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Simultaneous multiview light-sheet microscopy using two illumination and two detection arms with one- or two-photon illumination is coupled to a fast data acquisition framework and analysis pipeline for quantitative imaging and tracking of individual cells and the developing nervous system throughout a living fly embryo. A related paper by Krzic et al . is also in this issue. Live imaging of large biological specimens is fundamentally limited by the short optical penetration depth of light microscopes. To maximize physical coverage, we developed the SiMView technology framework for high-speed in vivo imaging, which records multiple views of the specimen simultaneously. SiMView consists of a light-sheet microscope with four synchronized optical arms, real-time electronics for long-term sCMOS-based image acquisition at 175 million voxels per second, and computational modules for high-throughput image registration, segmentation, tracking and real-time management of the terabytes of multiview data recorded per specimen. We developed one-photon and multiphoton SiMView implementations and recorded cellular dynamics in entire Drosophila melanogaster embryos with 30-s temporal resolution throughout development. We furthermore performed high-resolution long-term imaging of the developing nervous system and followed neuroblast cell lineages in vivo . SiMView data sets provide quantitative morphological information even for fast global processes and enable accurate automated cell tracking in the entire early embryo.

Structural and molecular imaging of the developing embryo can provide deep insights into the development of various pathologies, but few techniques enable the simultaneous detection of these parameters. We demonstrate the first use of combined optical coherence tomography and two-photon light sheet fluorescence microscopy (2P-LSFM) for simultaneous structural and molecular imaging. We aim to develop a multimodal high-resolution embryonic system that facilitates simultaneous structural and molecular embryonic imaging. We have developed a multimodal imaging system in which the optical coherence tomography (OCT) and light sheet illumination beams were optically co-aligned and scanned through the galvanometer-mounted mirrors and the same illumination objective. The swept-source OCT system provides a lateral resolution of and an axial resolution of . The 2P-LSFM light sheet thickness was , and the transverse resolution was . We have demonstrated the system's capabilities using fluorescent microbeads and fluorescently tagged mouse embryos. The co-alignment of the OCT and 2P-LSFM systems enables simple image registration and high-throughput multimodal imaging.

An optical phase-locked ultrasound lens integrated into a two-photon microscope enables continuous volumetric imaging of biological processes in vivo . In vivo imaging at high spatiotemporal resolution is key to the understanding of complex biological systems. We integrated an optical phase-locked ultrasound lens into a two-photon fluorescence microscope and achieved microsecond-scale axial scanning, thus enabling volumetric imaging at tens of hertz. We applied this system to multicolor volumetric imaging of processes sensitive to motion artifacts, including calcium dynamics in behaving mouse brain and transient morphology changes and trafficking of immune cells.

Among optical imaging techniques light sheet fluorescence microscopy is one of the most attractive for capturing high-speed biological dynamics unfolding in three dimensions. The technique is potentially millions of times faster than point-scanning techniques such as two-photon microscopy. However light sheet microscopes are limited by volume scanning rate and/or camera speed. We present speed-optimized Objective Coupled Planar Illumination (OCPI) microscopy, a fast light sheet technique that avoids compromising image quality or photon efficiency. Our fast scan system supports 40 Hz imaging of 700 μm-thick volumes if camera speed is sufficient. We also address the camera speed limitation by introducing Distributed Planar Imaging (DPI), a scaleable technique that parallelizes image acquisition across cameras. Finally, we demonstrate fast calcium imaging of the larval zebrafish brain and find a heartbeat-induced artifact, removable when the imaging rate exceeds 15 Hz. These advances extend the reach of fluorescence microscopy for monitoring fast processes in large volumes. Light sheet microscopy holds potential for imaging dynamics in 3D biological specimens, but is limited by scan speed and camera acquisition rate. Here the authors address both issues by developing speed-optimized Objective Coupled Planar Illumination and parallelizing image acquisition across cameras to achieve 40 Hz imaging over thick samples.

Intracellular endogenous fluorescent co-enzymes, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD), play a pivotal role in cellular metabolism; quantitative assessment of their presence in living cells can be exploited to monitor cellular energetics in Parkinson’s disease (PD), a neurodegenerative disorder. Here, we applied two-photon fluorescence lifetime imaging microscopy (2P-FLIM) to noninvasively measure the fluorescence lifetime components of NADH and FAD and their relative contributions in MPP + (1-methyl-4-phenylpyridinium) treated neuronal cells, derived from PC12 cells treated with nerve growth factor (NGF), to mimic PD conditions. A systematic FLIM data analysis showed a statistically significant (p < 0.001) decrease in the fluorescence lifetime of both free and protein-bound NADH, as well as free and protein-bound FAD in MPP + treated cells. On the relative contributions of the free and protein-bound NADH and FAD to the life time, however, both the free NADH contribution and the corresponding protein-bound FAD contribution increase significantly (p < 0.001) in MPP + treated cells, compared to control cells. These results, which indicate a shift in energy production in the MPP + treated cells from oxidative phosphorylation towards anaerobic glycolysis, can potentially be used as cellular metabolic metrics to assess the condition of PD at the cellular level.

Autofluorescence characteristics of the reduced nicotinamide adenine dinucleotide and oxidized flavin cofactors are important for the evaluation of the metabolic status of the cells. The approaches that involve a detailed analysis of both spectral and time characteristics of the autofluorescence signals may provide additional insights into the biochemical processes in the cells and biological tissues and facilitate the transition of spectral fluorescence lifetime imaging into clinical applications. We present the experiments on multispectral fluorescence lifetime imaging with a detailed analysis of the fluorescence decays and spectral profiles of the reduced nicotinamide adenine dinucleotide and oxidized flavin under a single excitation wavelength aimed at understanding whether the use of multispectral detection is helpful for metabolic imaging of cancer cells. We use two-photon spectral fluorescence lifetime imaging microscopy. Starting from model solutions, we switched to cell cultures treated by metabolic inhibitors and then studied the metabolism of cells within tumor spheroids. The use of a multispectral detector in combination with an excitation at a single wavelength of 750 nm allows the identification of fluorescence signals from three components: free and bound NAD(P)H, and flavins based on the global fitting procedure. Multispectral data make it possible to assess not only the lifetime but also the spectral shifts of emission of flavins caused by chemical perturbations. Altogether, the informative parameters of the developed approach are the ratio of free and bound NAD(P)H amplitudes, the decay time of bound NAD(P)H, the amplitude of flavin fluorescence signal, the fluorescence decay time of flavins, and the spectral shift of the emission signal of flavins. Hence, with multispectral fluorescence lifetime imaging, we get five independent parameters, of which three are related to flavins. The approach to probe the metabolic state of cells in culture and spheroids using excitation at a single wavelength of 750 nm and a fluorescence time-resolved spectral detection with the consequent global analysis of the data not only simplifies image acquisition protocol but also allows to disentangle the impacts of free and bound NAD(P)H, and flavin components evaluate changes in their fluorescence parameters (emission spectra and fluorescence lifetime) upon treating cells with metabolic inhibitors and sense metabolic heterogeneity within 3D tumor spheroids.

For generations researchers have been observing the dynamic processes of life through the lens of a microscope. This has offered tremendous insights into biological phenomena that span multiple orders of time- and length-scales ranging from the pure magic of molecular reorganization at the membrane of immune cells, to cell migration and differentiation during development or wound healing. Standard fluorescence microscopy techniques offer glimpses at such processes in vitro, however, when applied in intact systems, they are challenged by reduced signal strengths and signal-to-noise ratios that result from deeper imaging. As a remedy, two-photon excitation (TPE) microscopy takes a special place, because it allows us to investigate processes in vivo, in their natural environment, even in a living animal. Here, we review the fundamental principles underlying TPE aimed at basic and advanced microscopy users interested in adopting TPE for intravital imaging. We focus on applications in neurobiology, present current trends towards faster, wider and deeper imaging, discuss the combination with photon counting technologies for metabolic imaging and spectroscopy, as well as highlight outstanding issues and drawbacks in development and application of these methodologies. This review introduces the basic principle of fluorescence and two-photon excitation microscopy that is aimed at novice and advanced microscopists highlighting current techniques, trends, and practical guides for live imaging optimization.

Label-free imaging lets labs ‘see’ their samples less invasively than other techniques.

In this study we have applied high-spatial and temporal label-free imaging of individual live multidrug-resistant bacteria and bacteria-infected cells and animal tissue using two-photon fluorescence lifetime imaging microscopy (2p-FLIM). 2p-FLIM can identify and quantify fluorescence intensity and lifetimes among bacteria, infected cells, and tissues. We have implemented 2p-FLIM in combination with phasor plot analysis for quantifying molecular differences of NAD(P)H intensities and lifetimes for fast, sensitive, high-resolution, non-destructive imaging of live bacteria and bacteria-infected cells and tissues. We have further developed a coordinated workflow using 2p-FLIM for high-resolution temporal-spatial mapping of bacteria infected cells and tissues that can be performed for near real-time quantitation of NAD(P)H intensities and lifetimes for identifying changes in metabolism. 2p-FLIM may have broad applicability for characterizing microbial infection at the molecular, subcellular, cellular and tissue levels. The ability to quantitate and directly monitor changes in NAD(P)H metabolism in near real-time in bacteria cells and tissues during an infection, offers a potential mechanism for understanding microbial pathogenesis and evaluating therapeutic treatments that can be applied to multiple model systems. Overall, the application of this label-free imaging approach has the potential to address biomedical research needs and technical problems that occur broadly across multiple biological systems and diseases.