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We present experiments on cell cultures and brain slices that demonstrate two‐photon optogenetic pH sensing and pH‐resolved brain imaging using a laser driver whose spectrum is carefully tailored to provide the maximum contrast of a ratiometric two‐photon fluorescence readout from a high‐brightness genetically encoded yellow‐fluorescent‐protein‐based sensor, SypHer3s. Two spectrally isolated components of this laser field are set to induce two‐photon‐excited fluorescence (2PEF) by driving SypHer3s through one of two excitation pathways—via either the protonated or deprotonated states of its chromophore. With the spectrum of the laser field accurately adjusted for a maximum contrast of these two 2PEF signals, the ratio of their intensities is shown to provide a remarkably broad dynamic range for pH measurements, enabling high‐contrast optogenetic deep‐brain pH sensing and pH‐resolved 2PEF imaging within a vast class of biological systems, ranging from cell cultures to the living brain. [ABSTRACT FROM AUTHOR]

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, cell‐specific 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. [ABSTRACT FROM AUTHOR]

The lack of oxygen (O2) causes changes in the cell functioning. Modeling hypoxic conditions in vitro is challenging given that different cell types exhibit different sensitivities to tissue O2 levels. We present an effective in vivo platform for assessing various tissue and organ parameters in Danio rerio larvae under acute hypoxic conditions. Our system allows simultaneous positioning of multiple individuals within a chamber where O2 level in the water can be precisely and promptly regulated, all while conducting microscopy. We applied this approach in combination with a genetically encoded pH-biosensor SypHer3s and a highly H2O2-sensitive Hyper7 biosensor. Hypoxia causes H2O2 production in areas of brain, heart and skeletal muscles, exclusively in the mitochondrial matrix; it is noteworthy that H2O2 does not penetrate into the cytosol and is neutralized in the matrix upon reoxygenation. Hypoxia causes pronounced tissue acidosis, expressed by a decrease in pH by 0.4-0.6 units everywhere. Using imaging photoplethysmography, we measured in D.rerio fry real-time heart rate decrease under conditions of hypoxia and subsequent reoxygenation. Our observations in this experimental system lead to the hypothesis that mitochondria are the only source of H2O2 in cells of D.rerio under hypoxia.

Cellular antioxidant systems control the levels of hydrogen peroxide (H 2 O 2 ) within cells. Multiple theoretical models exist that predict the diffusion properties of H 2 O 2 depending on the rate of H 2 O 2 generation and amount and reaction rates of antioxidant machinery components. Despite these theoretical predictions, it has remained unknown how antioxidant systems shape intracellular H 2 O 2 gradients. The relative role of thioredoxin (Trx) and glutathione systems in H 2 O 2 pattern formation and maintenance is another disputed question. Here, we visualized cellular antioxidant activity and H 2 O 2 gradients formation by exploiting chemogenetic approaches to generate compartmentalized intracellular H 2 O 2 and using the H 2 O 2 biosensor HyPer to analyze the resulting H 2 O 2 distribution in specific subcellular compartments. Using human HeLa cells as a model system, we propose that the Trx system, but not the glutathione system, regulates intracellular H 2 O 2 gradients. Antioxid. Redox Signal. 31, 664-670.