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Choosing the microscope

Choosing the right fluorescent microscope for your application

While designing your experiment for fluorescence microscopy there is few questions you should ask yourself, here are some of them:

  • How deep in your sample do you need to image? How thick is your sample?
  • Is your sample live or fixed? If you want to do live experiment, are the microscopes around you equipped with environmental control?
  • What are the excitations and emissions wavelengths of your fluorophore? Are the microscopes available able to image these wavelengths?
  • How fast is the event you are trying to capture with the microscope?
  • How small are the objects you are trying to see? What resolution do you need?

Here is a general guideline for the different fluorescence microscopes that are commonly available in facilities:

Microscope Sample Thickness Living sample Speed Sensitivity
TIRF ~100nm +++ FAST ++
Widefield 1-10μm +++ FAST +
Widefield with deconvolution 5-20μm +++ FAST ++
Spinning disk confocal 10-50μm +++ HIGH ++
Laser scanning confocal 10-100μm + SLOW +++
Two-photon 10-100μm ++ SLOW ++
Lattice Light sheet >1mm +++ HIGH ++++

 

 

Sample thickness

This has nothing to do with how deep one can image (except for the light sheet microscope), how deep one is able to image depend mainly on the working distance of your objective. The sample thickness given here is an indication on which technique you should use depending of the thickness of the sample. However, how deep one will be able to image with a specific technique will also be dependent on your signal to noise ratio. For example, you can often image up to few hundreds microns without the need of a 2-photon if one has a "cleared" sample. Or, if one grow a single layer of cells on top of a 100μm transparent gel, you could probably image them with a wide-field microscope. If there is a good signal-to-noise, a spinning disk confocal will probably not work very well with a 50μm thick sample. In conclusion, try different technique to see which one is the appropriate for your sample.

Speed

All point scanning systems are inherently slower because one images sample point by point and scanner speed. For the other instruments the speed is dictated by the camera frame rates. Other parts of the microscope should also be considered. For example, to acquire multi-colors images, how fast can the microscope switch between the different colors? For a Z-stack, is there a Z-piezo? Can the software trigger the different part of the microscope? etc.

Sensitivity

  1. From the photons emitted by a fluorophore, how many reached the detectors. In a widefield microscope, this is mainly dependent on the light-gathering power of your objective where M is the magnification and NA the numerical aperture. In a confocal microscope, you also have to take into account that a lot of the light is rejected by the pinhole.

  2. The quantum efficiency of your detector, or its ability to convert photons into photon-electrons.