Traditional approaches to live-cell imaging are often based on short or long term time-lapse investigations designed to monitor cellular motility and dynamic events using common contrast enhancement techniques, including brightfield, differential interference contrast (DIC), Hoffman modulation contrast (HMC), phase contrast, and widefield fluorescence. However, modern techniques and newly introduced methodologies are extending these observations well beyond simply creating cinematic sequences of cell structure and function, thus enabling time-lapse imaging to be integrated with specialized modes for monitoring, measuring, and perturbing dynamic activities of tissues, cells, and subcellular structures.
A majority of the live-cell imaging investigations are conducted with adherent mammalian cells, which are positioned within 10 micrometers of the coverslip-medium interface. Increasingly, however, investigators are turning their attention to thicker animal and plant tissue specimens that can range in thickness from 10 to 200 micrometers. In virtually all live-cell imaging scenarios using widefield microscopy, out of focus information blurs the image and the constant churning of the cytoplasm creates limitations on exposure times. Both brightfield and fluorescence methods used for imaging thin as well as thicker animal tissues and plants must take into account the sensitivity of these specimens to light exposure and the problems associated with resolving features that reside more than 20 to 30 micrometers within the specimen.
Brightfield techniques are often less harmful to living cells, but methodology for observing specific proteins using transmitted illumination have not been widely developed. Generating a high-contrast chromatic (color) or intensity difference in a brightfield image is more difficult than identifying a luminous intensity change (in effect, due to fluorescence) against a dark or black background. Therefore, brightfield techniques find applications in following organelles or cell-wide behavior, while fluorescence methods, including confocal techniques, are generally used for following specific molecules.
Presented in Figure 1 is a schematic illustration of popular imaging modes in widefield and scanning modes of fluorescence microscopy. Widefield, laser scanning, spinning disk, and multiphoton techniques employ vastly different illumination and detection strategies to form an image. The diagram illustrates an adherent mammalian cell on a coverslip being illuminated with total internal reflection (TIRFM), laser scanning, multiphoton, and spinning disk confocal, in addition to traditional widefield fluorescence. The detection patterns for each technique are indicated in red overlays. In widefield, the specimen is illuminated throughout the field as well as above and below the focal plane. Each point source is spread into a shape resembling a double-inverted cone known as the point-spread function (PSF). Only the central portion of this shape resides in the focal plane with the remainder contributing to out-of-focus blur, which degrades the image.
In contrast the laser scanning, multiphoton, and spinning disk confocal microscopes scan the specimen with a tightly focused laser or arc-discharge lamp (spinning disk). The pattern of excitation is a point-spread function, but a conjugate pinhole in the optical path of the confocal microscopes prevents fluorescence originating away from the focal plane from impacting the photomultiplier or digital camera detector. The laser scanning confocal microscope has a single pinhole and a single focused laser spot that is scanned across the specimen. In the spinning disk microscope, an array of pinhole or slit apertures, in some cases fitted with microlens elements, are placed on a spinning disk such that the apertures rapidly sweep over the specimen and create an image recorded with an area array detector (digital camera). In the multiphoton microscope, the region at which photon flux is high enough to excite fluorophores with more than one photon resides at the in-focus position of the point-spread function. Thus, fluorophore excitation only occurs in focus. Because all fluorescence emanates from in-focus fluorophores, no pinhole is required and the emitted fluorescence generates a sharp, in-focus image.
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