Two-photon excitation microscopy: Why two is better than one
Two-photon microscopy is a fluorescence imaging technique that allows the visualisation of living tissue at depths unachievable with conventional (one-photon) fluorescence or confocal microscopy.
Also known as non-linear multiphoton, two-photon laser scanning or simply two-photon microscopy, it relies upon the principle of two-photon absorption.
How does it work?
First described by Maria Goeppert-Mayer in 1931, two-photon absorption is the concept that two photons of identical or different frequencies can excite a molecule from one energy state (usually the ground state) to a higher energy state in a single quantum event.
This state can be described physically as the jump of an electron from one atomic orbital to another that is less stable. The energy difference between the two states is equal to the sum of the energies of the two-photons absorbed.
For this to occur, the two photons must hit the molecule within 1 femtosecond of each other (10-15 seconds). This requires a focused laser that is able to produce very fast pulses of light (~80 MHz) with a lot of power (~150,000 W peak power).
The wavelength of light is proportional to its energy level. The shorter the wavelength the more energy it has. This is a linear relationship, such that a photon with a wavelength of 400 nm has twice the amount of energy as a photon that has a wavelength of 800 nm.
Traditional fluorescence microscopy uses a single photon to excite fluorescent dyes using mainly visible excitation wavelengths (390-700 nm). After excitation the electron will drop back down to its stable state and during this process it will release a photon of light with slightly less energy than the excitation photon (lost due to processes like vibrational relaxation).
In two-photon microscopy, two photons of light with double the wavelength are used to excite the same or similar fluorescent dyes. However, only one photon is released when the electron drops down to its more stable orbital. This will have the same wavelength as the equivalent one-photon fluorescence method (i.e. slightly longer than half the excitation wavelength).
The most commonly used fluorescent dyes have excitation spectra in the 400 to 500 nm range. The wavelengths used to excite the same dyes with two-photons therefore tend to be between about 800 and 1000 nm, in the infrared spectrum.
Why is this useful for imaging living tissues?
The laser and additional hardware necessary to create a two-photon microscope makes them fairly expensive in comparison to conventional fluorescence microscopes. So what is the advantage of using two-photons instead of one?
A key benefit of two-photon microscopy is its ability to restrict excitation to a tiny focal volume in thick samples. The objective focal point is the only space with a high enough photon density to ensure simultaneous presentation of two photons to the fluorophore. Effectively, this means there is no out of focus emission light and any light at the emission wavelength must have come from that single spot.
By scanning the laser over the region of interest it is possible to build up a 2D image of the fluorescence occurring in the living tissues. Carrying this out at various focal planes enables the formation of a high-resolution 3D model. Collected over time, these images or models can be used as the frame of a movie to visualise processes as they happen. The temporal resolution is limited by the scanning speed of the mirrors and the amount of time it takes to build up a significant fluorescent signal at each pixel (dwell time).
In other fluorescence techniques the laser will either excite the fluorescent dyes throughout a sample or, through a cone of light down to the focal plane (e.g. in confocal microscopy). This leads to more out of focus excitation, causing faster photobleaching (the gradual decline of most fluorophore's ability to fluoresce) and phototoxicity (the toxic effect of activated fluorophores on cells).
An additional benefit occurs through the use of infrared wavelengths. Light from the visible spectrum scatters a lot in biological tissue; severely limiting the depths it can penetrate with enough power to excite a fluorophore. The infrared lasers used for two-photon microscopy scatter much less. This means infrared laser light has enough power to excite fluorophores up to around 1 mm in living tissues. In comparison, single photon confocal microscopy can only penetrate to about 200 µm.
The first commercially available two-photon microscopes were introduced in 1996. Over 20 years later they have increased our understanding in numerous areas of biology. Now hundreds of labs around the world have adopted two-photon microscopy to overcome the limitations of alternative fluorescence microscopy techniques.
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