Press Acquire. This will launch a live acquisition mode.
Go to the . A popup named Waveform Parameter Settings will appear.
Uncheck Autoscale in Camera View under LUT and adjust the Min Counts and Max Counts if needed.
Set the wavelength’s Amplitude to
0.0.Adjust the wavelength’s Offset so the focus part of the image can be located perfectly in the center of the field of view.
Slowly adjust the wavelength’s Amplitude so it will be uniform across the whole field of view.
Adjust the wavelength’s Offset again slightly and make sure it is uniformly in focus across the whole field of view.
Go to Stage Control, set the Z position in Stage Positions to be
0.Find the region of interest by using the joystick or using the controls in the Stage Control tab.
Move along the Z axis with the joystick or the “Focus” in the Stage Control tab to one end of the region of interest. Under the Channels tab, in Stack Acquistion Settings (um), press Set Start Pos/Foc.
Go to Stage Control, change the Z position in Stage Positions to set the scan range. Be aware the range for z-piezo is 0 - 200. Going outside of the range will cause the stage to have issues.
Go back to Channels tab, in Stack Acquisition Settings (um), press Set End Pos/Foc.
Setup Step Size under the Channels, recommend 0.46 (CT-ASLM-V1) and 0.2 (CT-ASLM-V2) for isotropic imaging.
Under the Channels, make sure Enable is unchecked under Multi-Position Acquisition.
Under the Channels, make sure Save Data is checked under Timepoint Settings.
Select “Z-Stack” from the dropdown next to the Acquire button. Press Acquire. A popup named File Saving Dialog will appear.
Fill out the fields and press Acquire Data.
Tiling a sample larger than the field of view
This assumes you have already found the samples and ready to acquire data in either Stelzer mode or ASLM mode. (see Imaging a Z-Stack with Stelzer mode and Imaging a Z-Stack with ASLM mode).
Under Channels tab, press Launch Tiling Wizard. A popup named Multi-Position Tiling Wizard will appear.
Follow Imaging a Z-Stack with Stelzer mode to set up the start and end positions in Stack Acquisition Settings (um). At the same time, when pressing Set Start Pos/Foc to set up the start position, go to Multi-Position Tiling Wizard and press Set Z Start. When pressing Set End Pos/Foc to set up the end position, go to Multi-Position Tiling Wizard and press Set Z End.
Move the joystick or the “X Movement” in the Stage Control tab to the lower bound of the x-axis and press Set X Start in the Multi-PositionTiling Wizard popup. Navigate to the upper bound of the x-axis and press Set X End in the Multi-Position Tiling Wizard popup. Repeat for all axes except for z.
Press Populate Multi-Position Table. Navigate to the Multiposition tab and ensure the locations populated.
Under the Channels, make sure Enable is checked under Multi-Position Acquisition.
Under the Channels, make sure Save Data is checked under Timepoint Settings.
Select “Z-Stack” from the dropdown next to the Acquire button. Press Acquire.
Enter the sample parameters in the File Saving Dialog that pops up. Press Acquire Data.