CTAC ICBR SOP: Conducting a Z-stack Scan Using the Leica TCS SP5 Confocal Microscope

Materials Required:

  • LCS software
  • Computer
  • Leica TCS SP5 Confocal Microscope

Purpose:

To facilitate operation of the Leica TCS SP5 Confocal Microscope in conducting a Z-stack operation on a sample.

 

To perform a Z-stack Operation

Z stack allows for multiple optical sections to be cut through the structure of interest.

 

Open the “Z stack” window on the left side of the screen.  You should see a representation of a cube with two boxes labeled “Begin” and “End”.

Z Stack Window

  1. Click the “Live” button. (It will change to stop and be highlighted). Adjust the “Gain” and “Offset” if not done previously.
  1. The “Z POS” knob is used to set the beginning and end positions of the area to be sectioned. Turn “Z POS” knob to the right until the fluorescence starts to disappear.  This is the part of the sample closest to the coverslip.
  1. Click the black box labeled “Begin”. Turn “Z POS” knob to the left to find the end of the sample, when fluorescence begins to fade again.  This is the part of the sample closest to the slide. Click the “End” box.
  1. Click the “Stop” button to stop the scan.  The software will suggest the optimum number of sections for your chosen volume of sample when the “System Optimized” button is highlighted. This will often be more sections than you want. You can click the button “Nr. of Steps”, and enter the number of sections that you want to scan or enter the desired section size in “Z-step size” and the software will calculate the number of sections needed. Remember to set scan for “Line Average”, “Frame Average” or “Accumulations” if you need these.
  1. Click on the “Start” button to begin a Z stack series.

To view the series of sections click on the  button on the right side of the working window (right monitor screen).

The Z stack scan is finished when the “Start” button reappears.

 

IMPORTANT:  When you are finished return the Argon laser power to 0% and clean the objectives used with clean lens tissue paper.