This tutorial covers the use of the Leica SP8 confocals at the Advanced Light Microscopy Facility. The tutorial is broken up in several sections with side steps who describe specific topics in more detail.
Before you even start
Which (confocal) microscope should you use
Although this tutorial is about a confocal microscope, you should first decide if this is the best microscope to use for your experiment. To make a good choice, read the “Which Microscope to use” side step first.
To get the best results you should prepare your samples in the best way possible. Some tips and considerations can be found in the “Sample Preparation” sidestep.
Confocal Microscopy Experimental setup
- What is the detail level that you want to image (e.g. Number of Cells/Nuclei in Tissue, proteins on membrane, foci in nuclei, etc..). -> Choice of objective (10x overview of large tissue, small species, 20x details of large tissue, small species, organoide, spheroids, etc., 40x details of cell and surroundings, 63x details of singles cells)
- Noise level (a balance between keeping cells alive, bleaching, speed, dynamics, size and more), See sidestep “Fluorescent imaging trade-offs“
- Dynamics (live-cell, time-lapse)
- Tile-Scanning (large overviews with fine details)