A quick guide to light microscopy in cell biology

Abstract

Light microscopy is a key tool in modern cell biology. Light microscopy has several features that make it ideally suited for imaging biology in living cells: the resolution is well-matched to the sizes of subcellular structures, a diverse range of available fluorescent probes makes it possible to mark proteins, organelles, and other structures for imaging, and the relatively nonperturbing nature of light means that living cells can be imaged for long periods of time to follow their dynamics. Here I provide a brief introduction to using light microscopy in cell biology, with particular emphasis on factors to be considered when starting microscopy experiments.

FIGURE 1:

Schematic drawings of common microscopy techniques. (A) An inverted epifluorescence microscope. The sample sits between the slide and coverslip. The condenser lens delivers illumination for viewing light transmitted through the sample; the objective lens collects light from the sample and delivers excitation light for fluorescence microscopy. The filter cube consists of an excitation filter (blue), an emission filter (green), and a dichroic mirror (gray). The excitation and emission filters select the wavelengths that will illuminate the sample and be recorded on the camera, respectively, and the dichroic mirror reflects the excitation light to the sample while transmitting the emission light to the camera. An upright microscope has the same design, just rotated by 180°. (B) A confocal microscope. The excitation and emission pinholes are imaged onto the sample to define an illuminated point in the sample and to detect light from only that point. The lenses that image the pinholes onto the sample have been omitted for simplicity. The scan mirror scans the illuminated spot across the sample; because the scan mirror is in both the excitation and emission paths, the position of the spot detected from the sample is scanned in parallel with the excitation spot. (C) A light sheet microscope. An illumination objective, along with additional optics (not shown), is used to form a thin sheet of light that illuminates the sample. A detection objective images the light emitted from this sheet onto a camera.