A Microscopic View of the Store-Operated Calcium Entry-Pathway
Fluorescence microscopy techniques.
Advantages and uses
This technique measures the coexistence of signal at the same pixel location when at least two different fluorochromes are visualized by multichannel fluorescence microscopy . This method is helpful in answering the question of whether two or more fluorochromes are located in the same physical structure, within the diffraction limit.
(i) Examine multichannel signals. (ii) Experiments can be conducted with live and fixed cells. (iii) Detection with antigens and fluorescent proteins. (iv) Dynamic experiments with subcellular location. (v) Simple evaluation. (vi) Studies conducted at tissue and cellular levels.
(i) Not tested protein interactions. (ii) Does not provide direct proof of functional relationship.
FRET is a process by which energy is transferred from one fluorophore (donor) to another nearby molecule (acceptor) through a nonradioactive pathway. For FRET to occur 3 conditions must be met: (1) less than 10 nm of distance between donor and acceptor; (2) the emission spectrum of the donor must overlap the acceptor molecule excitation spectrum; (3) the emission dipole of the donor and excitation dipole of the acceptor must not be oriented perpendicular to each other [80, 95].
(i) Examine protein interactions and dynamics. (ii) Molecular ruler. (iii) Development of biomolecules sensors by FRET. (iv) Intramolecular transition studies. (v) Proteolysis studies. (vi) Live-cell molecular interactions. (vii) Several FRET microscopy techniques available. (viii) FRET goes beyond the resolution limit of light microscopy (diffraction limit).
(i) Lack of FRET not necessarily indicates lack of interaction, due to incorrect orientation (dipolar moment) of fluorophores. (ii) Limited number of interactions can be explored at once.
FRAP is used to assess the molecular mobility in living cells. Proteins of interest tagged with a fluorophore or fused to a fluorescent protein are subjected to irreversible bleaching in a region of interest (ROI) using a high-power laser illumination [96–98].
(i) Live-cell molecular dynamics. (ii) Studies of protein dynamics in a lipid raft context. (iii) Studies of lipids dynamics.
(i) Photodamage of the cells. (ii) Density of detectable molecules limited.
A technique to visualize protein interactions. It is based on structural complementation between two nonfluorescent fragments of a fluorescent protein .
(i) Irreversible BiFC complexes are useful to analyze weak or transient interactions. (ii) High sensitivity. (iii) Simplicity of analysis. (iv) BiFC does not require specialized equipment and postacquisition image processing.
(i) Irreversibility of BiFC complexes makes measurement of dynamic interaction impossible to assess. (ii) Overexpression of the protein of interest might force complementation, resulting in false-positives.
This microscopy offers spatial resolution beyond the diffraction limit of conventional microscopes by exploiting nonlinear phenomena. There are at least four approaches to reach superresolution: (1) stimulated emission depletion (STED), (2) structured illumination microscopy (SIM), (3) photoactivation localization microscopy (PALM), (4) stochastical optical reconstruction microscopy (STORM) [98, 99].
(i) -resolution in a range of 130–20 nm. (ii) -resolution in a range of 100–350 nm. (iii) Good for smaller or filamentous objects. (iv) Most techniques are not suitable for dynamic studies, because image acquisition is very slow (minutes).
(i) Dyes require special characteristics (photostability). (ii) Temporal resolution in minutes. (iii) Not suitable for live-cell imaging studies. (iv) Postprocessing imaging required (except for STED). (v) The performance critically depends on the labeling density and which biological structure is studied. Best suited for filamentous structures.
A powerful tool to examine a wide range of properties of individual molecules.
(i) Compatible with different kinds of microscopy: (1) confocal microscopy (2) superresolution microscopy (3) electron-microscopy (4) two-photon microscopy (5) TIRFM (6) epifluorescence (7) HILO (8) SPIM (ii) Live- and fixed-cells experiments. (iii) High spatial resolution for location and diffusion studies. (iv) Dynamic information with single particle tracking. (v) Conformational changes, vesicle movement, and oligomerization studies. (vi) Compatible with FRET, photobleaching, and photoactivation techniques. (vii) provide information about molecular kinetics.
(i) Very high signal-to-noise ratio. (ii) Dyes require photostability. (iii) High density of label and autofluorescence entails complications. (iv) It is limited by illumination depth and quenching of the signal.
Highly inclined and laminated optical sheet microscopy (HILO)
HILO uses a laser beam slightly below the critical angle to illuminate the sample in a laminated and inclined thin optical sheet, also called shallow angle fluorescence microscopy (SAFM).
(i) Increases the signal-to-background ratio. (ii) Illumination beam always passes through the center of the focal plane, which confers a powerful tool for three-dimensional imaging. (iii) Reduced photobleaching. (iv) Allows single molecule studies.
(i) Restricted visual plane. (ii) Limited penetration depth. (iii) Not suitable for molecular tracking.
SPIM illuminates the sample from one side in a focal plane where detection is done perpendicularly.
(i) Increases the signal-to-background ratio. (ii) Reduced photobleaching. (iii) Good penetration. (iv) Allows single molecule studies. (v) Fast acquisition. (vi) Phototoxicity limited.
(i) Requires time-consuming calibration of the system. (ii) Scattering light in the sample due to absorption by the specimen. (iii) Optical artifacts, like shadows and stripes, are common by the angle of illumination.
Total internal reflection fluorescence microscopy (TIRFM)
TIRFM is a surface selective imaging technique which illuminates a coverslip with a laser beam at an incident angle greater than critical angle, producing a nonpropagating electromagnetic field known as evanescent wave. The energy of evanescent wave decays exponentially with the distance, exciting fluorophores at distances not greater then 100 nm from the coverslip.
(i) Excellent tool to minimize background fluorescence. (ii) Dynamics of plasma membrane, like vesicle fusion, adhesion, and cell motion. (iii) -resolution in a range of 60–200 nm. (iv) Allows single molecule studies. (v) Live- and fixed-cells experiments.
(i) Limited studies in structures other than plasma membrane. (ii) Requires time-consuming calibration to find the critical angle of illumination. This procedure must be repeated for every excitation wavelength. (iii) Multichannel (multicolor) experiments are difficult to produce unless expensive equipment is acquired to automate the changing of the critical illumination angle.
This novel method for TIRFM uses an excitation light propagation via the coverslip to produce the evanescence wave, and uses the coverslip as a light guide.
In comparison with other systems of TIRFM, like objective or prism-based systems, LG-TIRFM confers the following advantages: (i) it can be used with dry, water, and oil immersion objectives, and a broad range of illuminators (not limited to laser excitation); (ii) excellent for multicolor experiments, and simple to setup; (iii) evanescence wave propagates in a large, evenly illuminated area; (iv) the excitation light does not enter the emission channel and does not interfere with detection, thus providing better signal/background ratios; (v) LG-TIRFM is compatible with open perfusion chambers, allowing simultaneous patch clamp or microinjections experiments; (vi) calibration of the system is much less demanding; (vii) low photobleaching in continuous laser excitation.
(i) Limited studies in structures other than plasma membrane. (ii) Expensive sapphire coverslip is required for oil-immersion objectives.
SPR is an optical technique to characterize interactions between different molecules. Through a monochromatic polarized light directed on a gold surface under total internal reflection, this produces an evanescence wave, which interacts with a sensor surface with a specific refractive index. When a molecule is covalently bound to the gold surface, it is possible to characterize in real-time the modifications in the refractive index similar to a signal measured in resonance units.
(i) Sensitive tool for identification and characterization of protein-protein interactions. (ii) Characterize antibody affinities. (iii) Protein-DNA interactions. (iv) Protein-lipids interactions. (v) Competitions and binding assays.
(i) Only for in vitro experiments. (ii) Not suitable for small analytes. (iii) High concentration of the analyte is required.
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