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Which microscope is best for my experiments?

Broad comparison

+Worst ++Better +++Even better ++++Best

Widefield microscopy illuminates the whole sample with light and collects the image onto a camera. 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 new Nikon ones, the Leica DMi8 and our EVOS is a very basic multi-user widefield.

Do you need high-resolution/super-resolution?

If you need better resolution than on a point scanning confocal you have a few options. See this review for a nice overview of the main super resolution techniques for cell biology.

Below 90 nm

To get below 90 nm (laterally/XY) requires a bigger investment in time/effort and at Warwick usually means a SMLM technique. Labs that are doing this are Nicole Robb with some ONI microscopes, Rob Cross and Nick Carter with a custom built setup, and in SLS Seamus Holden on custom setups. Alternatively, expansion microscopy is often an easier approach for biology labs and doesn't require specialist microscopes, Sam Dean group are doing this routinely. See here for an expansion microscopy review and here for a recent review on combining expansion microscopy with super resolution microscopy.

Around 100 nm

If you only need around 100 nm resolution then you have more microscope options. The only multi-user CAMDU microscope that can achieve this is the Nikon NSPARC that is part of the BioAFM system. Other WMS microscopes capable of around 100 nm resolution are the Royle lab's Nikon SoRa spinning disk and Unnikrishnan lab's Zeiss Airyscan 2. In SLS the light microscopy RTP has the Zeiss Elyra SIM and the Zeiss Airyscan. These all have their own strengths and weaknesses, see here for a technical review. In theory, choose the Zeiss Elyra SIM for the highest resolution and maybe the NSPARC to image deep. For live samples, I'm not sure which method is the best for speed and gentle imaging, possibly the Elyra SIM is more gentle and the Nikon SoRa is faster. Let me know if you know about this.

Good sample preparation is particularly important for all these techniques and includes using high-quality coverslips (#1.5H) and the appropriate mounting media. As always, you want lots of specific signal and low background, so optimising staining protocols is a must.

Comparison of super resolution techniques achieving around 100 nm

See the below table for comparison of Warwick microscopes that can achieve around 100 nm resolution. Some details were taken from this review, and some from the manufacturers. It's not easy making fair comparisons between different technologies so don't take it too literally, and sometimes these numbers are derived from super bright samples so may not be possible on a less optimal sample. If you want live imaging then this adds extra difficulty due to photo-toxicity and the need for bright labelling.

A note on naming: the Zeiss Lattice SIM is a completely different microscope to the Zeiss Lattice Lightsheet 7. The former is for super resolution (SIM, structured illumination microscopy) and the latter is for gentle/fast imaging (lightsheet microscopy). The Zeiss Lattice Lightsheet 7 is the Zeiss version of our 3i Lattice LightSheet Microscope. Another completely different microscopes is the Zeiss Lightsheet 7, which is for much larger samples (embryos to mouse brains), and in the same class as Bruker's MuVi SPIM.

Microscope Technique Lateral resolution (nm) Axial resolution (nm) Maximum imaging speed Maximum imaging depth Reference
Zeiss Elyra 7 with Lattice SIM SIM 120 300 5-50 fps maybe? Not sure, possibly the worst here

Zeiss whitepaper 2018

Zeiss Elyra 7 with Lattice SIM2 SIM plus further deconvolution 60 200 5-50 fps maybe? Not sure, possibly the worst here

Zeiss whitepaper 2021

Zeiss LSM 980 Airyscan Imaging scanning microscopy 120 350 ~4.7 fps. Can go to 47.5 fps at lower resolution Maybe <110 μm Zeiss whitepaper 2019
Zeiss LSM 980 Airyscan (jDCV) Imaging scanning microscopy plus further deconvolution 90 270 ~4.7 fps. Can go to 47.5 fps at lower resolution Maybe <110 μm Zeiss whitepaper, "Practical guide to deconvolution" 2021
Nikon AX R NSPARC Imaging scanning microscopy 100 300 Possibly faster than Airyscan due to resonant scanners >170 μm Nikon website and review.
Nikon SoRa Spinning disk optical reassignment 120 300 Fastest here, ~100-180fps Probably in between SIM and Airyscan/NSPARC Yokogawa website

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