Choosing the imaging modality before the instrument: why widefield microscopy collects light from the whole depth of the sample and why that is an advantage for thin, dim specimens and a problem for thick ones, when deconvolution recovers what a confocal would have rejected, where a darkfield microscope, a stereo microscope with digital camera, a stereo microscope camera or stereo microscope with camera arrangement, a digital stereo microscope and a digital microscope with camera answer contrast and working distance questions rather than resolution ones, what a digital microscope camera or microscope digital camera contributes to digital microscopy generally, what a microscope table or isolation platform contributes to any of them, and what has to be true of the sample before a modality choice can be made at all

Choosing the modality

1. Start from thickness and brightness. Thin and dim favours widefield with a sensitive camera, because it collects every photon. Thick favours optical sectioning. That single question settles most of the decision.
2. Consider deconvolution before buying sectioning. Computational deconvolution of a widefield stack recovers much of what sectioning would have rejected, at no cost in photons. For light sensitive samples it is frequently the better route.
3. Match contrast method to the specimen. Unstained, transparent and reflective samples need contrast rather than resolution: phase, differential interference, darkfield or reflected illumination. No amount of camera improves a specimen with no contrast.
4. Specify working distance for manipulation. Where the sample has to be reached, dissected or injected, working distance and stand geometry matter more than numerical aperture.
5. Isolate the stand where it needs it. Vibration limits resolution and long acquisitions more than most people expect. An isolation table is cheap next to the instrument and is frequently omitted.

Photons are the budget

Every modality is a way of spending a limited number of photons the sample can emit before it bleaches or dies. Sectioning throws some away in exchange for clarity; widefield keeps them and accepts blur.

Frame the choice that way and it usually settles itself, particularly for live and dim samples.

Contrast is a separate axis

Resolution and contrast are different problems, and unstained biological material is usually contrast limited. Phase, interference and darkfield methods address that directly and cost far less than a resolution upgrade.

Try a contrast method on the actual specimen before concluding that a better objective is needed.

Common questions

Is confocal always better than widefield?

No. Confocal rejects out of focus light, which is essential in thick samples and wasteful in thin ones, where widefield with a sensitive camera collects far more signal per unit of illumination.

What does deconvolution actually do?

It reassigns blurred light computationally using a model of the optics, which sharpens a widefield stack. It works well on well sampled data with a good point spread function and cannot invent information.

When is darkfield useful?

For unstained specimens and small scattering objects, where contrast rather than resolution is the limitation. It is a contrast technique and not a resolution improvement.

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