Widefield microscopy illuminates the whole sample with light and collects the fluorescence onto a camera. In general, widefield microscopy provides good images of "thin" samples. Widefield is generally easy to use and the efficient collection of light means it can be better than a spinning disk or point scanning confocals for dim samples. For thicker samples, the lack of optical sectioning means a widefield microscope might not be the best option. Defining what "thin" and "thick" means is a little tricky as it depends on the sample, but generally monolayers of cells are OK but you may want to use a different microscope for samples thicker than about 5-15 μm. Widefield images can be improved with deconvolution post-processing to reduce the amount of out of focus "blur" and also increase the resolution and contrast.
Multi-user widefields are the Nikon ones, the Leica DMi8 and the EVOS.
Spinning disk confocal uses multiple points of laser light to illuminate the sample and multiple pinholes to eliminate out-of-focus light. Spinning disks are generally used for high-speed, high resolution, optically sectioned imaging. They do, however, suffer from more out of focus light compared to point-scanning confocals in thick samples. It is hard to say what "thick" means as it depends on your sample and the needed quality of your images, but maybe above 30-50 μm you may consider using a point scanning confocal instead.
Point scanning confocal uses a single point of light to illuminate the sample and collects the fluorescence through a pinhole which blocks the out of focus light. The point of light is scanned or rastered across the sample (swept from side to side) to obtain an image. This provides superior optical sectioning compared to the spinning disk confocal, it blocks more out of focus light, especially deep into a sample. However, it is slower and has more photo-toxicity/bleaching.
Our multi-user point scanning confocals are Evident's FV3000 and the BioAFM.
Total internal reflection fluorescence microscopy (TIRFM) uses an evanescent wave to illuminate the bottom ~100 nm of the sample. It is excellent at preventing out of focus blur but is only suitable for 2D imaging close to the coverslip.
Lattice LightSheet Microscopy only illuminates the part of the sample that is in the focal plane of the detection objective, unlike confocals and widefields that expose the sample to light above and below the focal plane. This drastically reduces the photo-toxicity/bleaching for lightsheets and allows much faster imaging. The Lattice LightSheet Microscope uses a particularly thin sheet and provides "comparable" resolution to a confocal. Generally, our Lattice LightSheet Microscope is used when your sample is struggling with photo-toxicity/bleaching on a spinning disk. For fixed samples it is usually best to use a different microscope, with the exception being expansion microscopy where the labelling can be dim and easily bleached.
Multiphoton microscopy allows imaging at much greater depth into samples than confocal microscopy. It is the gold-standard for imaging in thick tissue. Once again, it is hard to say at what thickness you should consider multiphoton over confocal microscopy as it depends on your sample and what you need from the images. Very roughly, if you live sample is thicker than 40-100 μm you may consider using the multiphoton, but you could test both and compare. Similarly to point scanning confocals, the multiphoton scans a point across the sample and so is slower than spinning disk and widefield. For fixed samples, it is often better to use a point scanning confocal than the multiphoton - if you can prepare your sample appropriately the confocal will likely be just as good or better. This refers to appropriate mounting media and/or clearing methods.
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