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Current Protocols in Cytometry Mar 2020In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. For thicker samples, where the objective lens does...
In light microscopy, illuminating light is passed through the sample as uniformly as possible over the field of view. For thicker samples, where the objective lens does not have sufficient depth of focus, light from sample planes above and below the focal plane will also be detected. The out-of-focus light will add blur to the image, reducing the resolution. In fluorescence microscopy, any dye molecules in the field of view will be stimulated, including those in out-of-focus planes. Confocal microscopy provides a means of rejecting the out-of-focus light from the detector such that it does not contribute blur to the images being collected. This technique allows for high-resolution imaging in thick tissues. In a confocal microscope, the illumination and detection optics are focused on the same diffraction-limited spot in the sample, which is the only spot imaged by the detector during a confocal scan. To generate a complete image, the spot must be moved over the sample and data collected point by point. A significant advantage of the confocal microscope is the optical sectioning provided, which allows for 3D reconstruction of a sample from high-resolution stacks of images. Several types of confocal microscopes have been developed for this purpose, and each has different advantages and disadvantages. This article provides a concise introduction to confocal microscopy. © 2019 by John Wiley & Sons, Inc.
Topics: Animals; Drosophila; HeLa Cells; Humans; Larva; Microscopy, Confocal; Microtubules; Sample Size; Time Factors
PubMed: 31876974
DOI: 10.1002/cpcy.68 -
Cell Sep 2022Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range...
Genetically encoded voltage indicators are emerging tools for monitoring voltage dynamics with cell-type specificity. However, current indicators enable a narrow range of applications due to poor performance under two-photon microscopy, a method of choice for deep-tissue recording. To improve indicators, we developed a multiparameter high-throughput platform to optimize voltage indicators for two-photon microscopy. Using this system, we identified JEDI-2P, an indicator that is faster, brighter, and more sensitive and photostable than its predecessors. We demonstrate that JEDI-2P can report light-evoked responses in axonal termini of Drosophila interneurons and the dendrites and somata of amacrine cells of isolated mouse retina. JEDI-2P can also optically record the voltage dynamics of individual cortical neurons in awake behaving mice for more than 30 min using both resonant-scanning and ULoVE random-access microscopy. Finally, ULoVE recording of JEDI-2P can robustly detect spikes at depths exceeding 400 μm and report voltage correlations in pairs of neurons.
Topics: Animals; Interneurons; Mice; Microscopy; Neurons; Photons; Wakefulness
PubMed: 35985322
DOI: 10.1016/j.cell.2022.07.013 -
Neuroscience Research Jun 2022Holographic structured illumination combined with optogenetics enables patterned stimulation of neurons and glial cells in an intact living brain. Moreover, in vivo... (Review)
Review
Holographic structured illumination combined with optogenetics enables patterned stimulation of neurons and glial cells in an intact living brain. Moreover, in vivo functional imaging of cellular activity with recent advanced microscope technologies allows for visualization of the cellular responses during learning, emotion and cognition. Integrating these techniques can be used to verify the link between cell function and behavior output. However, there are technical limitations to stimulate multiple cells with high spatial and temporal resolution with available techniques of optogenetic stimulation. Here, we summarized a two-photon microscope combined with holographic system to stimulate multiple cells with high spatial and temporal resolution for living mice and their biological application.
Topics: Animals; Holography; Mice; Neurons; Optogenetics; Photic Stimulation; Photons
PubMed: 34740727
DOI: 10.1016/j.neures.2021.10.012 -
Sensors (Basel, Switzerland) Jan 2022In recent years, the biosensor research community has made rapid progress in the development of nanostructured materials capable of amplifying the interaction between... (Review)
Review
In recent years, the biosensor research community has made rapid progress in the development of nanostructured materials capable of amplifying the interaction between light and biological matter. A common objective is to concentrate the electromagnetic energy associated with light into nanometer-scale volumes that, in many cases, can extend below the conventional Abbé diffraction limit. Dating back to the first application of surface plasmon resonance (SPR) for label-free detection of biomolecular interactions, resonant optical structures, including waveguides, ring resonators, and photonic crystals, have proven to be effective conduits for a wide range of optical enhancement effects that include enhanced excitation of photon emitters (such as quantum dots, organic dyes, and fluorescent proteins), enhanced extraction from photon emitters, enhanced optical absorption, and enhanced optical scattering (such as from Raman-scatterers and nanoparticles). The application of photonic metamaterials as a means for enhancing contrast in microscopy is a recent technological development. Through their ability to generate surface-localized and resonantly enhanced electromagnetic fields, photonic metamaterials are an effective surface for magnifying absorption, photon emission, and scattering associated with biological materials while an imaging system records spatial and temporal patterns. By replacing the conventional glass microscope slide with a photonic metamaterial, new forms of contrast and enhanced signal-to-noise are obtained for applications that include cancer diagnostics, infectious disease diagnostics, cell membrane imaging, biomolecular interaction analysis, and drug discovery. This paper will review the current state of the art in which photonic metamaterial surfaces are utilized in the context of microscopy.
Topics: Biosensing Techniques; Microscopy; Optics and Photonics; Photons; Surface Plasmon Resonance
PubMed: 35161831
DOI: 10.3390/s22031086 -
Nature Methods Dec 2023Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a...
Despite the need for quantitative measurements of light intensity across many scientific disciplines, existing technologies for measuring light dose at the sample of a fluorescence microscope cannot simultaneously retrieve light intensity along with spatial distribution over a wide range of wavelengths and intensities. To address this limitation, we developed two rapid and straightforward protocols that use organic dyes and fluorescent proteins as actinometers. The first protocol relies on molecular systems whose fluorescence intensity decays and/or rises in a monoexponential fashion when constant light is applied. The second protocol relies on a broad-absorbing photochemically inert fluorophore to back-calculate the light intensity from one wavelength to another. As a demonstration of their use, the protocols are applied to quantitatively characterize the spatial distribution of light of various fluorescence imaging systems, and to calibrate illumination of commercially available instruments and light sources.
Topics: Fluorescence; Microscopy, Fluorescence; Fluorescent Dyes; Spectrometry, Fluorescence
PubMed: 37996751
DOI: 10.1038/s41592-023-02063-y -
Biochimica Et Biophysica Acta.... Apr 2020The higher plant chloroplast thylakoid membrane system performs the light-dependent reactions of photosynthesis. These provide the ATP and NADPH required for the... (Review)
Review
The higher plant chloroplast thylakoid membrane system performs the light-dependent reactions of photosynthesis. These provide the ATP and NADPH required for the fixation of CO into biomass by the Calvin-Benson cycle and a range of other metabolic reactions in the stroma. Land plants are frequently challenged by fluctuations in their environment, such as light, nutrient and water availability, which can create a mismatch between the amounts of ATP and NADPH produced and the amounts required by the downstream metabolism. Left unchecked, such imbalances can lead to the production of reactive oxygen species that damage the plant and harm productivity. Fortunately, plants have evolved a complex range of regulatory processes to avoid or minimize such deleterious effects by controlling the efficiency of light harvesting and electron transfer in the thylakoid membrane. Generally the regulation of the light reactions has been studied and conceptualised at the microscopic level of protein-protein and protein-ligand interactions, however in recent years dynamic changes in the thylakoid macrostructure itself have been recognised to play a significant role in regulating light harvesting and electron transfer. Here we review the evidence for the involvement of macrostructural changes in photosynthetic regulation and review the techniques that brought this evidence to light.
Topics: Acclimatization; Light; Photosynthesis; Thylakoids
PubMed: 31228404
DOI: 10.1016/j.bbabio.2019.06.011 -
Genetics Jul 2022Light microscopes are the cell and developmental biologists' "best friend," providing a means to see structures and follow dynamics from the protein to the organism... (Review)
Review
Light microscopes are the cell and developmental biologists' "best friend," providing a means to see structures and follow dynamics from the protein to the organism level. A huge advantage of Caenorhabditis elegans as a model organism is its transparency, which coupled with its small size means that nearly every biological process can be observed and measured with the appropriate probe and light microscope. Continuous improvement in microscope technologies along with novel genome editing techniques to create transgenic probes have facilitated the development and implementation of a dizzying array of methods for imaging worm embryos, larvae, and adults. In this review, we provide an overview of the molecular and cellular processes that can be visualized in living worms using light microscopy. A partial inventory of fluorescent probes and techniques successfully used in worms to image the dynamics of cells, organelles, DNA, and protein localization and activity is followed by a practical guide to choosing between various imaging modalities, including widefield, confocal, lightsheet, and structured illumination microscopy. Finally, we discuss the available tools and approaches, including machine learning, for quantitative image analysis tasks, such as colocalization, segmentation, object tracking, and lineage tracing. Hopefully, this review will inspire worm researchers who have not yet imaged their worms to begin, and push those who are imaging to go faster, finer, and longer.
Topics: Animals; Animals, Genetically Modified; Biological Phenomena; Caenorhabditis elegans; Fluorescent Dyes; Microscopy
PubMed: 35766819
DOI: 10.1093/genetics/iyac068 -
Communications Biology May 2023Light-sheet fluorescence microscopy has transformed our ability to visualize and quantitatively measure biological processes rapidly and over long time periods. In this... (Review)
Review
Light-sheet fluorescence microscopy has transformed our ability to visualize and quantitatively measure biological processes rapidly and over long time periods. In this review, we discuss current and future developments in light-sheet fluorescence microscopy that we expect to further expand its capabilities. This includes smart and adaptive imaging schemes to overcome traditional imaging trade-offs, i.e., spatiotemporal resolution, field of view and sample health. In smart microscopy, a microscope will autonomously decide where, when, what and how to image. We further assess how image restoration techniques provide avenues to overcome these tradeoffs and how "open top" light-sheet microscopes may enable multi-modal imaging with high throughput. As such, we predict that light-sheet microscopy will fulfill an important role in biomedical and clinical imaging in the future.
Topics: Microscopy, Fluorescence
PubMed: 37161000
DOI: 10.1038/s42003-023-04857-4 -
Light, Science & Applications Nov 2022EDITORIAL: "When all those around me are drunk, I alone am sober," lamented an ancient Chinese poet on fighting a lone and helpless cause. In the world of science a few...
EDITORIAL: "When all those around me are drunk, I alone am sober," lamented an ancient Chinese poet on fighting a lone and helpless cause. In the world of science a few decades ago, there was also a lone researcher who pursued the field of optical resolution despite suspicions and derisions. Unlike those who made their names young, this scientist only succeeded thanks to his own perseverance. He developed the 4Pi microscope and successfully increased the vertical resolution of traditional optical microscopes by 3-7 times. Once he sold the patent to a company, he invested the little personal money gained from the patent in breaking the Abbe diffraction limit. Despite all his hard work, his papers kept getting rejected by respected journals, and his work was subjected to mistrust and even criticism. This scientist is Stefan Hell, someone who is never afraid of difficulties and ever determined to forge ahead.Hell was awarded the Nobel Prize in Chemistry in 2014 for being the first to propose and demonstrate that the optical diffraction limit can be broken, and for successfully developing the STED super-resolution fluorescence microscope. Later Hell and his colleagues proposed and commercialized MINFLUX, which brought optical microscopy technology to the three-dimensional single-nanometer scale, opening the "post-superresolution era".Frank and sincere, persistent and humble, boldly innovative and resolute, Hell is a scientist, an entrepreneur, a mentor, and an everyday man who enjoys life.In this interview, we will reacquaint ourselves with the Nobel laureate, Stefan Hell.
PubMed: 36418300
DOI: 10.1038/s41377-022-01034-w -
Scientific Reports Apr 2022Recently developed descanned versions of the oblique light-sheet microscope promise to enable high-frame rate volumetric imaging in a variety of convenient preparations....
Recently developed descanned versions of the oblique light-sheet microscope promise to enable high-frame rate volumetric imaging in a variety of convenient preparations. The efficiency of these microscopes depends on the implementation of the objective coupling that turns the intermediate imaging plane. In this work, we developed a fully immersed coupling strategy between the middle and end objectives of the oblique light-sheet microscope to enable facile alignment and high efficiency coupling. Our design outperformed conventional designs that used only air objectives in resolution and light-collection power. We further demonstrated our design's ability to capture large fields-of-view when paired with a camera with built-in electronic binning. We simultaneously imaged the forebrain and hindbrain of larval zebrafish and found clusters of activity localized to each region of the brain.
Topics: Animals; Brain; Larva; Microscopy; Water; Zebrafish
PubMed: 35396532
DOI: 10.1038/s41598-022-09975-3