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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 -
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 -
Methods in Molecular Biology (Clifton,... 2013Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the...
Epi-fluorescence microscopy is available in most life sciences research laboratories, and when optimized can be a central laboratory tool. In this chapter, the epi-fluorescence light path is introduced and the various components are discussed in detail. Recommendations are made for incident lamp light sources, excitation and emission filters, dichroic mirrors, objective lenses, and charge-coupled device (CCD) cameras in order to obtain the most sensitive epi-fluorescence microscope. The even illumination of metal-halide lamps combined with new "hard" coated filters and mirrors, a high resolution monochrome CCD camera, and a high NA objective lens are all recommended for high resolution and high sensitivity fluorescence imaging. Recommendations are also made for multicolor imaging with the use of monochrome cameras, motorized filter turrets, individual filter cubes, and corresponding dyes being the best choice for sensitive, high resolution multicolor imaging. Images should be collected using Nyquist sampling and images should be corrected for background intensity contributions and nonuniform illumination across the field of view. Photostable fluorescent probes and proteins that absorb a lot of light (i.e., high extinction co-efficients) and generate a lot of fluorescence signal (i.e., high quantum yields) are optimal. A neuronal immune-fluorescence labeling protocol is also presented. Finally, in order to maximize the utility of sensitive wide-field microscopes and generate the highest resolution images with high signal-to-noise, advice for combining wide-field epi-fluorescence imaging with restorative image deconvolution is presented.
Topics: Cells, Cultured; Fluorescent Antibody Technique; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Image Processing, Computer-Assisted; Lighting; Microscopy, Fluorescence; Phalloidine; Staining and Labeling
PubMed: 23026996
DOI: 10.1007/978-1-62703-056-4_2 -
Optics Express Sep 2023A technical challenge in neuroscience is to record and specifically manipulate the activity of neurons in living animals. This can be achieved in some preparations with...
A technical challenge in neuroscience is to record and specifically manipulate the activity of neurons in living animals. This can be achieved in some preparations with two-photon calcium imaging and photostimulation. These methods can be extended to three dimensions by holographic light sculpting with spatial light modulators (SLMs). At the same time, performing simultaneous holographic imaging and photostimulation is still cumbersome, requiring two light paths with separate SLMs. Here we present an integrated optical design using a single SLM for simultaneous imaging and photostimulation. Furthermore, we applied axially dependent adaptive optics to make the system aberration-free, and developed software for calibrations and closed-loop neuroscience experiments. Finally, we demonstrate the performance of the system with simultaneous calcium imaging and optogenetics in mouse primary auditory cortex in vivo. Our integrated holographic system could facilitate the systematic investigation of neural circuit function in awake behaving animals.
Topics: Animals; Mice; Calcium; Holography; Photons; Software; Neurons
PubMed: 37859128
DOI: 10.1364/OE.498051 -
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 -
Applied Microbiology and Biotechnology Jun 2022While phototoxicity can be a useful therapeutic modality not only for eliminating malignant cells but also in treating fungal infections, mycologists aiming to observe... (Review)
Review
While phototoxicity can be a useful therapeutic modality not only for eliminating malignant cells but also in treating fungal infections, mycologists aiming to observe morphological changes or molecular events in fungi, especially when long observation periods or high light fluxes are warranted, encounter problems owed to altered regulatory pathways or even cell death caused by various photosensing mechanisms. Consequently, the ever expanding repertoire of visible fluorescent protein toolboxes and high-resolution microscopy methods designed to investigate fungi in vitro and in vivo need to comply with an additional requirement: to decrease the unwanted side effects of illumination. In addition to optimizing exposure, an obvious solution is red-shifted illumination, which, however, does not come without compromises. This review summarizes the interactions of fungi with light and the various molecular biology and technology approaches developed for exploring their functions on the molecular, cellular, and in vivo microscopic levels, and outlines the progress towards reducing phototoxicity through applying far-red and near-infrared light. KEY POINTS: • Fungal biological processes alter upon illumination, also under the microscope • Red shifted fluorescent protein toolboxes decrease interference by illumination • Innovations like two-photon, lightsheet, and near IR microscopy reduce phototoxicity.
Topics: Coloring Agents; Fungi; Light; Microscopy, Fluorescence; Photons
PubMed: 35599256
DOI: 10.1007/s00253-022-11967-2 -
BMJ Open Ophthalmology Jun 2023To compare subjective levels of comfort and visual experiences related to microscope light in patients undergoing their first cataract surgery with topical anaesthesia... (Randomized Controlled Trial)
Randomized Controlled Trial
Comparing microscope light-associated glare and comfort between heads-up 3D digital and conventional microscopes in cataract surgery: a randomised, multicentre, single-blind, controlled trial.
OBJECTIVE
To compare subjective levels of comfort and visual experiences related to microscope light in patients undergoing their first cataract surgery with topical anaesthesia using a digital microscope (the NGENUITY three-dimensional (3D) visualisation system) or a conventional microscope.
METHODS AND ANALYSIS
A prospective, randomised, single-blinded, parallel-group, multicentre, interventional study. Patients (n=128) were randomly assigned to one of two treatment groups: the experimental group (n=63) had surgery using the digital microscope and the control group (n=65) had surgery with a conventional microscope. The primary outcome was patients' subjective experience of glare from the microscope light during surgery on a numerical scale from 0 to 10. Key secondary outcomes were patients' subjective levels of comfort and visual experiences related to the microscope light.
RESULTS
The experimental group reported significantly lower levels of glare; median levels were 1.0 (0.0-4.0) for the experimental group vs 3.0 (0.0-6.0) for the control group (p=0.027). They also reported higher levels of comfort; median ratings were 8.0 (6.5-10.0) in the experimental group and 7.0 (5.0-9.0) in controls (p=0.026). There were no group differences in ratings of subjective pain or visual disturbances. Median microscope light intensity was lower in the experimental group than controls; 3425.0 (2296.0-4300.0) Lux vs 24 279.0 (16 000.0-26 500.0) Lux (p<0.0001), respectively.
CONCLUSION
Compared with conventional microscopes, the NGENUITY 3D visualisation system allows surgeons to operate with lower levels of light exposure, resulting in significantly less glare and improved comfort in patients undergoing cataract surgery.
TRIAL REGISTRATION NUMBER
NCT05085314.
Topics: Humans; Prospective Studies; Glare; Single-Blind Method; Cataract Extraction; Cataract
PubMed: 37493673
DOI: 10.1136/bmjophth-2023-001272 -
Scientific Reports Jun 2022Dynamic speckle illumination (DSI) has recently attracted strong attention in the field of biomedical imaging as it pushes the limits of interference microscopy (IM) in...
Dynamic speckle illumination (DSI) has recently attracted strong attention in the field of biomedical imaging as it pushes the limits of interference microscopy (IM) in terms of phase sensitivity, and spatial and temporal resolution compared to conventional light source illumination. To date, despite conspicuous advantages, it has not been extensively implemented in the field of phase imaging due to inadequate understanding of interference fringe formation, which is challenging to obtain in dynamic speckle illumination interference microscopy (DSI-IM). The present article provides the basic understanding of DSI through both simulation and experiments that is essential to build interference microscopy systems such as quantitative phase microscopy, digital holographic microscopy and optical coherence tomography. Using the developed understanding of DSI, we demonstrated its capabilities which enables the use of non-identical objective lenses in both arms of the interferometer and opens the flexibility to use user-defined microscope objective lens for scalable field of view and resolution phase imaging. It is contrary to the present understanding which forces us to use identical objective lenses in conventional IM system and limits the applicability of the system for fixed objective lens. In addition, it is also demonstrated that the interference fringes are not washed out over a large range of optical path difference (OPD) between the object and the reference arm providing competitive edge over low temporal coherence light source based IM system. The theory and explanation developed here would enable wider penetration of DSI-IM for applications in biology and material sciences.
Topics: Holography; Lenses; Lighting; Microscopy; Microscopy, Interference
PubMed: 35760816
DOI: 10.1038/s41598-022-14739-0 -
BMC Ophthalmology Jan 2023To evaluate the effect of the light intensity of the surgical microscope and illuminated chopper on the anterior chamber temperature.
BACKGROUND
To evaluate the effect of the light intensity of the surgical microscope and illuminated chopper on the anterior chamber temperature.
STUDY DESIGN
Experimental study.
METHODS
A model eye (Kitaro WetLab System; FCI Ophthalmics, Pembroke, MA, USA) was used in this experimental study. The illuminance of a surgical microscope (Leica M300; Leica Microsystems, Wetzlar, Germany) and illuminated chopper (iChopper NAM-25 GB; Oculight, Korea) with a light source (iVision; Oculight) was measured using an illuminometer. In addition, the temperature in the anterior chamber of the model eye filled with balanced salt solution when using the surgical microscope with a light intensity from level 1 to level 6 and the illuminated chopper at 99% light intensity was measured for 10 min.
RESULTS
The anterior chamber temperature was increased by 0.2, 0.5, 1.0, and 1.4 ℃ when using the surgical microscope at level 3 (10050 lux), 4 (16490 lux), 5 (24900 lux), and 6 (32500 lux), respectively, for 10 min. The illuminated chopper at 99% light intensity (14893 lux) positioned in the anterior chamber increased the anterior chamber temperature by 0.2° C after 10 min, which was equal to the increase in the temperature caused by the surgical microscope at level 3.
CONCLUSION
The photothermal effect of the illuminated chopper directly positioned in the anterior chamber appeared to be similar to that of a microscope with similar illuminance. Therefore, the illuminated chopper is safe in terms of anterior chamber temperature changes in cataract surgery.
Topics: Humans; Temperature; Light; Cataract Extraction; Microscopy; Anterior Chamber
PubMed: 36690966
DOI: 10.1186/s12886-023-02784-w -
The Journal of Histochemistry and... Feb 2011Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with... (Review)
Review
Light sheet fluorescence microscopy (LSFM) functions as a non-destructive microtome and microscope that uses a plane of light to optically section and view tissues with subcellular resolution. This method is well suited for imaging deep within transparent tissues or within whole organisms, and because tissues are exposed to only a thin plane of light, specimen photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal, or multiphoton microscopy. LSFMs produce well-registered serial sections that are suitable for three-dimensional reconstruction of tissue structures. Because of a lack of a commercial LSFM microscope, numerous versions of light sheet microscopes have been constructed by different investigators. This review describes development of the technology, reviews existing devices, provides details of one LSFM device, and shows examples of images and three-dimensional reconstructions of tissues that were produced by LSFM.
Topics: Animals; Image Processing, Computer-Assisted; Lasers; Light; Microscopy, Fluorescence; Specimen Handling
PubMed: 21339178
DOI: 10.1369/0022155410394857