Microscopic Imaging Classroom丨Application of Fluorescence Microscopy
Total internal reflection fluorescence microscopy (TIRFM) is a technique that utilizes the evanescent wave generated by a light beam propagating between two different refractive index media to probe the surface of fluorescently labeled living cells. In practice, when an incident laser beam encounters the interface between the coverslip and the aqueous medium containing the cells, it is reflected at the critical angle (total internal reflection). Because the energy of the evanescent wave decreases exponentially with the distance from the coverslip, only fluorophores within 10 nanometers of the surface (between 10 and 200 nanometers) are excited by the evanescent wave, while fluorophores farther away are largely unexcited. Affected. Thus, TIRFM results in high signal levels from fluorophores located near the coverslip, superimposed on a very dark background, providing an excellent signal-to-noise ratio. The extreme limitation of excitation depth is ideal for studying single molecules or membrane and organelle composition in adherent cells near the coverslip surface (see Figure 8(e)). Since excitation is restricted to thin regions near the coverslip, photobleaching and phototoxicity are also restricted to these regions, making TIRFM one of the most useful methods for long-term observation. The technique has become a fundamental tool for studying a wide range of phenomena in cell and molecular biology.
Deconvolution is an algorithm applied to a set of all-focus images acquired along the optical (Z) axis to enhance the photon signal in a stack of images for a given image plane or multiple focal planes. Microscopes must be equipped with high-precision motorized focus drives to guarantee image acquisition at precisely defined intervals between the focal planes of the sample. In a typical application (see Fig. 8(f)), the deconvolution process is used to deblur and remove out-of-focus light from a given focal plane using widefield fluorescence excitation and emission. The most complex application is to apply the deconvolution process to the entire image stack to generate projected views or 3D models. A set of widefield images used for deconvolution captures the theoretical maximum number of photons emitted by the sample. The deconvolution process redistributes the "blurred" intensities of photons emitted above and below the focal plane back to the original plane. Thus, deconvolution uses nearly all available emission intensities and provides an optimal light budget, making this technique the method of choice for very light-sensitive samples.
A variant of the resonance energy transfer phenomenon on fluorescence microscopy, fluorescence or Förster resonance energy transfer (FRET) is used to obtain quantitative temporal and spatial information on the binding and interaction of proteins, lipids, enzymes and nucleic acids in living cells. FRET can employ either constant-state or time-resolved techniques, but time-resolved FRET imaging has the advantage of more precisely mapping donor-acceptor distances. A standard widefield fluorescence microscope equipped with appropriate excitation and emission filters and a sensitive camera can be used for FRET imaging. Biosensors that sandwich an environmentally sensitive protein or peptide between two FRET-applicable fluorescent proteins are currently widely used in cell biology. These probes are readily imaged under wide-field fluorescence microscopy using sensitized emission FRET techniques combined with ratiometric analysis. In addition, spectral imaging and linear unmixing using laser scanning confocal microscopy can help monitor FRET phenomena in biosensors and other fluorescent protein applications.
Fluorescence lifetime imaging microscopy (FLIM) is a sophisticated technique capable of simultaneously recording the fluorescence lifetime and the spatial location of a fluorophore at every location in the image. This method provides a mechanism to study environmental parameters such as pH, ionic concentration, solvent polarity, noncovalent interactions, viscosity, and oxygen tension in single living cells and presents the data in spatial and temporal arrays. FLIM measurements of excited state lifetimes on the nanosecond scale are independent of local fluorophore concentrations, photobleaching effects, and path length (sample thickness), but are sensitive to excited state reactions such as resonance energy transfer. In fact, combining FLIM with FRET by monitoring the lifetime changes of fluorescent donors before and after resonance energy transfer is considered to be one of the best ways to study this phenomenon.
The mobility (transverse diffusion coefficient) of fluorescently labeled macromolecules and small fluorophores can be determined by fluorescence recovery after photobleaching (FRAP) technique. In FRAP, a very small selected area (a few micrometers in diameter) is intensely illuminated, usually with a laser, to produce complete photobleaching of the fluorophore in that area. The result is a dramatic reduction or annihilation of fluorescence. Following a photobleaching pulse, the rate and extent of fluorescence intensity recovery in bleached regions was monitored as a function of time at low excitation intensities to generate information on the kinetics of fluorophore repopulation and recovery (Figure 9). FRAP is usually performed using EGFP or other fluorescent proteins. Related photoactivation techniques are based on special synthetic caged fluorophores or similarly functional fluorescent proteins that can be activated by short pulses of UV or violet. Photoactivation and FRAP can be used as complementary techniques to determine mobility parameters.
In a technique related to FRAP, called fluorescence loss after photobleaching (FLIP), a defined fluorescent region within a living cell undergoes repeated photobleaching by intense irradiation. If all fluorophores were able to diffuse into the area being photobleached during the time period measured, this would result in a complete loss of fluorescent signal throughout the cell. By calculating the rate at which fluorescence disappears from the entire cell, the diffusional mobility of the target fluorophore can be determined. In addition, FLIP can readily identify the location and nature of any diffusional barriers between individual compartments of cells, such as the barrier between the soma and axon of a neuron.
Fluorescence correlation spectroscopy (FCS), mainly used in laser scanning confocal microscopy or multiphoton microscopy, is a method designed to determine kinetic information such as chemical reaction rates, diffusion coefficients, Techniques for molecular weight, flow rate and aggregation. In FCS, a small volume (approximately one femtometer; the diffraction-limited focus of the laser) is illuminated with a focused laser beam to record autofluorescence intensity fluctuations induced by the dynamics of fluorescent molecules as a function of time in the volume occupied by fluorescent molecules (Fig. 10). Relatively small fluorophores diffuse rapidly in the illuminated volume, producing short bursts of random intensity. Conversely, larger complexes (fluorophores bound to macromolecules) move more slowly, producing longer, more persistent time-dependent fluorescence intensity patterns.
When fluorescently labeled structures are densely packed and overlap in specific regions of living cells, their dynamics and spatial distribution are difficult to analyze. Fluorescence spot microscopy (FSM) is a technique compatible with almost all imaging modalities that takes advantage of very low concentrations of fluorescently labeled subunits, reduces out-of-focus fluorescence, and improves visibility of labeled structures and their dynamics in thick regions. FSMs are implemented by labeling only a fraction of the entire structure of interest. In this sense, FSM is similar to performing FCS over the entire field of view, although it places more emphasis on spatial patterns rather than quantitative temporal analysis. Fluorescent spot microscopy is particularly useful in determining the mobility and aggregation of cytoskeletal elements such as actin and microtubules in hyperactive cells.
Stimulated emission depletion microscopy (STED) is an emerging super-resolution technique with spatial resolution far beyond the diffraction limit, using ring-shaped depleted light to surround a smaller beam of excitation light to obtain sub-50 nm on-axis to the resolution. The technique relies on excitation of fluorophores with synchronized laser pulses and spatially coordinated circular STED pulses that deplete the emitted light, suppressing fluorescence of excited molecules around the laser scanning focus. Fluorescence generated at the periphery of the spot is suppressed, but not at the center of the spot, thus significantly reducing the size of the fluorescent spot and correspondingly significantly increasing the resolution. STED has proven to be a useful tool for high-resolution detection of living cells. Other emerging super-resolution techniques, such as photoactivated localization microscopy (PALM) and structured light illumination microscopy (SIM), will also become fundamental tools for live cell imaging in the near future.
The increasing use of genetically encoded fluorescent proteins and advanced synthetic fluorophores for live-cell imaging opens the door to new optical modalities for monitoring temporal dynamics and spatial relationships. Microscopists now have a complete set of tools to observe and record image data of cellular processes occurring over a wide range of timescales and at multiple resolutions. Slower events can be easily observed and recorded using laser scanning confocal microscopy, while faster kinetic events can be obtained using spinning disk technology. In addition, multiphoton microscopy enables deep imaging in thick tissues, and total internal reflection techniques enable probing of membrane surfaces with confocal precision. Advanced fluorescence methods, such as FRET, FLIM, FRAP, FCS, FSM, SIM, PALM, and STED, can be used to monitor protein-protein interactions at resolutions often better than those allowed by the diffraction limit. With advances in fluorophore, microscope and detector technology, a wider world will be brought "under the microscope".
