Микроскопия с использованием полного внутреннего отражения: принципы и применение.

A total internal reflection fluorescence microscope (TIRFM) is a type of microscope with which a thin region of a specimen, usually less than 200 nanometers can be observed. TIRFM is an imaging modality which uses the excitation of fluorescent cells in a thin optical specimen section that is supported on a glass slide. The technique is based on the principle that when excitation light is totally internally reflected in a transparent solid coverglass at its interface with a liquid medium, an electromagnetic field, also known as an evanescent wave, is generated at the solid liquid interface with the same frequency as the excitation light. The intensity of the evanescent wave exponentially decays with distance from the surface of the solid so that only fluorescent molecules within a few hundred nanometers of the solid are efficiently excited. Two dimensional images of the fluorescence can then be obtained, although there are also mechanisms in which three dimensional information on the location of vesicles or structures in cells can be obtained.

Widefield fluorescence was introduced in 1910 which was an optical technique that illuminates the entire sample. Confocal microscopy was then introduced in 1960 which decreased the background and exposure time of the sample by directing light to a pinpoint and illuminating cones of light into the sample. In the 1980s, the introduction of TIRFM further decreased background and exposure time by only illuminating the thin section of the sample being examined. This idea was then extended by Daniel Axelrod at the University of Michigan, Ann Arbor in the early 1980s as TIRFM. A TIRFM uses an evanescent wave to selectively illuminate and excite fluorophores in a restricted region of the specimen immediately adjacent to the glass water interface. The evanescent electromagnetic field decays exponentially from the interface, and thus penetrates to a depth of only approximately 100 nm into the sample medium. Thus the TIRFM enables a selective visualization of surface regions such as the basal plasma membrane (which are about 7.5 nm thick) of cells. Note, however, that the region visualized is at least a few hundred nanometers wide, so the cytoplasmic zone immediately beneath the plasma membrane is necessarily visualized in addition to the plasma membrane during TIRF microscopy. The selective visualization of the plasma membrane renders the features and events on the plasma membrane in living cells with high axial resolution. TIRF can also be used to observe the fluorescence of a single molecule, making it an important tool of biophysics and quantitative biology. TIRF microscopy has also been applied in the single molecule detection of DNA biomarkers and SNP discrimination. Cis geometry (through objective TIRFM) and trans geometry (prism and lightguide based TIRFM) have been shown to provide different quality of the effect of total internal reflection. In the case of trans geometry, the excitation lightpath and the emission channel are separated, while in the case of objective type TIRFM they share the objective and other optical elements of the microscope. Prism based geometry was shown to generate clean evanescent wave, which exponential decay is close to theoretically predicted function. In the case of objective based TIRFM, however, the evanescent wave is contaminated with intense stray light. The intensity of stray light was shown to amount 10–15% of the evanescent wave, which makes it difficult to interpret data obtained by objective type TIRFM

Key differences between objective based (cis) and prism based (trans) TIRFM are that prism based TIRFM requires usage of a prism/solution interface to generate the evanescent field, while objective based TIRFM does not require a prism and utilizes a cover slip/solution interface to generate the evanescent field. Typically objective based TIRFM are more popularly used, however have lowered imaging quality due to stray light noise within the evanescent wave.

TIRFM is predicated on the optical phenomena of total internal reflection, in which waves arriving at a medium interface do not transmit into medium 2 but are completely reflected back into medium 1. Total internal reflection requires medium 2 to have a lower refractive index than medium 1, and for the waves must be incident at sufficiently oblique angles on the interface. An observed phenomena accompanying total internal reflection is the evanescent wave, which spatially extends away perpendicularly from the interface into medium 2, and decays exponentially, as a factor of wavelength, refractive index, and incident angle. It is the evanescent wave which is used to achieve increased excitation of the fluorophores close to the surface of the sample, and diminished excitation of superfluous fluorophores within solution. For practical purposes, in objective based TIRF, medium 1 is typically a high refractive index glass coverslip, and medium 2 is the sample in solution with a lower refractive index. There may be immersion oil between the lens and the glass coverslip to prevent significant refraction through air.

For complex fluoroscope microscopy techniques, lasers are the preferred light source as they are highly uniform, intense, and near monochromatic. However, it is noted that ARC LAMP light sources and other types of sources may also work. Typically the wavelength of excitation beam is designated by the requirements of the fluorophores within the sample, with most common excitation wavelengths being in the 400–700 nm range for biological samples. In practice, a lightbox will generate a high intensity multichromatic laser, which will then be filtered to allow the desired wavelengths through to excite the sample. For objective based TIRFM, the excitation beam and fluoresced emission beam will be captured via the same objective lens. Thus, to split the beams, a dichromatic mirror is used to reflect the incoming excitation beam towards the objective lens, and allow the emission beam to pass through into the detector. Additional filtering may be required to further separate emission and excitation wavelengths.

When excited with specific wavelengths of light, fluorophore dyes will reemit light at longer wavelengths (which contain less energy). In the context of TIRFM, only fluorophores close to the interface will be readily excited by the evanescent field, while those past ~100 nm will be highly attenuated. Light emitted by the fluorophores will be undirected, and thus will pass through the objective lens at varying locations with varying intensities. This signal will then pass through the dichromatic mirror and onward to the detector.

Glass cover slips typically have a reflective index around , while the immersion oil refractive index is a comparable The medium of air, which has a refractive index of , would cause refraction of the excitation beam between the objective and the coverslip, thus the oil is used to buffer the region and prevent superfluous interface interactions before the beam reaches the interface between coverslip and sample.

The objective lens numerical aperture (NA) specifies the range of angles over which the system can accept or emit light. To achieve the greatest incident angles, it is desirable to pass light at an off axis angle through the peripheries of the lens.

The back focal plane (also called "aperture plane") is the plane through which the excitatory beam is focused before passing through the objective. Adjusting the distance between the objective and BPF can yield different imaging magnification, as the incident angle will become less or more steep. The beam must be passed through the BPF off axis in order to pass through the objective at its ends, allowing for the angle to be sufficiently greater than the critical angle. The beam must also be focused at the BPF because this ensures that the light passing through the objective is collimated, interacting with the cover slip at the same angle and thus all totally internally reflecting. This coating is designed to have high reflectivity for shorter wavelengths and high transmission for longer wavelengths. While the filter transmits the selected excitation light (shorter wavelength) through the objective and onto the plane of the specimen, it also passes emission fluorescence light (longer wavelength) to the barrier filter and reflecting any scattered excitation light back in the direction of the laser source. This maximizes the amount of exciting radiation passing through the filter and emitted fluorescence beam that is detected by the detector.

The barrier filter mainly blocks off undesired wavelengths, especially shorter excitation light wavelengths. It is typically a bandpass filter that passes only the wavelengths emitted by the fluorophore and blocks all undesired light outside this band. More modern microscopes enable the barrier filter to be changed according to the wavelength of the fluorophore's specific emission. The electrical signal is then convoluted with a point spread function (PSF) to sample the original signal. As such, image resolution is highly dependent on the number of detectors and the point spread function will determine the image resolution.

Most fluorescence imaging techniques exhibit background noise due to illuminating and reconstructing large slices (in the z direction) of the samples. Since TIRFM uses an evanescent wave to fluoresce a thin slice of the sample, there is inherently less background noise and artifacts. However, there are still other noises and artifacts such as poisson noise, optical aberrations, photobleaching, and other fluorescence molecules. Poissonian noise are fundamental uncertainties with the measurement of light. This will cause uncertainties during the detection of fluorescence photons. This noise may cause misrepresentation of the object at incorrect pixel locations. Optical aberrations can arise from diffraction of fluorescence light or microscope and objective misalignment. Diffraction of light on the sample slide can spread the fluorescence signal and result in blurring in the convoluted images. Similarly, if there is a misalignment between the objective lens, filter, and detector, the excitation or emission beam may not be in focus and can cause blurring in the images. The fluorescing substances will always degrade to some extent by the energy of the exciting radiation and will cause the fluorescence to fade and result in a dark blurry image. Photobleaching is inevitable but can be minimized by avoiding unwanted light exposure and using immersion oils to minimize light scattering. This technique, called the maximum likelihood method, is being further improved by algorithms to improve its performance speed.