Two-photon excitation microscopy
A multi-photon microscope (English Multi- Photon Laser Scanning Microscope, MPLSM also: multi- photon microscopy, MPM ) is a special light microscope from the group of laser scanning microscopes.
Images are formed by one is used by two different physical phenomena:
With the help of a strong, focused laser beam while nonlinear optical effects are produced, based on the interaction of several simultaneously incoming in a molecule photons ( light particles). Therefore, the strength of the signal generated does not increase linearly with the number of incident photons, but with the square (with two-photon - effects ) or to the third power (when three-photon - effects).
The operation of a multi-photon microscope is similar to that of a confocal laser scanning microscope. However, while the confocal laser scanning microscopy has a penetration depth depending on the preparation of 50-80 microns, can use the multi -photon microscopy deeper areas, such as 200 microns, in very favorable cases even up to 1000 microns ( = 1 mm) can be achieved. Wherein images of living tissues, are possible for the imaging are inaccessible otherwise.
- 2.1 Basics
- 2.2 Second Harmonic Generation
- 2.3 Third Harmonic Generation
- 3.1 1931-1990
- 3.2 Since 1990
Multi- photon fluorescence microscopy
The most common multi-photon technique is the two- photon fluorescence microscopy, sometimes just referred to as two-photon microscopy. In the conventional fluorescence microscopy, an electron by the absorption of a photon in each case excited in a fluorescent molecule, that is placed in a higher energy state. In the two- photon fluorescence excitation of the electron microscopy is determined by the simultaneous absorption of two photons generated ( two-photon absorption). And an excitation with three or more simultaneously incoming photons is possible.
Principle
Fluorescence occurs when dyes absorb incoming photons and re-emit in the sequence to another photon. Due to the incoming, " stimulating " photon, an electron is raised to a higher energy level, ie the energy temporarily stored in such a way. In normal fluorescence microscopy, this excitation is done by exactly one photon. The electron remains for a few nanoseconds at the higher energy level before it falls back while sending out a new, longer-wavelength, lower energy photon. When excited with blue light such as, usually green fluorescence, for example with fluorescein obtained.
This single photon excitation may be replaced by two or more photons, if they have the same amount of energy. Thus, dark red or infrared light are used to produce green fluorescence. In addition, both photons must simultaneously (within an attosecond = 10-18 s ) arrive, since no stable intermediate energy level exists.
In the normal fluorescence microscopy the exciting photon has a shorter wavelength, i.e., a higher frequency, and thus more energy than the emitted photon. The wavelength difference of the two photons is referred to as a Stokes shift. On the contrary, is excited in the multi-photon excitation with photons per photon have a considerably longer wavelength, lower frequency and hence less energy than the emitted photons. This is only possible because lead two or more stimulating photons to produce only an emitted photon. In two- photon excitation the excitation wavelength is approximately twice the excitation wavelength normally used in the three- photon excitation, a three- fold.
Technical implementation
In order to achieve a simultaneous arrival of two or more photons at the focal point of the stimulable electrons, very high photon densities are required. This can only be achieved, if a pulsed laser is used to mode coupling. The special feature of this type of laser is that very short (eg 0.14 ps = 0.14 10 - 12s), intense laser pulses are emitted, which are repeatedly eg 80 million times per second. The intervals between two pulses in the given example are thus 12.5 ns ( = 12,500 ps) long, so that the total energy generated in the laser can be delivered in a fraction of the time.
The titanium used for the excitation in the rule: sapphire lasers are expensive (about 150,000 euros ) and therefore represent a major barrier to the widespread adoption dar. Ti: Sa laser can be set to wavelengths from about 700 nm to about 1050 nm be. More wavelengths can be created through the use of an " optical parametric oscillator " (OPO). This is the Ti: "pumped " Sa laser and can then generate wavelengths up to about 1300 nm. This also red and dark red fluorescent dyes in two-photon mode can be excited.
As in a confocal laser scanning microscope, the laser beam is focused by the microscope objective to a point of the preparation. By moving mirror in the light path (scan mirror;. Engl to scan = scanning ), the laser beam is in its position changed so that the focal point moves through the specimen, so this is scanning. The resulting fluorescence is collected by the lens, split spectrally dichroic beam splitter and directed to the detector ( photomultiplier primarily, but also avalanche photodiodes ). The detectors measure the brightness of each pixel so successively. At no point is created in the microscope a complete picture of the preparation, it is assembled only in the control computer.
Due to the complexity of complete units are currently offered only by a few manufacturers. In Europe, these are currently (as of 2011 ) six, Carl Zeiss, LaVision Biotec, Leica Microsystems, Nikon, Olympus and Till Photonics. Since the scanning and detection technology of a multi-photon microscope of a confocal laser scanning microscope is very similar, there are some companies providing such extensions, as well as working groups to upgrade such microscopy your own with an appropriate excitation laser and appropriate filters for multiphoton microscope.
Benefits
As described above, the generation of the two-photon effect requires very high photon density, can achieve only a pulsed laser. But even in this case, it is only at the focal point to a sufficiently high photon density to produce a fluorescence excitation, but not above and below (see figure): Outside the focal plane, the same amount of excitation photons distributed to a strong increase in diameter of the jet cone. However, two -photon excitation is dependent on the square of the light intensity so that the light intensity outside of the focal plane, in contrast to other fluorescence microscopes, for the generation of fluorescence is no longer sufficient.
This leads to practical advantages:
Higher Harmonic Generation
In addition to playing in the fluorescence multiphoton microscopy Second and Third Harmonic Generation (SHG and THG; literally produce the second (or third ) harmonic; the Germans: frequency doubling and frequency tripling ) play a role. They are summarized as Higher harmonic generation ( HHG ).
This generation of light having a lower wavelength is not physically related to multi-photon fluorescence excitation. HHG occurs but under similar lighting conditions such as multiphoton fluorescence, namely, ( only ) with very strong excitation light. Therefore HHG signal is observed at the focal point of a pulsed laser, but not above or below it. The sections outlined above, technical implementation 'and' benefits ' shall apply accordingly. The necessary equipment is largely the same, so that for example a device that was built for two-photon fluorescence microscopy, usually also allows second harmonic generation.
Basics
Light is electromagnetic radiation, it has in accordance with an electric field. This field occurs in interactions with the irradiated matter. When the pulsed laser is focused onto a specimen in the multiphoton microscope, these interactions lead to the creation of a "harmonious ". The wavelength of the " second harmonic " is exactly half of the incident light that the "Third Harmonic " at THG exactly one-third.
In contrast to fluorescence at HHG no energy in the specimen remains, a fading of the signal does not occur. Phototoxic effects can, however, occur independently of the HHG effect due to the simultaneous excitation of autofluorescence or by absorption. Also, a too high intensity of the excitation light directly lead to the destruction of preparations.
Second Harmonic Generation
The generation of an SHG signal, ie frequency doubling is only possible when there are variations, the electrical properties of the irradiated molecule in all directions, so if it asymmetrically, more non- centro symmetric, is.
The SHG signal propagates primarily in the "forward" direction, as the incident light beam also: The individual phases of the forward-facing photons ( the SHG signal ) are usually in phase ( coherent), so that the waves of produced different molecules amplify. In other directions, the waves cancel each other out partially (destructive interference). The units of the forward and after facing backward signal will also depend on the structure of the irradiated molecules. The strength of the resultant signal is further dependent on the polarization direction of the incident laser light. For objects with a longitudinal structure (e.g. muscle fibers), this results in a dependence of the signal strength of the orientation of the polarization plane of the laser to the specimen.
From the formation mechanism follows that SHG is produced very efficiently at some periodic structures, for example of urea crystals. In biological tissues arises about collagen fibers and on myosin in smooth muscle. SHG facilitated by the orientation of the specimen, even if mostly fluorescence to be observed.
Since the shortest wavelength of light that can be recorded on a microscope, typically lies in the blue region, a wavelength of about 800 nm is used for the generation of signals SHG.
Third Harmonic Generation
When the excitation wavelength is about 1200 nm, also third harmonic generation ( THG; frequency tripling ) can be observed or absorb with filters for visible light. In contrast to SHG THG is not dependent on the presence of non - centro symmetric structures. At sufficiently high intensity of the incident light THG can be caused in principle in any fabric, but the strength of the signal depends on the material (see frequency doubling ). Contrasting GHG images arise when different optical density structures adjacent to each other, for example, cells and blood plasma.
History and applications
1931-1990
The physical principle of the fluorescence excitation of a molecule by several photons was first predicted in 1931 by Maria Goeppert- Mayer. The first experimental observation of two-photon fluorescence excitation took place in 1961, soon after the development of the first laser. Microscopy with two-photon fluorescence excitation succeeded in 1990 for the first time.
SHG effect was observed immediately after the development of the laser 1960. Microscopically, it was first used in 1974, initially in a conventional light microscope without scanning technique: Robert Hellwarth and Paul Christensen ( University of Southern California, Los Angeles) to investigate the structure of zinc selenide polycrystals. 1978 SHG generated by JN Gannaway and CJR Sheppard for the first time with a scanning microscope. They were therefore the first to be able to restrict the signal produced on the focal plane. Also, they studied crystals. The continuous-wave lasers used in this set but in the preparation, so much energy is released that biologics are destroyed. Only with the introduction of pulsed lasers, this problem was solved because only so that the average energy input is sufficiently low.
Probably the first application of SHG in a biological specimen succeeded in 1980 by the group of Isaac friend at Bar- Ilan University in Ramat- Gan, Israel, by examining collagen in tendons of rats. Here, a Nd: YAG laser with a fixed wavelength of 1064 nm, and collected in the signal forward direction. Although First was the intensity depending on the angle of incidence of the laser beam are measured, an image but initially could not be created. However, this was the same group on the same property in 1986.
Since 1990
After Denk et al. In 1990, the first time had demonstrated two-photon fluorescence microscopy, it took only four more years until it was possible to carry it out on live animals ( intravital microscopy) to examine, in this case, to increase blood flow and behavior of white blood cells in the kidney.
The advantages of a multiphoton microscope, in particular the high penetration depth come to take particular in tissues in which the structural differences between the upper and lower tissue layers are present: the deeper layers of tissue are available for other types of microscopy, not or only to a fixed, cut specimens. Local operations can not otherwise be observed therefore in living organs. Examples are live investigations in different brain layers, the observation of various immune system cells in lymph nodes, investigations, such as tumor cells can invade adjacent tissues and studies on muscle cells in the intact heart. In the examples mentioned was two-photon fluorescence and partly also used SHG.
While the observation of fluorescence in flat preparations ( single cells, tissue sections ) may well take place in normal fluorescence microscope or confocal laser scanning microscopes, HHG is possible only with a multi-photon microscope. In addition to the collagen fibers and muscle myosin mentioned above also lead strength and to a lesser extent cellulose to SHG.
In addition, it is possible to use specific dyes that produce, for example, SHG and stain biomembranes. In 1996, with such a dye that is sensitive to the membrane potential, first published SHG on living cells. In other SHG membrane dyes the SHG signal is lost when two such labeled membranes come together, suddenly a Centro symmetry occurs. This process can therefore be detected very sensitively.
SHG microscopy suffered for a long time with very long recording times from minutes to hours per image. This changed in 1999, when it became possible to produce SHG on a laser scanning microscope equipped with a Ti: Sa laser was equipped.
THG has been used in only a few published biomedical studies. One reason for this is that in conventional titanium: sapphire laser, which are usually used for multi-photon microscopes, the maximum available wavelength ( below 1100 nm ) are not sufficient to generate GHG in the visible range. To UV light, however, can not be taken with the usual instrumentation, so that used for other GHG excitation laser. Previous applications were for example the observation of lipid droplets or the observation of hydroxyapatite crystals in enamel. At an excitation wavelength above 1200 nm, the observation signals in the greenhouse and SHG blue signals in the red part of the spectrum is possible. This was for example used to represent intact mouse embryos in three dimensions.