Three‐photon‐resonance‐enhanced third‐harmonic generation for label‐free deep‐brain imaging: In search of a chemical contrast

Abstract

Within the past decade, nonlinear Raman microscopy has earned a well‐deserved status of a gold‐standard technology for chemically selective imaging. Even though second‐ and third‐harmonic microscopy is much less demanding on a laser source and multifrequency beam arrangement, it is increasingly falling behind nonlinear Raman scattering as a method of bioimaging because it offers no mechanism whereby imaging could be made chemically specific. Here, we show, however, that such a mechanism does exist, helping harmonic‐generation microscopy overcome its no‐chemical‐specificity handicap. We demonstrate that, with the laser wavelength tuned to a three‐photon resonance with the Soret band of hemoglobin, third‐harmonic generation provides a chemically specific method for a high‐contrast imaging of red blood cells in a broad class of biological systems, including live brain. Moreover, third‐harmonic generation imaging can be conveniently combined with second‐harmonic microscopy on a compact laser platform, providing, as our experiments on rat brain show, a powerful resource for three‐dimensional, cell‐specific label‐free deep‐brain imaging.

Single‐beam optogenetic multimodal χ(3)/χ(5) nonlinear microscopy and brain imaging

“…With the laser wavelength set at λ 0 ≈ 1.25 μm, the THG response from RBCs is resonantly enhanced due to a three‐photon resonance with the Soret band of hemoglobin. [ 51 ] 3PEF microscopy, on the other hand, provides a powerful tool to image SypHer3s‐stained astrocytes along with their processes and endfeet but does not necessarily offer means to resolve individual RBCs. A combination of 3PEF and THG imaging modalities is thus central for the microscopy of gliovascular interfaces, [ 52 ] with 3PEF and THG providing two signals needed to image, respectively, the endfeet of astrocyte processes and blood vessels (Figure 4c)—two essential constituents of a gliovascular interface.…”

Exploring the future of regenerative medicine: Unveiling the potential of optical microscopy for structural and functional imaging of stem cells

“…This technique holds an advantage as it can be applied for living, dynamic, biological, and non-biological specimens [120]. One of the major disadvantages of THG is its weak capability toward chemical specificity [121]. With better development, THG can be then implemented in field research and clinical applications [117].…”

Recent advances in linear and nonlinear Raman spectroscopy. Part XIV

“…Lanin et al [ 113 ] performed three‐photon‐resonance‐enhanced third‐harmonic generation for label‐free deep‐brain imaging in search of a new form of chemical contrast. They demonstrate that with the laser wavelength tuned to a three‐photon resonance with the Soret band of hemoglobin, third‐harmonic generation provides a chemically specific method for a high‐contrast imaging of red blood cells in a broad class of biological systems, including live brain.…”