The resolution of a microscope can be defined as the smallest distance at which two small objects can still be seen as separate objects. This lecture discusses various criteria for resolution, the factors that influence resolution in the lateral and axial planes, and how to sample an image adequately using a camera or confocal microscope, such that the full optical resolution is retained.
- You have a 100×1.4na objective, no additional magnification in your microscope and can choose between 4 different cameras, varying in pixel size. Which camera should you choose to fulfill the Nyquist sampling criterium, assuming that you resolution limit is described by the Rayleigh criterium and you work with light of 500nm?
- 16 micron pixels
- 10 micron pixels
- 6.5 micron pixels
- 4.0 micron pixels
- You use a 40×1.4na objective on your confocal microscope and are imaging a red fluorescent protein emitting at ~575nm. You are using an image size of 512×512 pixels. To satisfy Nyquist sampling (using the Rayleigh criterium for resolution) you should scan an area no larger than:
- 64×64 mm
- 128×128 microns
- 64×64 microns
- 40×40 microns
- Which change(s) in your imaging setup will increase the resolution of your sample for GFP fluorescence?
- Higher wavelength
- Higher refractive index of immersion medium
- Lens with higher numerical aperture
- Lens with lower numerical aperture
- Lens with better chromatic correction
Jeff Lichtman’s interest in how specific neuronal connections are made and maintained began while he was a MD-PhD student at Washington University in Saint Louis. Lichtman remained at Washington University for nearly 30 years. In 2004, he moved to Harvard University where he is Professor of Molecular and Cellular Biology and a member of the… Continue Reading