Multiphoton Microscopy Applications

Three-Photon Imaging

Three-photon microscopy improves the imaging depth of multiphoton microscopy, making it one of the most promising recent advances in multiphoton microscopy technology

Bruker's Ultima 2Pplus multiphoton microscope is equipped standard to enable three-photon imaging experiments. Lens coatings throughout the optical path are designed for using extended wavelength laser sources, with special care taken in the design of the system to allow for short laser pulse-widths at the sample, which is critical for efficient three-photon excitation. Customers continue to successfully publish exciting three-photon experiments on Bruker multiphoton microscopes, and we hope to grow the community using this technique.

Diving Deeper: Three-Photon Microscopy and Imaging

What is three-photon microscopy?

Three-photon microscopy is a fluorescence microscopy technique where, in contrast to two-photon microscopy, the fluorophore absorbs three photons almost simultaneously. These excitation photons have longer wavelengths and lower energy than those used to excite the same fluorophore in one- or two-photon excitation microscopy.


How do researchers benefit from using three-photon microscopy?

Two-photon excitation microscopy has facilitated fundamental discoveries in life science research. Unfortunately, two-photon technology suffers from some limitations. Specifically, when imaging at and below 500 µm, the background signal increases and the contrast decreases — making it impossible to resolve either structures or physiological activity. Conversely, the physical characteristics of the excitation light in three-photon excitation microscopy offer deeper imaging with superior confinement of the excitation, thereby providing better contrast and signal-to-background ratio (SBR) than two-photon microscopy.

Three-photon imaging in the mouse visual cortex in vivo. The z-stack progresses dorso-ventrally, from the pial surface to below the white matter. The final z-slice is 1.1 mm below the pial surface. Blood vessels are shown in magenta and neuronal cell bodies labeled with a calcium dye are shown in yellow. Fibers and processes including the white matter are shown in cyan. Data courtesy of Dr. Prakash Kara, U. Minnesota. See additional three-photon publications from the Kara lab here:

Life Science Research Applications of Three-Photon Microscopy

Imaging in larger animals

Two-photon microscopy of the cortex in cats and monkeys allows imaging only up to layers II and III. More complete study of cortical circuits requires access to layer VI, which is well below 1 mm deep in brain tissue. Three-photon microscopy enables structural imaging of cortical columns and functional imaging in larger animals like rats, cats, ferrets, macaques.

Imaging through un-thinned skull

The ability to image neurons through the un-thinned skull is an ideal approach for experiments where any surgically-induced inflammation or brain swelling would compromise the validity of the study. In addition, this approach facilitates everyday sample preparation.

Non-invasive imaging through opaque cuticle

Recent study shows that three-photon microscopy can be used for non-invasive imaging through an opaque cuticle in popular model insects like flies. Therefore, this technology could be also applied to non-invasive imaging in ants and mosquitos another invertebrate models.

Imaging throughout an animal's lifespan

Three-photon microscopy enables new opportunities to study the development of zebrafish from newborn to adulthood.

Deeper imaging in the spinal cord and brain stem

Three-photon microscopy enables deeper imaging than two-photon microscopy in a mouse spinal cord and brainstem.

Deeper imaging in non-sparsely labeled brain tissue

Because three-photon microscopy provides greater image contrast than two-photon microscopy, it is an excellent choice for imaging in densely labeled scattering brain tissue like Thy1-GFP mice.

Three-color imaging in non-sparsely labeled brain tissue

Research based on multicolor three-photon fluorescence (THG, GCaMP, and TexasRed) imaging with single-wavelength excitation deep in the mouse brain has been published recently upon 1340 nm wavelength excitation.

Intravital imaging by label-free autofluorescence multi-harmonic microscopy

Simultaneous imaging of autofluorescence (FAD and NADH) and second/third harmonic generation from a variety of array of cellular and extracellular components in living tissue, such as tumor cells, immune cells, and vessels can provide more complete insights into the physiology and pathology of disease.

Practical Considerations for Three-Photon Microscopy

What are the limitations of three-photon microscopy?

The primary obstacle to the popularization of three-photon imaging has been the predicted increase in water absorption at the long wavelength window, as this effect might lead to overheating of the sample. However, researchers have found that the optimum wavelength for three-photon imaging is a trade-off between absorption and scattering in the 1300 nm and 1700 nm windows. Specifically, there is 2x more absorption but also almost 2x less scattering at 1300 nm, and as a result, imaging requires less laser power to be used. This demonstrates that there is enough room to accommodate research goals while complying with the physical constraints of three-photon microscopy. The convenience of three-photon imaging further balances out these constraints, as many common indicators used in two-photon microscopy can be easily re-purposed for use in three-photon imaging.

It is also important to consider the cost of the special laser sources and imaging lenses suitable for three-photon imaging. Typically, two lasers are needed for this application. To increase the probability of three-photon absorption, researchers must use a light source with a high photon density. The most common approach is based on a high-power laser pumping an optical parametric amplifier (OPA), which takes light of one wavelength from a femtosecond laser and turns it into light of two different wavelengths. The resulting beam, called the idler beam, that demonstrates a longer wavelength than the initial pumped laser is ideal for three-photon imaging. OPA is a low repetition laser (~1-4 MHz) and due to this characteristic, imaging with a resonant scanner at 30 Hz speed is not allowed. Typical imaging rates are less than 10 Hz with smaller fields-of-view, which could improve with the advancement of laser technology.

Raw images from three-photon GCaMP6 imaging ~800μm below the pial surface of the cat visual cortex. Prakash Kara lab, U.Minnesota

Available fluorophores for three-photon microscopy

Fluorescent protein

1P excitation (nm) Emission (nm) 3P excitation (nm)
mOrange 548 560 1645
tdTomato, Ds-Red 555 583 1665
mCherry, Jred 587 610 1755
Crimson-EM 590 650 1770
GFP, GCaMP 488 525 1300