Two-Photon Microscopy

By Shelley Farrar Stoakes, M.Sc., B.Sc.Reviewed by Dr. Tomislav Meštrović, MD, Ph.D.

Two-photon microscopy (also known as two-photon fluorescence light microscopy) was invented in 1990 as a new method of imaging live cells and tissues in three-dimensions. Conventional fluorescence microscopy illuminates a specimen through the processes of excitation and emission.

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In this conventional method excitation occurs when a shorter wavelength light is absorbed by an electron of the fluorophore (a fluorescent molecule which re-emits light upon exposure to a photon), so the photon (a particle which conveys light) changes the condition of the fluorophore from the ground state to a higher energy excited state. Emission is when the fluorophore returns to its lower energy state by emitting a photon at a longer wavelength.

Two-photon microscopy differs from conventional fluorescence microscopy as the excitation wavelengths of the two photons are longer than the resulting emitted light. The method reduces the risk of photo bleaching and photo toxicity that may limit the application of conventional fluorescence microscopy for living specimens. This is because it optimizes the process and detects more single photons per excitation event.

Principles of Two-Photon Microscopy

The theoretical foundation of two-photon microscopy is two-photon excitation. This is where a fluorophore is excited by the simultaneous absorption of two photons. The two photons are less energetic and have a longer wavelength than conventional excitation with a single photon.

The wavelengths of the two photons in two-photon microscopy are usually in the infrared spectral range whilst the single photon in conventional fluorescence microscopy is in the ultraviolet or blue/green spectral range. The simultaneous absorption of the two photons by the fluorophore is dependent on the combined energy of the two photons being greater than the gap between the energy of the grounded and excited states.

Two-photon microscopy produces fluorescence in the area where the laser beam is tightly focused. This is because the two photons that are absorbed and excite the fluorophore increase the probability of emission quadratically with excitation density. Therefore, excitation mainly occurs at the microscope focal volume where photon density is high. Of the total fluorescence signal emitted, 80% is derived from a region one micrometer thick about the focal point.

The Structure of Two-Photon Microscopes

The structure of two-photon microscopes consists of combining the fundamentals of two-photon absorption with the application of a laser scanner. The laser is focused to a tight area on the specimen plane and then scanned in parallel over the sample.

As the laser comes into contact with the fluorophores in the sample, the fluorescence photons are generated in the small focal volume and detected by photodetectors. The signal produced is mapped to individual pixels of the image via a computer.

The structure of the fluorescence detection path differs between conventional fluorescence microscopes and two-photon microscopes. For conventional fluorescence microscopes the light passes through scan mirrors and through a pinhole before detection.

In two-photon microscopy, the objective lens collects all the fluorescence photos that constitute the emitting signal - therefore, a detector pinhole is not required. A further difference is the type of laser utilized. Two-photon microscopy lasers provide a rapid stream of pulses that can achieve the required high photon density and flux.

Advantages of Two-Photon Microscopy

One of the main advantages to two-photon microscopy is that the long excitation wavelengths from the two photons are less damaging to the specimen. Long excitation wavelengths also provide deeper penetration of tissues, on average between five and twenty times deeper than conventional fluorescent microscopes.

Furthermore, a deeper penetration is produced due to a reduced scattering and a reduction in the absorption of light by chromophores (i.e. a light absorbing group of atoms and electrons responsible for the color of a compound). Because the excitation occurs near the focal plane, tissue damage is reduced to the surrounding area. A further advantage to the concentrated area of the laser is that resulting images have fewer blurs, therefore avoiding difficulties in interpretation caused by light that is out-of-focus.

Further Reading

• All Fluorescence Microscopy Content
• How do Epifluorescence Microscopes Work?
• GFP-tagging in Fluorescence Microscopy
• Advances in Fluorescence Microscopy
• Wide-field Fluorescence Microscopy