École Polytechnique images blood cells with TSFG microscopy

09 Feb 2023

Third-harmonic technique adds new capabilities to deep-tissue imaging.

Imaging of red blood cells (RBCs) and blood vessels is an increasingly valuable biophotonics application, with two-photon (2P) microscopy often the technique of choice.

These techniques allow deeper imaging with longer wavelength excitation and reduced photodamage compared to classical microscopy, and are continually being refined. A project at MIT and Harvard University, for example, has tackled the inherently limited throughout of a non-linear 2P imaging workflow compared to conventional wide-field techniques.

Some hurdles still remain, however, including limitations on the imaging times achievable, and the risk of interference from any out of focus background signals present in the field of view.

A project at France's École Polytechnique has now developed a new label-free imaging method for red blood cells and oxygenation, by extending the non-linear imaging operation to third-order principles. The work was reported in Light: Science & Applications.

The research builds on the increasing availability of high power laser amplifiers and sources in relevant wavelength ranges for imaging of blood vessels, which bring third-harmonic generation (THG) effects into play. These THG effects are potentially better at identifying the interfaces between different component parts of biological systems, the points at which optical properties change.

The Paris project, based at the École's Laboratory for Optics & Biophotonics, set out to develop a functional imaging method based on characterizing the wavelength dependence of THG signals from red cells, along with a spectral imaging scheme compatible with microscopy of moving objects such as RBCs flowing inside blood vessels. It then applied the method to the imaging of zebrafish and human RBCs.

The resulting spectroscopic version of THG microscopy could be particularly relevant for probing the absorption properties of non-fluorescent objects, potentially yielding specific contrast from red blood cells during in vivo examination.

Neuroscience and physiology applications

One key aspect of the team's method is its simultaneous measurement of signals from both THG and a further multiplexed non-linear phenomenon termed third-order sum frequency generation (TSFG), arising when more than one excitation wavelength is used.

The project found that if two femtosecond pulse trains are used for excitation, then three or four distinct THG and TSFG signals can be stimulated and detected, effectively allowing a multicolor THG imaging operation. Initial trials on zebrafish showed that the method allowed label-free RBC-specific contrast at depths exceeding 600 microns in live adult animal brains.

A further discovery was that that the oxygenation state of hemoglobin could also be indicated by the same method, specifically through analysis of the ratio of signals measured at 401 and 433 nanometers. Both the imaging of cells and the determination of oxygenation were then successfully observed with human blood as well.

The team expects that its new method could be readily implemented on existing three-photon microscope systems, adding a label-free hemoglobin-specific contrast to these platforms, and providing a way to separate blood from other visible structures under examination.

"This brings important perspectives for multiphoton imaging based on third-order contrast, and it should open the way to a variety of applications in neuroscience and physiology," commented the team in its paper.