Which microscope is best suited for your application?
- First, how thick is your sample?
- Second, is it living and moving: do you need to record dynamic events?
Here are the short, general, answers to which microscope to use:
- Thick samples (10 – 70 µm) are best imaged using a point-scanning confocal.
- Thin samples (1 – 10 µm) are best imaged using a widefield deconvolution microscope.
- Photo-sensitive samples should be imaged using the structured illumination microscope.
- Very sensitive samples should be imaged using the EM-CCD on the live-cell microscope.
- Very thick tissue (70 – 250 µm) should be imaged using a multi-photon microscope.
- Moving samples should be imaged with a Nipkow spinning-disc confocal or a dedicated live-cell microscope.
Points to consider:
High NA objectives (normally also high magnification objectives) have a limited depth of field – the distance between the upper and lower planes of the in-focus region – which is often a micrometer or less. Fluorescently-stained specimens are self-luminous. Thus specimens having a thickness greater than about 5 µm will produce images in which most of the light is contributed by regions that are not in exact focus. Hence the need to optically section your sample.
- Do you need live cell imaging – inverted systems are best for this. Can your (fixed) sample be imaged inverted?
- If the sample is fluid, image with Petri-dishes or well-chambers with a coverslip bottom.
- Do you need to provide heating and/or CO2 over any length of time?
- Do your fluorescent probes match the available lasers and/or filter blocks?
- Better optical sectioning in LSCM than WFM.
- If using LSCM, overstain thicker samples to provide better S/N
- If zooming is important, use the LSCM.
- Use the widefield microscopes DeltaVision/Optigrid/live-cell) if your sample is photo-sensitive.
- WFM with deconvolution is less good for rapidly moving samples.
- Consider fluorescent protein overlap – LSCM good at spectral separation
- EM CCD on Leica live-cell fastest instrument but it has limited fluorophore sets
- Have you got a lot of slides to process – consider the Optigrid.
There is always a trade-off between photo-damage and collecting a high S/N ratio. Or, to put it another way: between (a) acquisition speed (b) sensitivity and (c) resolution. See the ‘iron triangle’ in the sidebar on the live-cell imaging page.
A gain in speed involves a sacrifice of both speed and resolution, and so on.
For more a more detailed answer on which microscope to use, read the bullet point list given in the downloadable PDF file (see right-hand box).