to set "begin" and clockwise 
to set the "end" point.Collecting a Z-Series
Z-Wide
Projecting a Z-Series
Collecting an XZ-Image
Collecting a series for 3D reconstruction
Collecting a Z-Series
Click on the series button to get the z-series menu.
Drag the yellow plane in the middle of the cube up to the start position. Alternatively, turn the z-position knob on the panel box counterclockwise (focusing toward the coverslip).
Check begin.
Drag the yellow box down to the end point of the z-series. If using the panel box z-position, turn clockwise (focusing toward the slide) to find the end point.
As you "focus" check that gain and offset are adequate throughout the sample. Adjust if necessary.
Check the end box.
Once the end points have been selected they will be displayed in red and green with the relative positions and the total distance.
The galvanometer driven stage has a travel of only 170 microns. Use z-wide for greater travel.
Select either galvo or z-wide with the z-scan button.
The microscope needs to have the upper limit set for z-wide. Use the end point to set this limit by holding in the upper limit button. This setting may need to be removed later if you can't focus the sample.
Set the focus on the panel box to z-wide
Focus counterclockwise to set "begin" and clockwise to set the "end" point.
When using z-wide it may be easier to find the endpoints by focusing with the fine focus knob on the microscope.
Select the Sect button.
Options are given for selecting the number of sections from 1-50 or selecting "Others" and getting a menu showing the # Sections and the Step size.
The optical sectioning thickness must be considered in relation to the step size for subsequent reconstructions:
-avoid using a step size that is larger than the optical sectioning thickness
-the optical sectioning thickness depends on the objective in use, the pinhole and the excitation wavelength
-to get an idea what the optimal optical section thickness is for a given objective look at the xz-resolution chart available in the preferences of the 
software
-it is better to oversample (smaller step size and more images - but more bleaching) than to undersample for subsequent 3D reconstruction. Shannon's sampling theorum would have you sample at 1/2 the smallest detail or resolution.
Change either the number of sections or the step size and "Calculate" the other value.
The minimum step size for the galvanometer driven stage (not z-wide) is 41 nanometers.
Set the number of frames to average for each image.
When ready to collect images, use the "Series" button.
Projecting a Z-Series
Project all of the images by selecting the View button and one of the projection buttons (Avg., Max., or Trans.)
Project part of a z-series by first selecting the images with ITool 
using the menu shown below and then using the projection methods shown above:
Sequential scans may be useful in the following situations:
Select each fluorophore individually from the available methods. Check the parameters in the "Beam Path Setting" menu. Use only the lasers needed for a particular method. Use one dichroic for all the methods in the sequence. 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 (offset), pinhole, etc. to optimize the image. Then save the method.
Example of setting up for a triple label confocal sequential scan: select the FITC method. Start scanning and adjust image acquisition paramenters. Stop the scan. Save the method. Then selct the TRITC method, scan, optimize the image, stop the scan and save the method. Do the same with the CY5 method.
1. Check the "Sequential Scan" box at the bottom of the Beam Path Setting menu to open the sequential scan settings menu.
2. Select each fluorophore from the available methods list.
3. Add it to the sequential methods column by clicking on the Add button.
Parameters should all be checked (except Frame-Average).
When collecting a z-series, the method can be changed between stacks or between frames (usually better). Check the selection under Mode.
When ready to collect images, use the "Series" button.
Sequential scans with 2-photon and confocal:
Sequential scans combining 2-photon acquisition with confocal imaging should be collected with the same dichroic to avoid any lateral shift between the images. Set the dichroic for the confocal images to be collected.
Change the pinhole setting for the 2-photon image so it is at the largest aperture. The pinhole is not necessary for 2-photon imaging.
Keep the ND at 6ND for the 2-photon image. If the ND filter isn't changed for the 2-photon image during the sequential scan then include this setting in the confocal parameters. It has no effect on confocal acquisition.
Collecting an XZ-Image
The "old" TCS NT software is better for many XZ applications as it lets you select the y-position of the line scanned.
Select the line for xz imaging after collecting an xy image and adjusting gain, offset, pinhole, etc. on this image. As indicated by the three arrows on the left side of the above image:
With XY selected under Mode, a white line appears on the scanned XY image (shown at right on the above image).
The line can be moved to select the object to be scanned in the xz direction. Line 1. corresponds to the first xz image below and line 2. to the second xz image below.
Before scanning, change the mode to xz. The stage drops by a preset distance to collect the image.
1.
XZ image taken at a line indicated by position 1. above. 2.
XZ image taken at a line indicated by position 2. above. Collecting a series for 3D reconstruction Read the section on collecting a z-series Additional considerations for collecting a z-series for use with Volocity on the Mac in the Keck Center:
-Collect high quality images that are bright but not saturated. Use a sufficient number of averages to minimize noise.
-Volocity reads the Leica .tif files. It is easier to save in this format.
-Images saved as raw files can't be opened directly into Volocity. They can be opened in ImageJ and saved as a .tiff stack. Note the XYZ dimensions of your images in the Leica software as this will need to be input into that file's properties in Volocity.
