SMI uses optical microscopy, and since most of the biological molecules are translucent for visible light, some kind of labeling is required. For detection of single molecules, labeling with fluorophore is effective, because it provides high-contrast imaging, which is essential both for detection of small signals from a single molecule and for detection of specific species of molecules in crowded conditions of living cells.
Labeling by fluorescent proteins has expanded the application of SMI in living cells.
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Fluorescence signal from single fluorophores is small but enough to be imaged individually when recent high-sensitivity video cameras, like EM-CCD or CCD equipped with a multi-channel plate image intensifier are used. Standard temporal resolution of SMI in living cells is several tens of ms, and in some cases, under strong illumination, ms sampling has been achieved gathering hundreds of photons from a single fluorophore per single video frame.
For detection of small signals from single molecules, background rejection is essential. Total internal reflection [ 1 ] and oblique illumination [ 18 ] are useful technologies of wide-field fluorescence microscopy to realize effective background rejection and can be used for SMI in living cells Figure 1 [ 19 ].
Single-molecule imaging in living cells. Schemes of total internal reflection A and oblique illumination B microscope and a single-molecule image of tetramethylrhodamine-labeled EGF on the surface of living HeLa cells under an oblique illumination microscope C are shown. Detection of singlemolecules can be examined by single-step photobleaching D.
Figure 2 shows the optical setup of our TIR microscope for SMI, which was home-built based on a commercial inverted fluorescence microscope. Solid-state continuous wave lasers in different emission wavelengths are used for illumination according to the species of fluorophore.
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Between the lasers and the microscope, a two-dimensional beam scanning system is constructed. This system is composed by a pair of diagonallypositioned Galvanometer-scanning mirrors and a telescope that inserted between the two scanning mirrors. Therefore, the specimen is illuminated from every direction during the acquisition of single frames.
Thus, the system achieves pseudo-circular illumination. Circular illumination is better in TIR-microscopy than the illumination from a fixed single direction that is usually used in commercial TIR system, especially for observations of biological samples having anisotropic structure [ 19 ]. It also reduces effects of spatially inhomogeneous illumination pattern often caused by the strong coherence of laser beams. There are several methods to construct circular illumination path using only static optical elements or a rotatory moving mirror with a fixed angle, however, using a pair of Galvanometer, the incident angle of the illumination beam to the specimen is easily adjusted to the best position for each specimen by changing the amplitude of vibration of the scanner mirrors.
Optical path of a total internal reflection fluorescence microscope for single-molecule imaging. The illumination laser beams are introduced into an inverted fluorescence microscope from the epi-illumination path. Between the microscope and lasers, a two-demensional beam scanning system is inserted to achieve pseudo-circular illumination see text for details. The violet nm laser is used for photoconversion of fluorophores. Galvanometer scanner mirror, L: Both the chemical fluorophores and fluorescent proteins are applicable as the probes of SMI in living cells. To obtain good contrast against autofluorescence of cells and optics, fluorophores with relatively longer wavelength emissions are generally better.
Among the fluorescent proteins, as far as we know, eGFP is the best for SMI because of relatively good photostability. Protein tags, like HaLo and SNAP, which can be conjugated covalently with chemical fluorophores after expression in living cells, are useful, because chemical fluorophores are generally more photostable than the fluorescent proteins, and colors of fluorescence emission can be changed according to the purpose of the experiment.
When an especially strong and stable long observation time signal is required, Q-dot or other fluorescent beads will be used. In such cases, steric hindrance and multivalency should be controlled carefully. For SMI, cells are cultured on a coverslip and set on the microscope. Since contamination of small dust on the coverslip prevents SMI, the coverslip must be washed thoroughly before transfer of cells onto it [ 21 ].
Usage of glass coverslips not conventional plastic cell culture dishes that has the high refractive index was necessary to achieve total internal reflection; however, some cell culture dishes or chambers made of plastic with the refractive index similar to that of glass 1. One day or more before the observation, the culture medium should be replaced to one that does not contain phenol red to reduce background fluorescence.
The culture medium used during observation should also not contain phenol red. When proteins tagged with fluorescent proteins, like GFP, are expressed and observed in cells, conditions for the transfection of cDNAs should be carefully controlled to avoid overexpression that prevents SMI see section 3.
Similarly, when HaLo or SNAP tag is used, staining should be carried out with a much lower concentration of fluorescent regents than that recommended by the manufactures. Spatial filtering of the images is also used to improve image quality. But, one must be careful to use any temporal and spatial filtering, because they sometimes do not preserve the linearity of signal intensity.
Background subtraction is usually carried out before quantification of single-molecule signals. In cells, because background signals are highly inhomogeneous, the background levels should be determined locally. After the appropriate pretreatments, the position and signal intensity are determined for individual single-molecule images. For this purpose, fitting with a two-dimensional Gaussian distribution is usually used. Fitting functions can include background signals instead of the pretreatment of background subtraction. We usually use a Gaussian distribution on an inclined background plane as the fitting function [ 21 ].
Positions of the molecule can be determined as the centroid of the distribution with sub-pixel spatial resolution.
Signal intensity can be calculated by integration of the distribution function. Accuracy of these parameters depends on the measurement system and should be determined statistically from the repeated measurements of the same single molecules. There are several criteria to judge whether single molecules are really detected or not [ 4 ].
Single-step photobleach is the most convenient and used criterion Figure 1D. To distinguish photobleach from disappearance by the movements of molecules, like release into the solution, illumination intensity should be changed. Photobleaching rate, but not the rate of most of other phenomena, depends on the illumination intensity. Because the size of fluorophores is much smaller than the spatial resolution of the optical microscope, the profile of single-molecule images must be the point spread function of the optics. The intensity distribution of single molecules should be Gaussian, because the photon emission from a fluorophore is a Poisson process; however, when the intensities are small, the distribution becomes binominal or sometimes looks as a log-normal distribution.
Photobleach of the fluorophore seems to be the most serious problem in SMI. In typical conditions, the emission photon numbers from a single chemical fluorophore, including TMR and Cy3, before photobleach is less than 1 million, and those from fluorescent proteins are several times smaller.
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If a video rate movie is taken, single molecules could be observed only for about 10 s in average. Signal intensity photon flux of single fluorophores is limited, because the fluorescence emission cycle requires a finite time. The fluorescence lifetime, which is the rate-limiting parameter under strong enough illumination, of typical fluorophores used in SMI is about 1 ns, meaning that the maximum photon flux is about 10 9 s However, strong illuminations that cause such high-rate emission induce higher-order excitation that could be the reason of undesired photochemical reactions.
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Practical photon emission rate is no more than about 10 6 s This means that because thousands of photons are required to acquire a snapshot of SMI, temporal resolution of SMI is difficult to be improved to more than 1 ms. Accuracy of position detemination depends on the signal intensity. When more than 10, photons are obtained on the camera for a single frame, the centroid of a single-molecule image can be determined with 1 nm of accuracy [ 22 ].
Such high accuracy cannot be obtained with a temporal resolution better than subseconds. The special resolution of the optical microscopy is worse than nm. This limits the densities or the concentrations of the molecule to be observed, because in dense conditions, images of molecules overlap to inhibit single-molecule detection. Concentrations of most cell signaling proteins are thought to be within these limits, but those of structural proteins could exceed these limits.
Durations and intervals of molecular interactions contain information about reaction kinetics. On-times and off-times can be measured for single events using SMI. Dual-color SMI Figure 3 is possible to detect on-times, but in practice, due to photobleach, it is difficult to detect successive multiple on-times for a single molecule and not easy to detect even a single off-time. Basal cell surface was observed using a dual-colour total internal reflection fluorescence microscope.
More practical single-molecule measurements of on- and off-times are achieved for the interactions between a soluble molecule and a molecule stably attached on stationary structures. Because of the rapid Brownian movements in solution, soluble molecules cannot be observed as clear fluorescent spots and can only be imaged when they associate with fixed or slowly moving molecules. Therefore, in vitro SMI measurements, interactions between fluorescently labelled soluble molecules and a non-labeled molecule fixed on the substrate are often observed [ 1 , 11 ].
In such cases, because different soluble molecules interact with a fixed molecule in turn, photobleach has minimal effect. Similar measurements can be achieved in living cells when interactions are observed between a fluorescently labelled soluble molecule either in the extracellular solution or in the cytoplasm and molecules in the membrane or cytoskeleton structures.
However, even in living cells, measurements of the waiting times of the first association after some perturbation and measurements of off-times are usually possible for kinetic analyses. In other words, the on-time distribution represents the reaction rate to produce the dissociation state with time after the formation of the association state. Here, the reaction is assumed to proceed according to a simple mass action model. Different from the kinetic analyses in conventional biochemical techniques that deal with the concentration changes, the reaction rate equations in SMI describe state changes of a single molecule with time; i.
By fitting the on-time distribution with equation 3 , the best-fit value for k is obtained. This procedure is similar to that used in a conventional biochemical analysis based on ensemble-molecule measurements. Preparation of Chemotactically Competent Dictyostelium Cells. Modeling the self-organized phosphatidylinositol lipid signaling system in chemotactic cells using quantitative image analysis. Statistical analysis of lateral diffusion and reaction kinetics of single molecules on the membranes of living cells. Self-organization of the phosphatidylinositol lipids signaling system for random cell migration.
Statistical analysis of lateral diffusion and multistate kinetics in single-molecule imaging. Single-molecule imaging techniques to visualize chemotactic signaling events on the membrane of living Dictyostelium cells. Methods in molecular biology Clifton, N. Topics Discussed in This Paper. Transduction machine learning Chemotactic Factors Signal Transduction.
Kinetics Signal-to-noise ratio Cell signaling. Citations Publications citing this paper. Showing of 4 extracted citations. Todd Washington , Maria Spies Methods in enzymology Exploration of the spontaneous fluctuating activity of single enzyme molecules. Anne Schwabe , Timo R. Maarleveld , Frank J.