Microscopy Basics

NCI Optical Microscopy Laboratory

Optical Microscopy Basics

Wide-field microscopy

This is the technical term for a conventional fluorescence microscope. Typically the entire field of view of the specimen is illuminated with excitation light and then all of the emitted fluorescence is collected from the same field of view. Wide-field imaging is valuable for several reasons. A good wide-field microscope today will come with a very sensitive camera that detects very low light levels in the specimen. The instrument is also equipped with simple software that allows easy collection of images. Wide-field is often valuable in quantitative measurements since the light collected in a single focal plane contains all of the signal from the entire 3D specimen.

Confocal microscopy

The confocal microscope improves contrast in a specimen and enables optical sectioning. All of this is achieved by an altered optical design in which a point (or a series of widely spaced points) in the specimen is imaged with excitation light and then only the in-focus emitted light is detected, typically by a pinhole in front of the detector that rejects any fluorescence from out-of-focus regions of the specimen. The rejection of out-of-focus light greatly improves image contrast, and it also provides the optical equivalent of slicing the specimen into sections, hence the term “optical section”.

Deconvolution microscopy

Deconvolution is an alternative approach that also improves contrast and provides optical sectioning. Deconvolution removes out-of-focus light by obtaining a wide-field image of the specimen and then using computational methods to distinguish between the in-focus and out-of-focus light. In many cases, particularly with dimmer or light-sensitive specimens, deconvolved images are superior to confocal images. This is because in confocal microscopy the out-of-focus light is rejected, whereas in deconvolution microscopy the out-of-focus light is restored to its source of origin in the image. In addition, many deconvolution microscopes have more sensitive detectors than what is available on most confocal microscopes. However, deconvolution is somewhat more complicated to perform than obtaining a confocal image.

Two photon microscopy

Another approach to optical sectioning is two photon microscopy. In this approach the excitation light is restricted to a small diffraction limited spot in x,y and z. As such, out-of-focus regions of the specimen are not excited in the first place, so there is no out-of-focus light to be removed. Two photon microscopy is especially well suited for imaging of thicker specimens (>10 um) since there is much less scattering of the light in this technique and in many cases it is possible to image with reasonable clarity for several hundred microns into a specimen. For thinner specimens, for example, tissue culture cells there is usually not a significant advantage over confocal or deconvolution microscopy, except when imaging dyes that require excitation in the uv range. This can be accomplished more easily on a two photon