Table 1.
Light microscopy techniques described in this review
| Microscopy technique | Principle | Advantages and limitations |
|---|---|---|
| Confocal Laser Scanning Microscopy (CLSM) | The sample is scanned point-by-point by a focused laser beam (raster scanning), out-of-focus signal is removed by an adjustable iris (i.e. pinhole), and an image is built up pixel-by-pixel by collecting the emitted light via sensitive point detectors (e.g. PMTs) | Versatile technique, as it works with both thick and thin samples and with many different objective magnifications (i.e. variable pinholes), can produce thin optical sections, can spectrally separate different fluorophores, and the focused laser beam is compatible with photoactivation or targeted photobleaching. However, the application of an intense and focused laser beam can lead to photodamage and photobleaching, and scanning the entire sample in 3D is relatively slow |
| Spinning Disk Confocal Microscopy (SDCM) | Excitation light passes through a series of pinholes on a rotating disk so that only the imaged pixels are illuminated at a given time, out-of-focus light is also removed by those pinholes and light is collected in parallel on sensitive array detector(s) (e.g. EMCCD or sCMOS camera) | Faster and more gentle imaging than CLSM at the expense of z-resolution (i.e. optical section not as thin as with CLSM), difficult to perform spectral imaging, need additional dedicated equipment for photomanipulation. Not as versatile as CLSM because it has fixed pinholes that are not adjustable to various objective magnifications |
| TIRFM | The laser beam hits the coverslip/medium interface at a critical angle, leading to its total refraction, which locally emits a shallow evanescent wave (∼100–200 nm). As a result, only the portion of the cell in close contact with the coverslip is illuminated | Because there is no out-of-focus light, TIRF microscopes can be coupled with highly sensitive cameras, thereby allowing very fast acquisition as well as single molecule imaging. TIRFM increases the resolution in depth (basically determined by the thickness of the evanescent wave); however, this technique is limited to the cell cortex |
| Variable Angle Epifluorescence Microscopy (VAEM/VA-TIRF) | Variation of the TIRF technique that uses a subcritical angle for the laser beam, which does not lead to total refraction, but instead partial (inclined) illumination of the sample | VA-TIRF/VAEM is sometimes referred to as the “dirty” TIRF technique. It is a compromise between a deeper excitation into the sample and a less contrasted image |
| Light Sheet Fluorescence Microscopy (LSFM) | The whole field-of-view is illuminated by a laser light sheet (i.e. thin slice of light of a few hundreds of nanometers to few micrometers) perpendicularly to the direction of the detection | LSFM is very rapid and gentle in terms of phototoxicity and photobleaching, thus it allows long term imaging, or fast 4D imaging. Like SDCM, LSFM cannot perform spectral imaging easily and needs additional dedicated equipment for photomanipulation. Sample mounting can be difficult and often highly specialized, which means that LSF microscopes are often dedicated to specific applications and not highly versatile |
| Two-photon Excitation Microscopy (TPEM) | Simultaneous excitation of a fluorophore by two photons with longer wavelength than the emitted light. It typically uses tunable femtosecond pulsed laser with a raster scanning as in CLSM | Two-photon microscopy is used for deep tissue imaging, as near infrared light minimize scattering in the tissue and only the fluorophores in the focal plan are activated. High laser energy can destroy the cell by overheating, which is a potential drawback, but it can be used to generate very precise cell ablation deep in the tissue. Many dyes are excited by the same wavelength in TPEM, which can generate strong background and reduces the choice of fluorophores for multicolor imaging |
| PALM | Super-resolution microscopy technique based on stochastic activation of photo-activatable fluorescent proteins, which allows their precise localization. Images are reconstituted by iterative cycles of activation, acquisition, and photobleaching | PALM has a very high lateral resolution (∼20–30 nm) and is a single molecule imaging technique (as such, it is often performed in TIRF, which is a very sensitive imaging technique). However, it is very slow because it requires iterative image acquisition, and the cells receive a lot of laser power (photodamage). It also requires dedicated transgenic lines expressing photo-activatable or photo-switchable fluorescent protein fusions, and multicolor imaging is limited. PALM also requires a lot of post-acquisition processing |
| Structured Illumination Microscopy (SIM) | Super-resolution imaging technique that uses interference patterns created by a grid. It requires several images (with translations and rotations of the grid) and post-processing to compute a super-resolved image | SIM roughly double the resolution limit of light microscopy (∼120 nm laterally, 300 nm axially). It can be done in 3D and with multiple fluorophores and is compatible with classical fluorescent proteins. Because several images need to be acquired, it can be slow, it requires image post-processing and somewhat long illumination time (photobleaching). The increase in resolution is not as high as in PALM. Note that it can be coupled with TIRF (TIRF-SIM) to increase the contrast |
| SCLIM | Spinning disk microscopy with several paralleled array detectors and post processing (i.e. deconvolution) | SCLIM is equipped with three array detectors (i.e. cameras), and as such it is fast and can acquire several channels simultaneously, making it a solution of choice to study rapid processes such as membrane trafficking. However, it relies heavily on image post-processing, and the increase in lateral resolution is due to the deconvolution algorithm and is thus modest |
| Stimulated Emission Depletion (STED) microscopy | Scanning of the sample by two different laser pulses: a first excitation pulse (excitation laser), and a second doughnut-shaped pulse (depletion laser) for the selective deactivation of the fluorophore. The focal spot is raster scanned, like in CLSM | Lateral resolution of ∼50–70 nm (>500 nm axially), can be rapid but in a small field-of-view, deep imaging compared with other super-resolution techniques (10- to 15-µm deep) and does not require image post-processing. Has not been extensively used in live imaging in plants, likely due to high phototoxicity (high-intensity depletion laser) and photobleaching |
| Fluorescence Recovery After Photobleaching (FRAP) | Technique used to study fluorescent molecule diffusion based on the bleaching of a population of fluorophores and the subsequent quantitative analysis of the fluorescence recovery. | FRAP is a popular technique to study molecule diffusion because it can be performed on most CLSM and with standard fluorescent protein fusions. It provides information on the diffusion of an ensemble of molecules, but diffusion coefficient calculation requires complicated models (and thus is quite indirect). |
| Single Particle Tracking (SPT) | Technique aiming at tracking single fluorescent particles (e.g. single molecules or single objects such as vesicles or microtubule tips) to analyze their dynamics. Can be coupled with PALM (i.e. sptPALM) to obtain super-resolved localization of diffusing individual molecules | SPT techniques are a direct measure of diffusion and they tend to be very accurate for relatively slow diffusing molecules/structures compared to other techniques. They rely on complex image post-processing: automated tracking algorithms. These algorithms work well only if individual structures are well-defined/isolated from each other |
| FRET | Energy transfer between a donor and acceptor fluorescent protein that happens when they are in close proximity (i.e. less than 10 nm) and at the correct orientation with respect to each other | FRET is typically used as a ruler to study molecular proximity, for example to study protein-protein interactions, or intramolecular conformational changes in the case of ratiometric biosensors. It is a very powerful technique, as it can detect dynamic molecular interactions in vivo. FRET can be measured on a variety of microscopes (including CLSM and widefield microscopy). However, it is difficult to accurately measure in practice. In addition, it is difficult to predict a priori how well FRET will work between two interacting molecules, and it has to be tested empirically |
| FLIM | Technique based on the exponential decay rate of a fluorophore, which requires the use of a pulsed illumination source | FLIM is often used to accurately measure FRET, since the fluorescent lifetime of the donor decreases upon energy transfer. It can also be used to differentiate fluorophores with otherwise overlapping spectra and can (for example) help to filter out autofluorescence. Although they are becoming more and more accessible, most FLIM systems are complex to use both in terms of image acquisition and analyses |