• Nico Stuurman shows us the technique of measuring a point spread function – how to prepare a bead slide and image the beads in a microscope to obtain its point spread function.

• It is important to know how to clean objective lenses because dirt and residue on the surface of objective lenses and filters can degrade image quality. This tip describes how to inspect lenses and filters for dirt and how to clean them properly with solvent so that these expensive components will not be damaged in the process.

• Focusing is the first step in microscopy. This video provides some tips on how to find the right focal plane.

• Koehler illumination is the prefered method for obtaining even illumination of the specimen and is a critical alignment procedure in microscopy. This lab goes through a step-by-step procedure of aligning the lamp and condenser to achieve Koehler illumination.

• To image living cells with a microscope, they must stay alive. Here we discuss how to control the temperature, humidity, and atmosphere (CO2 concentration) around your cells so that they survive during live cell imaging. We discuss both simple and more complex systems for environmental control.

• Ron Vale illustrates how illumination of a specimen can cause photobleaching and protein damage and gives us a tip about minimizing damage from fluorescence. Ways to minimize such problems include strategies for minimizing light exposure and removing molecular oxygen from in vitro reactions.

• In this video about camera calibration, Nico Stuurman shows you how to measure a camera dark image, calculate the readout noise, estimate the dark current, get a camera flat field image using a plastic cup and a cell phone screen, and estimate the photon conversion factor and full well capacity by measuring a photon transfer curve.

Please contact Dr. Ulrike Boehm (boehmu at janelia.hhmi.org) if you would like to add an additional useful microscopy tip and / or trick.