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

Abstract

We demonstrate single-beam optogenetic multimodal nonlinear-optical microscopy that combines third-harmonic generation (THG) and three-photon-excited fluorescence (3PEF)two nonlinear-optical processes related to the third-and fifth-order susceptibilities, χ (3) and χ (5). A carefully tailored unamplified short-pulse output of mode-locked solid-state lasers is shown to provide an ample parameter space for the optimization of such a single-beam multimodal microscopy, enabling subcellular-resolution, cellspecific imaging using genetically encoded fluorescent-protein-based reporters in a vast variety of biological systems and settings, ranging from HeLa to brain cells. Experiments on brain slices and cell cultures presented in this paper demonstrate the potential of short-pulse 3PEF/THG microscopy for a subcellular-resolution, high-contrast imaging of HeLa-line cancer-cell derivatives, as well as mitochondrial and somatic intracellular structures within deep-brain neurons and astrocytes. As a step beyond the state of the art in optical brain imaging, single-beam subcellular-resolution, cell-specific optogenetic 3PEF/THG imaging of fundamental functional brain units is experimentally demonstrated. One fundamental question that this work brings up for the future nonlinear absorption and Raman/hyper-Raman studies is whether the Herzberg-Teller corrections, needed for an accurate description of two-photon absorption in fluorescent proteins (FPs), would be also sufficient for an adequate treatment of higher-n n-photon absorption in FP-based systems or models that include other quantum pathways would be necessary for the analysis of FP agents for higher-n nonlinear microscopy.

Real‐time fiber‐optic recording of acute‐ischemic‐stroke signatures

“…When excited via a suitable 2-or 3-photon pathway [1][2][3][70][71][72][73][74][75], the fluorescence readout has been shown to provide spatial and temporal resolution that is substantially higher than the spatial and temporal resolution attainable with CT, PET, LSI, OCT, or PAI. However, attenuation of optical radiation due to absorption and scattering by brain tissues severely limits the imaging depths accessible via optical imaging.…”

Single‐beam dual‐color alternate‐pathway two‐photon spectroscopy: Toward an optical toolbox for redox biology

“…Viewed in a broader context, this idea leads us to rethink the potential of other multicolor nonlinear-optical processes, such as multiphoton-absorption-driven fluorescence [26,27] and photochromism, [28] as well as transient refraction/ absorption, [29][30][31] and to explore whether a single-beam implementation of these methods could help advance their real-life applications. The thrust for such an extension is now stronger than ever, as the range of applications of multicolor nonlinear processes continues to grow, extending from canonical, all-optical time-resolved studies [31][32][33][34] to multimodal bioimaging, [35,36] optical data storage, [37] neuroscience, [38] attosecond physics, [39][40][41][42] and innovative pump-probe approaches combining conventional lasers with advanced free-electron radiation sources, [43,44] as well as ultrashort optical and electron pulses. [45] Here, we extend the Motzkus-Silberberg ideas of single-beam nonlinear optics to multicolor multiphoton absorption spectroscopy.…”

Development and applications of nonlinear optical spectroscopy: 18th ECONOS/38th ECW meeting in Rouen (France)

“…The data revealed clear correlations that are believed to be of general nature and offer wide applicability. Lanin et al [ 2 ] demonstrate an approach for single‐beam optogenetic multimodal nonlinear‐optical microscopy. For this purpose, they combined third‐harmonic generation and three‐photon‐excited fluorescence.…”