Two-photon laser microscope - a laser microscope that allows you to observe living tissue at a depth of more than one millimeter using the phenomenon of fluorescence . A two-photon microscope is a type of multiphoton fluorescence microscope . Its advantages compared with a confocal microscope are its high penetrating power and low degree of phototoxicity [1] .
A two-photon microscope was first designed by Winfred Denk in the laboratory of VV Webb at Cornell University [2] . He combined the idea of two-photon excitation with laser scanning.
Principle of Operation
A two-photon laser microscope is based on the physical principle described by Maria Göppert-Mayer in her doctoral dissertation [3] in 1931.
The process of two-photon excitation occurs as follows: two photons with low energy excite a fluorophore (a molecule capable of fluorescence or a part of a molecule) during a single quantum event. The result of this excitation is the subsequent emission of excited fluorescence photon molecules. The energy of a fluorescent photon is greater than the energy of exciting photons.
The probability that both excitation photons will be absorbed by one molecule is very small. Therefore, a large flux of exciting photons is needed, which can be obtained using a laser emitting photons with a high pulse repetition rate (80 MHz). The most commonly used fluorophores have an excitation spectrum in the range of 400-500 nm, while the wavelength of the exciting laser is in the range of 700-1000 nm (infrared wave region). If the fluorophore absorbs two photons simultaneously, then it will receive enough energy to go into an excited state. Further, the excited fluorophore will emit one photon (in the visible part of the spectrum), the wavelength of which depends on the type of fluorophore.
Since the absorption of two photons is necessary for the fluorophore to become excited, the probability of the emission of a secondary photon by the fluorophore is proportional to the square of the excitation intensity. Therefore, fluorescence will be stronger when the laser beam is clearly focused and not scattered. The maximum fluorescence is observed in the focal volume (the volume where the laser beam is focused) and shows a sharp decrease in the area outside the focus.
Design
In a two-photon microscope, the infrared laser beam is focused using a collecting lens. Usually a high-frequency 80 MHz sapphire laser is used, which emits a pulse with a duration of 100 femtoseconds, which provides a high photon flux density, which is necessary for two-photon absorption.
The light emitted by the fluorescent sample is amplified by a highly sensitive photomultiplier . Since the light receiver is single-channel, the light intensity observed in a given focal volume forms one pixel of the image. In order to obtain a two-dimensional pixel image, scanning is performed in the focal plane of the sample.
Advantages and disadvantages
The use of infrared light to excite the fluorophore in the studied tissues has its advantages [4] :
- Long waves scatter less than short ones, which provides high spatial resolution .
- Exciting photons have little energy, therefore, they are less destructive to tissues (which prolongs the life of the tissue under study).
But there are some disadvantages:
- Laser operation requires expensive optical instruments to provide pulse intensity.
- The two-photon absorption spectrum of a fluorophore can vary greatly, in contrast to the single-photon absorption spectrum.
- A beam with a wavelength of more than 1400 nm is significantly absorbed by water in living tissues.
Notes
- ↑ Denk W., Strickler J., Webb W. Two-photon laser scanning fluorescence microscopy (Eng.) // Science. - 1990. - Vol. 248 , no. 4951 . - P. 73-6 .
- ↑ Denk W., Svoboda K. Photon upmanship: why multiphoton imaging is more than a gimmick (English) // Neuron : journal. - Cell Press 1997. - Vol. 18 , no. 3 . - P. 351-357 .
- ↑ Göppert-Mayer M. Über Elementarakte mit zwei Quantensprüngen (neopr.) // Ann Phys. - 1931.- T. 9 . - S. 273-295 .
- ↑ Helmchen F., Denk W. Deep tissue two-photon microscopy (Eng.) // Nat Methods : journal. - 2005. - Vol. 2 , no. 12 . - P. 932-940 .