SignificanceFluorescence lifetime imaging microscopy (FLIM) of the metabolic co-enzyme nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a popular method to monitor single-cell metabolism within unperturbed, living 3D systems. However, FLIM of NAD(P)H has not been performed in a light-sheet geometry, which is advantageous for rapid imaging of cells within live 3D samples.AimWe aim to design, validate, and demonstrate a proof-of-concept light-sheet system for NAD(P)H FLIM.ApproachA single-photon avalanche diode camera was integrated into a light-sheet microscope to achieve optical sectioning and limit out-of-focus contributions for NAD(P)H FLIM of single cells.ResultsAn NAD(P)H light-sheet FLIM system was built and validated with fluorescence lifetime standards and with time-course imaging of metabolic perturbations in pancreas cancer cells with 10 s integration times. NAD(P)H light-sheet FLIM in vivo was demonstrated with live neutrophil imaging in a larval zebrafish tail wound also with 10 s integration times. Finally, the theoretical and practical imaging speeds for NAD(P)H FLIM were compared across laser scanning and light-sheet geometries, indicating a 30 × to 6 × acquisition speed advantage for the light sheet compared to the laser scanning geometry.ConclusionsFLIM of NAD(P)H is feasible in a light-sheet geometry and is attractive for 3D live cell imaging applications, such as monitoring immune cell metabolism and migration within an organism.
SPAD array sensors support higher-throughput fluorescence lifetime imaging microscopy (FLIM) by transitioning from laser-scanning to wide-field geometries. While a SPAD camera in epi-fluorescence geometry enables wide-field FLIM of fluorescently labeled samples, label-free imaging of single-cell autofluorescence is not feasible in an epi-fluorescence geometry because background fluorescence from out-of-focus culture medium masks cell autofluorescence and biases lifetime measurements. Here, we address this problem in a proof-of-concept implementation by integrating the SPAD camera in a light-sheet illumination geometry to achieve optical sectioning and limit out-of-focus contributions, enabling label-free wide-field FLIM of single-cell NAD(P)H autofluorescence.
We present a multiphoton excitation based hyperdimensional imaging microscope (HDIM). We demonstrate how this system may be used for metabolic readouts, how to evaluate samples with a very low photon budget (<50 photons/pixel), and how to combine such systems with real-time visualizations. We demonstrate how this HDIM approach can help us with metabolic interpretations in two use cases: cancer cells using NAD(P)H and in plant cells using chlorophyll fluorescence.
SignificanceAdvanced digital control of microscopes and programmable data acquisition workflows have become increasingly important for improving the throughput and reproducibility of optical imaging experiments. Combinations of imaging modalities have enabled a more comprehensive understanding of tissue biology and tumor microenvironments in histopathological studies. However, insufficient imaging throughput and complicated workflows still limit the scalability of multimodal histopathology imaging.AimWe present a hardware-software co-design of a whole slide scanning system for high-throughput multimodal tissue imaging, including brightfield (BF) and laser scanning microscopy.ApproachThe system can automatically detect regions of interest using deep neural networks in a low-magnification rapid BF scan of the tissue slide and then conduct high-resolution BF scanning and laser scanning imaging on targeted regions with deep learning-based run-time denoising and resolution enhancement. The acquisition workflow is built using Pycro-Manager, a Python package that bridges hardware control libraries of the Java-based open-source microscopy software Micro-Manager in a Python environment.ResultsThe system can achieve optimized imaging settings for both modalities with minimized human intervention and speed up the laser scanning by an order of magnitude with run-time image processing.ConclusionsThe system integrates the acquisition pipeline and data analysis pipeline into a single workflow that improves the throughput and reproducibility of multimodal histopathological imaging.
Autofluorescence-based optical imaging methods have advanced cellular metabolism research as a technique to non-invasively measure metabolic shifts temporally and spatially. In this work, we present metabolic investigations using fluorescence anisotropy in two well-defined plant and animal models: 1) A set of flavonoid-deficient Arabidopsis thaliana mutants which shows unique fluorescence polarization properties with varying degrees of flavonoid accumulation. 2) The cancer metabolic deformities manifested in animal cells using real-time fluorescence anisotropy based metabolic readout (4fps). These studies demonstrated the potential of using fluorescence anisotropy in a systematic way to study metabolism and cellular heterogeneity.
The excited state lifetime of a fluorophore together with its fluorescence emission spectrum provide information that can yield valuable insights into the nature of a fluorophore and its microenvironment. However, it is difficult to obtain both channels of information in a conventional scheme as detectors are typically configured either for spectral or lifetime detection. We present a fiber-based method to obtain spectral information from a multiphoton fluorescence lifetime imaging (FLIM) system. This is made possible using the time delay introduced in the fluorescence emission path by a dispersive optical fiber coupled to a detector operating in time-correlated single-photon counting mode. This add-on spectral implementation requires only a few simple modifications to any existing FLIM system and is considerably more cost-efficient compared to currently available spectral detectors.
Spinal cord injury (SCI) causes permanent paralysis below the damaged area. SCI is linked to neuronal death, demyelination, and limited ability of neuronal fibers to regenerate. Regeneration capacity is limited by the presence of many inhibitory factors in the spinal cord environment. The use of poly(lactide-co-glycolide) (PLG) bridges has demonstrated the ability to sustain long-term regeneration after SCI in a cervical hemisection mouse model. Critically, imaging of regenerating fibers and the myelination status of these neuronal filaments is a severe limitation to progress in SCI research. We used a transgenic mouse model that selectively expresses fluorescent reporters (eGFP) in the neuronal fibers of the spinal cord. We implanted a PLG bridge at C5 vertebra after hemisection and evaluated in live animals’ neuronal fibers at the bridge interface and within the bridge 8 weeks postimplant. These in vivo observations were correlated with in situ evaluation 12 weeks postimplantation. We sectioned the spinal cords and performed fluorescent bioimaging on the sections to observe neuronal fibers going through the bridge. In parallel, to visualize myelination of regenerated axons, we exploited the characteristics of the third-harmonic generation arising from the myelin structure in these fixed sections.
Atomic force microscopes (AFM) provide topographical and mechanical information of the sample with very good axial resolution, but are limited in terms of chemical specificity and operation time-scale. An optical microscope coupled to an AFM can recognize and target an area of interest using specific identification markers like fluorescence tags. A high resolution fluorescence microscope can visualize fluorescence structures or molecules below the classical optical diffraction limit and reach nanometer scale resolution. A stimulated emission depletion (STED) microscopy superresolution (SR) microscope coupled to an AFM is an example in which the AFM tip gains nanoscale manipulation capabilities. The SR targeting and visualization ability help in fast and specific identification of subdiffraction-sized cellular structures and manoeuvring the AFM tip onto the target. We demonstrate how to build a STED AFM and use it for biological nanomanipulation aided with fast visualization. The STED AFM based bionanomanipulation is presented for the first time in this article. This study points to future nanosurgeries performable at single-cell level and a physical targeted manipulation of cellular features as it is currently used in research domains like nanomedicine and nanorobotics.
Instruments with single-molecule level detection capabilities can potentially benefit a wide variety of fields, including medical diagnostics. However, the size, cost, and complexity of such devices have prevented their widespread use outside sophisticated research laboratories. Fiber-only devices have recently been suggested as smaller and simpler alternatives, but thus far, they have lacked the resolution and sensitivity of a full-fledged system, and accurate alignment remains a critical requirement. Here we show that through-space reciprocal optical coupling between a fiber and a microscope objective, combined with wavelength division multiplexing in optical fibers, allows a drastic reduction of the size and complexity of such an instrument while retaining its resolution. We demonstrate a 4×4×18 cm3 sized fluorescence correlation spectrometer, which requires no alignment, can analyze kinetics at the single-molecule level, and has an optical resolution similar to that of much larger microscope based devices. The sensitivity can also be similar in principle, though in practice it is limited by the large background fluorescence of the commonly available optical fibers. We propose this as a portable and field deployable single molecule device with practical diagnostic applications.
In India, the pedagogy of science education is “believe what text book says”. Providing schools with appropriate teaching materials to enhance teaching has always been a challenge in a developing country like India. Generally it is not possible for a normal school in India to afford the expensive teaching materials to teach through demonstrations and experiments. Thus students are forced to believe what text book says rather than learning concepts through experiments. The International School of Photonics SPIE (International Society for Optical Engineering) student chapter came up with ‘Optics kit’ to supplement the teaching of optics in school level. ‘Optics kit’, developed with indigenously procured components, could be sold at an affordable prize for an average Indian School. The chapter is currently selling the kit for less than $20. The content of the kit is at par with many kits already available commercially in developed countries, and the price is just 10% compared to those kits. The kit is aimed to higher secondary level students in India, where students are taught Ray optics and basics of Wave Optics. The content of the kit is developed based on this syllabus. The Optics Kit contains simple optical elements like lens, grating, polarizer, mirror, diode laser etc. The kit can be used to demonstrate optics phenomena like interference, diffraction, polarization etc. The kit was developed based on the feedback gathered by the chapter through its outreach activities. The syllabus for the kit was developed through thorough discussion with educational experts in the field of Physics. The student community welcomed the optics kit with overwhelming enthusiasm and hence the project proved to be successful in giving an opportunity for students to “See and Believe” what they are learning.
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