Light Microscopy Techniques for Live Cell Imaging

Abstract

Since the earliest examination of cellular structures, biologists have been fascinated by observing cells using light microscopy. The advent of fluorescent labeling technologies plus the plethora of sophisticated light microscope techniques now available make studying dynamic processes in living cells almost commonplace. For anyone new to this area, however, it can be daunting to decide which techniques or equipment to try. Here, we aim to give a brief overview of the main approaches to live cell imaging, with some mention of their pros and cons.

Virus trafficking – learning from single-virus tracking

“…In the figure, part (b) shows imaging geometries. In the epi-fluorescence geometry (left panel), a collimated light beam illuminates a large sample area (~ 100 μm in linear dimension) 81 . Fluorescence emission from the sample is collected by a high numerical aperture objective and detected by a CCD camera.…”

“…Confocal microscopy (middle panel) uses a focused light beam and spatial filtering techniques (pinholes in excitation and detection paths) to eliminate out-of-focus background fluorescence, but at the cost of signal reduction 82 . For the observation of fast dynamics in live cells, a spinning disk confocal setup is often preferred over the relatively slow scanning confocal microscope, which relies on rotating mirrors to scan a focused laser beam in the imaging plane and a point detector, such as avalanche photodiode or photomultiplier tubes, for signal acquisition 81 . The spinning disk confocal microscope relies on a pair of rapidly rotating discs, one with an array of pinholes and the other with correspondingly aligned micro-lenses.…”

Oxidized-1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine induces vascular endothelial superoxide production: Implication of NADPH oxidase

“…NADPH and NADH are collectively referred to as NAD(P)H [16], and emit a broadband blue fluorescence centered at 450-460 nm while their oxidized forms, namely NAD + and NADP + , are not fluorescent [17]. In this context, the fluorescence properties of NAD(P)H permit identification and characterization of NADPH levels in the presence of agents that change the cellular redox state [18,19].…”

Comparing video‐rate STED nanoscopy and confocal microscopy of living neurons

“…In particular, studying dynamics opened up an enormous field of application not only in biology [1] but also in specific areas in the material sciences [2]. However, one drawback of standard optical microscopy is that the resolution is limited to 200-300 nm in the focal plane, due to the wave nature of light [3][4][5].…”