Three-Photon Infrared Stimulation of Endogenous Neuroreceptors in Vivo.

To interrogate neural circuits and crack their codes, in vivo brain activity imaging must be combined with spatiotemporally precise stimulation in three dimensions using genetic or pharmacological specificity. This challenge requires deep penetration and focusing as provided by infrared light and multiphoton excitation, and has promoted two-photon photopharmacology and optogenetics. However, three-photon brain stimulation in vivo remains to be demonstrated. We report the regulation of neuronal activity in zebrafish larvae by three-photon excitation of a photoswitchable muscarinic agonist at 50 pM, a billion-fold lower concentration than used for uncaging, and with mid-infrared light of 1560 nm, the longest reported photoswitch wavelength. Robust, physiologically relevant photoresponses allow modulating brain activity in wild-type animals with spatiotemporal and pharmacological precision. Computational calculations predict that azobenzene-based ligands have high three-photon absorption cross-section and can be used directly with pulsed infrared light. The expansion of three-photon pharmacology will deeply impact basic neurobiology and neuromodulation phototherapies.

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas.

Retinal dystrophies are a leading cause of blindness worldwide. Extensive efforts are underway to develop advanced retinal prostheses that can bypass the impaired light-sensing photoreceptor cells in the degenerated retina, aiming to partially restore vision by inducing visual percepts. One common avenue of investigation involves the design and production of implantable devices with a flexible physical structure, housing a high number of electrodes. This enables the efficient and precise generation of visual percepts. However, with each technological advancement, there arises a need for a reliable and manageable ex vivo method to verify the functionality of the device before progressing to in vivo experiments, where factors beyond the device's performance come into play. This article presents a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer (GCL) following electrical stimulation. Specifically, the following steps are outlined: (1) fluorescently labeling the rat retina using genetically encoded calcium indicators, (2) capturing the fluorescence signal using an inverted fluorescence microscope while applying distinct patterns of electrical stimulation, and (3) extracting and analyzing the calcium traces from individual cells within the GCL. By following this procedure, researchers can efficiently test new stimulation protocols prior to conducting in vivo experiments.

Volumetric imaging of fast cellular dynamics with deep learning enhanced bioluminescence microscopy.

Bioluminescence microscopy is an appealing alternative to fluorescence microscopy, because it does not depend on external illumination, and consequently does neither produce spurious background autofluorescence, nor perturb intrinsically photosensitive processes in living cells and animals. The low photon emission of known luciferases, however, demands long exposure times that are prohibitive for imaging fast biological dynamics. To increase the versatility of bioluminescence microscopy, we present an improved low-light microscope in combination with deep learning methods to image extremely photon-starved samples enabling subsecond exposures for timelapse and volumetric imaging. We apply our method to image subcellular dynamics in mouse embryonic stem cells, epithelial morphology during zebrafish development, and DAF-16 FoxO transcription factor shuttling from the cytoplasm to the nucleus under external stress. Finally, we concatenate neural networks for denoising and light-field deconvolution to resolve intracellular calcium dynamics in three dimensions of freely moving Caenorhabditis elegans.

Few-cycle all-fiber supercontinuum laser for ultrabroadband multimodal nonlinear microscopy.

Temporally coherent supercontinuum sources constitute an attractive alternative to bulk crystal-based sources of few-cycle light pulses. We present a monolithic fiber-optic configuration for generating transform-limited temporally coherent supercontinuum pulses with central wavelength at 1.06 µm and duration as short as 13.0 fs (3.7 optical cycles). The supercontinuum is generated by the action of self-phase modulation and optical wave breaking when pumping an all-normal dispersion photonic crystal fiber with pulses of hundreds of fs duration produced by all-fiber chirped pulsed amplification. Avoidance of free-space propagation between stages confers unequalled robustness, efficiency and cost-effectiveness to this novel configuration. Collectively, the features of all-fiber few-cycle pulsed sources make them powerful tools for applications benefitting from the ultrabroadband spectra and ultrashort pulse durations. Here we exploit these features and the deep penetration of light in biological tissues at the spectral region of 1 µm, to demonstrate their successful performance in ultrabroadband multispectral and multimodal nonlinear microscopy.

Analysis of intracellular protein dynamics in living zebrafish embryos using light-sheet fluorescence single-molecule microscopy.

Single-molecule microscopy techniques have emerged as useful tools to image individual molecules and analyze their dynamics inside cells, but their application has mostly been restricted to cell cultures. Here, a light-sheet fluorescence microscopy setup is presented for imaging individual proteins inside living zebrafish embryos. The optical configuration makes this design accessible to many laboratories and a dedicated sample-mounting system ensures sample viability and mounting flexibility. Using this setup, we have analyzed the dynamics of individual glucocorticoid receptors, which demonstrates that this approach creates multiple possibilities for the analysis of intracellular protein dynamics in intact living organisms.

Interference effects in quantum-optical coherence tomography using spectrally engineered photon pairs.

Optical-coherence tomography (OCT) is a technique that employs light in order to measure the internal structure of semitransparent, e.g. biological, samples. It is based on the interference pattern of low-coherence light. Quantum-OCT (QOCT), instead, employs the correlation properties of entangled photon pairs, for example, generated by the process of spontaneous parametric downconversion (SPDC). The usual QOCT scheme uses photon pairs characterised by a joint-spectral amplitude with strict spectral anti-correlations. It has been shown that, in contrast with its classical counterpart, QOCT provides resolution enhancement and dispersion cancellation. In this paper, we revisit the theory of QOCT and extend the theoretical model so as to include photon pairs with arbitrary spectral correlations. We present experimental results that complement the theory and explain the physical underpinnings appearing in the interference pattern. In our experiment, we utilize a pump for the SPDC process ranging from continuous wave to pulsed in the femtosecond regime, and show that cross-correlation interference effects appearing for each pair of layers may be directly suppressed for a sufficiently large pump bandwidth. Our results provide insights and strategies that could guide practical implementations of QOCT.