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Cold Spring Harbor Protocols Oct 2014Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain... (Review)
Review
Fluorescence microscopy is a major tool with which to monitor cell physiology. Although the concepts of fluorescence and its optical separation using filters remain similar, microscope design varies with the aim of increasing image contrast and spatial resolution. The basics of wide-field microscopy are outlined to emphasize the selection, advantages, and correct use of laser scanning confocal microscopy, two-photon microscopy, scanning disk confocal microscopy, total internal reflection, and super-resolution microscopy. In addition, the principles of how these microscopes form images are reviewed to appreciate their capabilities, limitations, and constraints for operation.
Topics: Animals; Fluorescence; Humans; Microscopy, Fluorescence; Optical Devices
PubMed: 25275114
DOI: 10.1101/pdb.top071795 -
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 -
Histopathology Jan 2017Colour is central to the practice of pathology because of the use of coloured histochemical and immunohistochemical stains to visualize tissue features. Our reliance... (Review)
Review
Colour is central to the practice of pathology because of the use of coloured histochemical and immunohistochemical stains to visualize tissue features. Our reliance upon histochemical stains and light microscopy has evolved alongside a wide variation in slide colour, with little investigation into the implications of colour variation. However, the introduction of the digital microscope and whole-slide imaging has highlighted the need for further understanding and control of colour. This is because the digitization process itself introduces further colour variation which may affect diagnosis, and image analysis algorithms often use colour or intensity measures to detect or measure tissue features. The US Food and Drug Administration have released recent guidance stating the need to develop a method of controlling colour reproduction throughout the digitization process in whole-slide imaging for primary diagnostic use. This comprehensive review introduces applied basic colour physics and colour interpretation by the human visual system, before discussing the importance of colour in pathology. The process of colour calibration and its application to pathology are also included, as well as a summary of the current guidelines and recommendations regarding colour in digital pathology.
Topics: Color; Coloring Agents; Humans; Image Interpretation, Computer-Assisted; Microscopy; Pathology; Staining and Labeling
PubMed: 27607349
DOI: 10.1111/his.13079 -
Journal of Biomedical Optics Jun 2018Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100-...
Total internal reflection microscopy (TIRF) has been a powerful tool in biological research. The most valuable feature of the method has been the ability to image 100- to 200-nm-thick layer of cell features adjacent to a coverslip, such as membrane lipids, membrane receptors, and structures proximal-to-basal membranes. Here, we demonstrate an alternative method of imaging thin-layer proximal-to-basal membranes by placing a sample on a high refractive index coverslip covered by a thin layer of gold. The sample is illuminated using the Kretschmann method (i.e., from the top to an aqueous medium). Fluorophores that are close to the metal surface induce surface plasmons in the metal film. Fluorescence from fluorophores near the metal surface couple with surface plasmons allowing them to penetrate the metal surface and emerge at a surface plasmon coupled emission angle. The thickness of the detection layer is further reduced in comparison with TIRF by metal quenching of fluorophores at a close proximity (below 10 nm) to a surface. Fluorescence is collected by a high NA objective and imaged by EMCCD or converted to a signal by avalanche photodiode fed by a single-mode optical fiber inserted in the conjugate image plane of the objective. The system avoids complications of through-the-objective TIRF associated with shared excitation and emission light path, has thin collection thickness, produces excellent background rejection, and is an effective method to study molecular motion.
Topics: Astrocytes; Fluorescence; Fluorescent Dyes; Gold; Humans; Microscopy, Fluorescence; Surface Plasmon Resonance
PubMed: 29935016
DOI: 10.1117/1.JBO.23.6.060502 -
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 -
Cells Nov 2022In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the... (Review)
Review
In bio-medical mobile workstations, e.g., the prevention of epidemic viruses/bacteria, outdoor field medical treatment and bio-chemical pollution monitoring, the conventional bench-top microscopic imaging equipment is limited. The comprehensive multi-mode (bright/dark field imaging, fluorescence excitation imaging, polarized light imaging, and differential interference microscopy imaging, etc.) biomedical microscopy imaging systems are generally large in size and expensive. They also require professional operation, which means high labor-cost, money-cost and time-cost. These characteristics prevent them from being applied in bio-medical mobile workstations. The bio-medical mobile workstations need microscopy systems which are inexpensive and able to handle fast, timely and large-scale deployment. The development of lightweight, low-cost and portable microscopic imaging devices can meet these demands. Presently, for the increasing needs of point-of-care-test and tele-diagnosis, high-performance computational portable microscopes are widely developed. Bluetooth modules, WLAN modules and 3G/4G/5G modules generally feature very small sizes and low prices. And industrial imaging lens, microscopy objective lens, and CMOS/CCD photoelectric image sensors are also available in small sizes and at low prices. Here we review and discuss these typical computational, portable and low-cost microscopes by refined specifications and schematics, from the aspect of optics, electronic, algorithms principle and typical bio-medical applications.
Topics: Microscopy; Point-of-Care Systems; Lenses; Algorithms; Microscopy, Interference
PubMed: 36429102
DOI: 10.3390/cells11223670 -
Scientific Reports Jan 2023In this contribution, we demonstrate a wide-field hyperspectral mid-infrared (MIR) microscope based on multidimensional single-pixel imaging (SPI). The microscope...
In this contribution, we demonstrate a wide-field hyperspectral mid-infrared (MIR) microscope based on multidimensional single-pixel imaging (SPI). The microscope employs a high brightness MIR supercontinuum source for broadband (1.55 [Formula: see text]-4.5 [Formula: see text]) sample illumination. Hyperspectral imaging capability is achieved by a single micro-opto-electro-mechanical digital micromirror device (DMD), which provides both spatial and spectral differentiation. For that purpose the operational spectral bandwidth of the DMD was significantly extended into the MIR spectral region. In the presented design, the DMD fulfills two essential tasks. On the one hand, as standard for the SPI approach, the DMD sequentially masks captured scenes enabling diffraction-limited imaging in the tens of millisecond time-regime. On the other hand, the diffraction at the micromirrors leads to dispersion of the projected field and thus allows for wavelength selection without the application of additional dispersive optical elements, such as gratings or prisms. In the experimental part, first of all, the imaging and spectral capabilities of the hyperspectral microscope are characterized. The spatial and spectral resolution is assessed by means of test targets and linear variable filters, respectively. At a wavelength of 4.15 [Formula: see text] a spatial resolution of 4.92 [Formula: see text] is achieved with a native spectral resolution better than 118.1 nm. Further, a post-processing method for drastic enhancement of the spectral resolution is proposed and discussed. The performance of the MIR hyperspectral microsopce is demonstrated for label-free chemical imaging and examination of polymer compounds and red blood cells. The acquisition and reconstruction of Hadamard sampled 64 [Formula: see text] 64 images is achieved in 450 ms and 162 ms, respectively. Thus, combined with an unprecedented intrinsic flexibiliy gained by a tunable field of view and adjustable spatial resolution, the demonstrated design drastically improves the sample throughput in MIR chemical and biomedical imaging.
Topics: Microscopy; Lighting; Optical Devices; Equipment Design
PubMed: 36609672
DOI: 10.1038/s41598-022-26718-6