Fluorescence microscope

Fluorescence microscopy is a special form of optical microscopy. It is based on the physical effect of the fluorescence in the fluorescent dyes ( fluorochromes ) are excited with light of a wavelength and thus emit a few nanoseconds later, light of a different wavelength. By specific filter to ensure that only the emitted light is observed.

Some substances are themselves fluorescent ( autofluoreszent ), a coloring is not then required. Others can be specifically stained, so that structures shown to be examined very selectively and with a high contrast with certain fluorescent dyes. Also immunofluorescence has been widely used in biomedical applications, a special form of immunohistochemistry, are coupled with the specific antibodies with fluorescent dyes. Fluorescence in situ hybridization is a technique for the microscopic detection of larger DNA fragments. These possibilities make the fluorescence microscopy at a very versatile method.

In addition to the classical fluorescence microscopy, there are some advanced microscopic applications such as confocal laser scanning microscopy and multi-photon fluorescence microscopy. Also different methods that improve the resolution by bypassing the Abbe limit, are fluorescence microscopic Art

• 4.1 confocal Microscopy
• 4.2 2 -photon microscopy
• 4.3 Super Resolution Microscopy Vertico SMI / SPDMphymod
• 4.4 Photoactivated Localization Microscopy
• 4.5 STED microscopy
• 4.6 TIRF microscopy
• 4.7 Lens Microscopy

Principle

Physical requirements

The function of a fluorescence microscope is based on the following conditions and physical conditions:

• In preparation to be examined contains fluorescent dyes called fluorochromes that can be excited by light of a specific wavelength to emit light.
• The so- excited fluorochromes emit light that has a longer wavelength by the Stokes shift as the excitation light. ( Exception: Multi -photon fluorescence excitation, see below and Articles Two -photon absorption and multiphoton microscope. )
• Excitation and emission light have to be sufficiently different wavelengths to optically separate.

The size of the objects under investigation may well be due to their own luminescence at sufficiently high contrast below the resolution limit of a light microscope.

Basic structure

The basic structure of a typical fluorescence microscope corresponds to a reflected light microscope. The object to be observed is illuminated through the lens. (Hence the correct term is Auflichtfluoreszenzmikroskop or epifluorescence microscope ). The light sources are lamps with very high luminosity used, such as mercury vapor lamps, metal halide lamps or xenon gas discharge lamps. Most light sources are lit over the entire visible spectrum and in the ultraviolet range.

• By a suitably selected optical filter, the excitation filter, only the portion of the light generated is transmitted, containing the necessary for excitation of the fluorescent dye wavelengths. The region of the spectrum in which the fluorescent dye lights must not be passed by the excitation filter.
• A beam splitter reflects the excitation light to the lens, after which the preparation is produced fluorescence. This is a longer wavelength than the excitation light.
• The proportion of the fluorescent light which is collected by the lens, in turn, passes to the beam splitter. Through its particular characteristics of this longer-wavelength light is, however, the direction of the eyepiece or detector passed (and not mirrored). Excitation light that is reflected in the specimen is, however, largely directed back to the lamp.
• A small portion of the reflected excitation light still gets in the preparation, however, the direction of the eyepiece or detector. Since the intensity of the fluorescence is very weak compared to the excitation, and therefore is a further optical filter, called notch filter or the emission filter is required to eliminate this residual excitation light.

Careful selection of the properties of the filters used can be determined which fluorochromes are visible and which are not so that can be detected by changing the filters one more fluorochromes.

History

That structures under the microscope show a luminous phenomenon when they are irradiated with short-wavelength light, was first observed in 1904 by August Köhler. Fluorescence microscopy has been derived from this observation and developed at Carl Zeiss from August Koehler and Henry Siedentopf and presented its application as " luminescence " on April 4, 1908 by A. Köhler for the first time to the public at a microscopy course. From about 1908, the first microscopes for excitation of fluorochromes were developed at Carl Zeiss. Significant contributions to the development came in 1911 also from the Viennese optician Karl Reichert.

Applications

In biology, fluorescence microscopes are widely used. In the simplest case, cell components are made ​​visible, the. Naturally fluoresce ( autofluorescence ) This is possible especially in plants, because chlorophyll and other plant pigments have a natural ability to fluoresce. In general, this property is undesirable, however, because non-specific autofluorescence of various cell components leading to high background signals ( see also TIRF ). The most important applications are based on the specific staining of individual cell components, usually of certain proteins. These are previously either by specific binding of fluorescently labeled ligands labeled ( eg phalloidin -FITC for actin, DAPI for DNA) specific antibodies ( immunofluorescence ) or by fusion with a fluorescent protein such as GFP. From the fluorescence microscopic image indicator of the localization of the protein can then be taken into the cell (e.g., the nucleus, the cytoplasm, bound to the membrane or exported to outside ) or cell components to be visualized by their specific proteins (e.g., actin cytoskeleton by, microtubules through tubulin ). Also, interactions of proteins with each other can be observed. Through gene expression of a marker protein such as GFP under the control of a specific promoter and cell types can be identified that can not be safely addressed solely on the basis of their light microscopic appearance. Depending on the method used and the tracking of individual processes in living cells is possible.

Today there is a wide range of fluorescent markers, marking methods and microscopy techniques.

Special methods of fluorescence microscopy

Confocal microscopy

In confocal microscopy, as well as in their specific form of confocal laser scanning microscopy ( CLSM), passes through the use of a pinhole only fluorescent light of a tiny part of the excited sample in the microscope. The volume from which fluorescence is then read, is limited as to be less than one femtoliter ( 0.000 000 000 000 001 liters). This has the distinct advantage that the background signal also fluorescent components can be hidden from higher or deeper layers of the preparation. In this way, a microscope in multilayer preparations, for example, whole leaves or animal embryos possible. By scanning lying one above the other layers and subsequent reconstruction of the individual images thus obtained can also be created three-dimensional images.

2 -photon microscopy

In the 2 -photon microscopy, the effect is exploited that the fluorescence excitation can take place take place by absorption of a high-energy photon by the simultaneous absorption of two lower-energy photons, which means that instead of short-wavelength light (eg 375 nm) light of longer wavelength (z. , 750 nm) can be used for excitation. 2-photon effect requires a high photon flux density, as it exists only in the center of a pulsed laser beam, ie, the sample is excited to fluoresce only in this area. In contrast to confocal microscopy where the readout volume is restricted here, the excitation volume is limited, resulting in a much more gentle handling of the often sensitive biological samples. In addition, longer wavelength laser light has a higher depth of penetration into biological samples such as tissues, and results in less chemical bond breaks as e.g. short-wave laser light (UV). By just about doubled wavelength of the excitation light, the fluorescence with a wavelength shorter than the excitation causing it. The size of the light spot produced but are still subject to the diffraction limit. Typical excitation sources are pumped titanium - sapphire laser, the pulses in the 90-120 fs range, and tunable from about 750 nm to 1100 nm (near infrared NIR).

Super Resolution Microscopy Vertico SMI / SPDMphymod

The Vertico SMI microscopy (SMI stands for Spatially Modulated Illumination) is an optical nanoscopy that can accommodate all living cells with wide-field images. The 3D Vertico SMI is based on a combination of localization microscopy SPDM with structured illumination SMI.

SPDM ( Spectral Precision Distance Microscopy ) is a light- optical method of fluorescence microscopy, with which to " optically isolated " particles (such as individual molecules) position, distance and angle measurements below the optical diffraction limit are widely available. " Optically isolated " means that at any given time only a single particle / molecule in a specified by the conventional optical resolution area (typically 200-250 nm in diameter ) is registered. This is possible if the particles located in such an area / molecules have different spectral labels (e.g., have different colors, or other useful differences in the light emission point ).

A further development is the SPDMphymod technology with which the blinking behavior of normal fluorescent dyes such as the green fluorescent protein (GFP) can be used for single molecule detection. In addition to normal fluorescent dyes from the GFP group, Alexa dyes can also fluorescein used in ophthalmologic diagnostics are used. This method can be dispensed with the use of special photoactivatable or photoswitchable fluorescent dyes.

Photoactivated Localization Microscopy

The Photoactivated Localization Microscopy (short: PALM) is based on the light-driven, stochastic switching on and off of individual Fluoreszensmoleküle. The switching on and off is carried out over a certain period may be made ​​in the individual images. Adjacent molecules are distributed on the pictures and can be mapped on a subsequent computer calculation with a resolution beyond the diffraction limit.

STED microscopy

In STED microscopy ( STED stands for English Stimulated Emission Depletion) the diffraction limit is clearly overcome. The excitation is followed by a subsequent redshift, ring-shaped light beam. The excited molecules via stimulated emission at the edges to fall back to the normal state. The emitting volume decreases thereby and the resolution of the microscope increases. It provides for, inter alia, changes in living cells observed live.

TIRF microscopy

TIRF microscopy (English Total internal reflection fluorescence microscopy) is a method of fluorescence microscopy to examine structures that are very close (about 200 nm ) are located at the contact surface of the preparation. Thus, a significantly higher contrast arises because little material is excited to fluorescence. This is achieved by making the object carrier is irradiated at an angle which is large enough that total internal reflection occurs. Since the radiation still penetrates partially into the slide, but it is weakened at the same time becoming stronger with increasing distance ( so-called evanescent wave), it reaches only a few layers of fluorescent material.

Lens Microscopy

The lens microscopy (or English Single Plane Illumination Microscopy, SPIM) uses to illuminate the sample, a second lens, the perpendicular to the observation lens (ie, in the focal plane ) a very limited layer of the sample illuminates (called a lens and a light sheet ). Can be observed only from the illuminated region then fluorescence. This method allows to improve the axial resolution of a normal fluorescence microscope when the light sheet is thinner than the depth of field of the microscope. In addition, light is strongly suppressed from outside the focal plane, since there is excited little or no fluorescence.