Three-Photon Microscopy Reveals Immune Cell Behavior in Real Time

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A research group from Cornell University has used three-photon microscopy to image the full depth of the popliteal lymph node in a living mouse — reportedly for the first time. The researchers successfully observed the dynamic interactions of immune cells in the lymph node in real time and at micron-scale resolution.

The three-photon imaging technique for visualizing lymph nodes could provide the same level of precision for cancer biology and other biomedical research fields, according to the team.

The group is led by professor Chris Xu, the inventor of three-photon microscopy, a technique that uses fluorescent markers and laser illumination to acquire high-resolution images in 3D. Xu’s group previously used three-photon microscopy to image the brains of mice and zebra fish with resolution up to 2 mm.

As challenging as the brain is to image, it is even more difficult to capture images of a lymph node. “Compared to the brain, the lymph node is much more opaque,” Xu said. “In the brain, our technique now can go to 2 mm. And in the lymph node, the best we can do is a little bit less than a millimeter, because the lymph node is at least twice as dense as the brain.”

Research team member Kibaek Choe reoptimized the three-photon imaging process to make it applicable for an anatomical environment loaded with blood vessels and cells instead of neurons. The researchers used long-wavelength (1300 to 1700 nm) three-photon imaging to visualize blood vessels, lymphatic vessels, and migrating T cells and B cells in the mouse lymph node in vivo, over the node’s entire depth of 600 to 900 μm. They also imaged blood vessels in the mouse’s spleen at a depth of 570 µm.

The team measured CD8+ T cells and CD4+ T cell motility in the T cell zone, without reporting signal loss and at submicron spatial resolution. The motility of naïve CD4+ T cells in the T cell zone during lipopolysaccharide-induced inflammation was dependent on depth.

A 3D reconstruction shows the distribution of CD8+ T cells (red) and CD4+ T cells (green), along with lymphatic sinuses (purple), captured through three-photon microscopy in a mouse lymph node. Courtesy of Cornell University.
A 3D reconstruction shows the distribution of CD8+ T cells (red) and CD4+ T cells (green), along with lymphatic sinuses (purple), captured through three-photon microscopy in a mouse lymph node. Courtesy of Cornell University.
In addition to providing clarity and high resolution, the three-photon imaging technique provides the ability to simultaneously tag cells and vessels in the lymph node with four different fluorescent dyes. In the future, this capability will allow immunologists to observe how the cells interact with each other and with their environment, the researchers said.

“The type of dynamic tracking we did is exactly what immunologists would like to do,” Xu said. “Now for the first time, we can see things people have never seen before deep inside a lymph node, from the top to the bottom, in a living, breathing animal in real time. I think it will open doors for immunology.

“It’s very hard to image very deep into the tissue. And when a lymph node inflames, it becomes much bigger and it’s even harder to go through. At this point, we know this technology fundamentally can go maybe another factor of two deeper than what we have shown. I think we have some really interesting directions to push it all the way through.”

Xu’s group partnered with professor Ari M. Melnick at Weill Cornell Medicine.

The research was published in Nature Immunology (

Published: February 2022
three-photon microscopy
Three-photon microscopy is an advanced imaging technique that belongs to the family of multi-photon microscopy methods. It is used in biological and medical research to capture high-resolution images of biological specimens, particularly in thick tissues, without causing significant damage. This technique takes advantage of the nonlinear optical processes that occur when multiple photons interact with a fluorophore to achieve fluorescence imaging. Key concepts behind three-photon microscopy...
Fluorescence is a type of luminescence, which is the emission of light by a substance that has absorbed light or other electromagnetic radiation. Specifically, fluorescence involves the absorption of light at one wavelength and the subsequent re-emission of light at a longer wavelength. The emitted light occurs almost instantaneously and ceases when the excitation light source is removed. Key characteristics of fluorescence include: Excitation and emission wavelengths: Fluorescent materials...
Research & TechnologyeducationCornellAmericasMicroscopymultiphoton microscopyThree-Photon MicroscopyimmunologyBiophotonicsmedicalImagingbiomedical imagingChris Xudyefluorescencefluorescent dyesfluorescent dye moleculesBioScan

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