Collecting a Z-Series
Use the "ZPOS" knob on the far right of the panel box to focus.
Focus clockwise to the plane where you want to start the zseries.
Click on the "Begin" button.
Use the "ZPOS" knob to focus counterclockwise through the sample to where you want the zseries to end. As you focus check that gain and offset are adequate throughout the sample. Adjust if necessary.
Click on the "End" button.
Select the #Sect. button and click on "user defined" from the menu.
Type in the step size. Then select calculate. The program will determine the number of sections from the thickness defined when focusing with ZPOS and the step size. Adjust if needed.
Set the number of accumulations for each image.
When ready to collect images, use the "Series" button.
Sequential scans may be useful in the following situations:
First select each fluorophore individually from the available methods. Check the parameters in the "filters menu".
Select one dichroic for ALL of the fluorophores - never change the dichroic when collecting multiple labels. Select the DD (dual) dichroic for samples using 488 and 568 excitation. Use the TD (triple) dichroic for samples using any 633 excitation.
Scan the sample and adjust the gain, black level, pinhole, etc. to optimize the image for each wavelength to be used.
Save each method.
For example, select 1.FITC and scan the sample. Adjust the gain, black level, pinhole, etc. to optimize the image. Save the method. Do the same with the 1.TRITC and 1.CY5 settings. Use only one laser at a time.
Click on the "Seq." button in the filter menu to open the sequential scan settings menu.
Example of a sequential scan collecting triple labels with the confocal:
Select the fluorophores from the available methods list.
For example, select a FITC method and add it to the sequential methods column by clicking on the Add button. Then select and add a TRITC method and a CY5 method.
The first selection will be "channel 1" and green, the second method "channel 2" and red and the third choice will be "channel 3" and blue.
Check the "Activate sequential scan" box at the top of the menu. (The "Activate sequential scan" box must be unchecked to collect individual methods.)
Select "add channels" for each seq. method. When collecting a z-series, the method can be changed between stacks or between frames (usually better).
Activate stored method parameters should all be checked (except filterwheels - don't change the dichroic for multiple labeling).
Close the menu.
When ready to collect images, use the "Series" button.
Example of a sequential scan combining 2-photon acquisition with confocal imaging:
The acquisition parameters of each method should have been selected and saved as in the example for triple labels above.
Remember to use the same dichroic for both confocal and 2-photon acquisition. Open the pinhole in the 2P method and use an appropriate pinhole for the confocal method (0.5-1.3?).
Select the fluorophores/method from the available list.
For example, if collecting a double FITC/TRITC confocal image and a DAPI 2-photon image, select the 1.FITC/TRITC method and add it to the sequential methods column by clicking on the Add button. Then select and add the 2P-DAPI method.
Channel 1 will be the green, FITC image and channel 2 will be the red, TRITC image. The blue, 2P-DAPI image will be in channel 3.
Check the "Activate sequential scan" box at the top of the menu. (The "Activate sequential scan" box must be unchecked to collect individual methods using the series button.)
Select "add channels" for each seq. method. When collecting a z-series, the method can be changed between stacks or between frames (usually better).
Activate stored method parameters should all be checked.
Close the menu.
When ready to collect images, use the "Series" button.