Multiphoton microscopy moves forward

Jimmy Fong explores how advances in a modern microscopy technique are enabling emerging biological applications

Confocal microscopy has dominated life sciences research since the late 1980s. By rastering a laser beam and relying on a pinhole to block the out-of-focus emission, confocal microscopy can generate thin optical sections and has remained the standard technique for imaging both fixed and living samples for many years. Nevertheless, confocal microscopy has certain constraints, especially in terms of how deep it can image into a sample. These limitations have spurred development and rapid adoption of multiphoton microscopy (MPM), which is a related technique, but that can produce 3D images of biological structures and processes at unprecedented depths.

Principles of two-photon microscopy

In confocal microscopy, a single photon of visible light is absorbed by a fluorophore, causing it to emit a fluorescent photon. By contrast, in two-photon microscopy (the most widely used form of MPM), two photons are absorbed simultaneously by the fluorescent molecule. In this case, the two photons used to excite the molecule are of half the energy, or twice the wavelength, of its single photon counterpart to bridge the same energy transition and induce fluorescence.

The two-photon effect, in comparison to single-photon excitation, requires higher powered, pulsed lasers that deliver the energy to the molecule almost simultaneously. These ultrashort laser pulses are focused to a single point within the sample, where it is the only spot that has sufficient photon flux to cause fluorescence. In other parts of the specimen, where photons are present at much lower densities, two-photon absorption generally does not occur, minimising unwanted fluorescence and out-of-focus background signal. This translates to high signal-to-noise imaging and optical sectioning during acquisition.

In general, the most common reason MPM is chosen for an experiment is because of its depth penetration. The longer wavelengths of near infrared light are scattered less than visible photons, and in MPM a pinhole is not necessary since the excitation is confined to a focus spot. This allows more fluorescent photons, especially scattered ones, to be detected since there is not a pinhole restricting the emission.

Recent advances in multiphoton microscopy technology

Typically, two-photon microscopy relies on exciting fluorophores at a single focal point at a time. To build an entire 3D image, galvanometric (galvo) mirrors are typically used to vary mirror angles and scan the beam across the sample. By having two mirrors for the x and y plane, it becomes possible to create a 2D image. One deficiency of these scanning galvo mirrors, however, is the limited speed at which they can move, due to motor heating during repeated acceleration and deceleration. With the advent of resonant galvo mirrors that operate at a fixed frequency, shorter acquisition times and faster frame rates are now possible.

The dynamics of many biological events, such as immune cell interactions or neuron firing, occur over timescales of milliseconds or faster. Capturing these events is aided by resonant scanners in the two lateral dimensions, but also by advancements in scanning in the axial direction. Fast piezo-electric stages for objective lenses and electrically tunable lenses have been incorporated in MPM to quickly change the imaging focal plane to capture cellular events in three dimensions.

Optical stimulation is also offering new potential for studying responses to light, as in activation of neural circuits. With developments in the field of optogenetics, neurons can be manipulated to express both light-sensitive channel proteins, called opsins, and neural activity reporters, such as GCaMP calcium indicators. When stimulated with certain wavelengths, the opsin channels open, causing the illuminated neuron to fire. The GCaMP reporter can then be imaged allowing neural activity to be monitored in response to the specific activation.

Advancements in stimulation technologies have enabled activation to move beyond single-cell activation and provide the ability to selectively excite ensembles of neurons in 3D. This is made possible by spatial light modulators (SLMs), which split a single laser beam into a holographic pattern of spots to stimulate multiple cells at the same time. Looking forward, as two-photon microscopy has become well adopted, emerging three-photon microscopy offers the potential to image even deeper into samples. As further infrared wavelengths are scattered even less, three-photon excitation has the promise to be able to image several millimetres into the brain.

Evolving applications for multiphoton microscopy technology

Many of the applications of two-photon microscopy are in neurophysiology, where it is being used to image both explanted samples of brain tissue and neurons in the brains of living animals. Imaging in these tissues is often correlated to electrical measurements of activity to paint a more complete picture. Optogenetics with MPM is another area where studies can be conducted to link animal behaviour to the processing in the brain. Combining these techniques and technologies together with more efficient opsins and improved reporters will serve to create more opportunities for highly sophisticated neural investigations.

In the years since its development, MPM has become a preferred means for imaging biological processes in living cells, tissues and animals. Offering unmatched penetration depth and low phototoxicity, a wealth of applications for MPM will continue to emerge in neurology, cell biology, embryology, and immunology. As the development of MPM continues to accelerate, many more applications are undoubtedly just around the corner.

Jimmy Fong is with Bruker




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