Widefield microscopy and choosing an imaging modality

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, and a dark field microscope is one answer

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.

A c mount microscope camera and the adapter behind it

A c mount microscope camera uses a standard thread, which is what makes cameras interchangeable, and the part that decides the image is the adapter between the port and the sensor, since it sets the magnification and whether the field is filled. A sensor larger than the port's field will vignette however good it is.

A microscope video camera and what it is for

A microscope video camera is chosen for frame rate and live latency rather than for pixel count, because its job is watching and teaching rather than measuring, and a camera that is sharp at one frame a second is useless for following a moving specimen. Where measurement matters, exposure control and a stated pixel size are the specifications.

Common questions

Is confocal always better than wide field microscopy?

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 a darkfield microscope 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.

Browse by service class

• Compare Gene synthesis on advertised prices