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Journal of Microscopy Oct 2018When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent...
UNLABELLED
When studying microtubules in vitro, label free imaging of single microtubules is necessary when the quantity of purified tubulin is too low for efficient fluorescent labelling or there is concern that labelling will disrupt function. Commonly used techniques for observing unlabelled microtubules, such as video enhanced differential interference contrast, dark-field and more recently laser-based interferometric scattering microscopy, suffer from a number of drawbacks. The contrast of differential interference contrast images depends on the orientation of the microtubules, dark-field is highly sensitive to impurities and optical misalignments. In addition, all of these techniques require costly optical components such as Nomarski prisms, dark-field condensers, lasers and laser scanners. Here we show that single microtubules can be imaged at high speed and with high contrast using interference reflection microscopy without the aforementioned drawbacks. Interference reflection microscopy is simple to implement, requiring only the incorporation of a 50/50 mirror instead of a dichroic in a fluorescence microscope, and with appropriate microscope settings has a similar signal-to-noise ratio to differential interference contrast and fluorescence. We demonstrated the utility of interference reflection microscopy by high-speed imaging and tracking of dynamic microtubules at 100 frames per second. In conclusion, the optical quality of interference reflection microscopy falls within the range of other microscope techniques, being inferior to some and superior to others, depending on the metric used and, with minimal microscope modification, can be used to study the dynamics of unlabelled microtubules.
LAY DESCRIPTION
The cytoskeleton gives a cell its shape and plays a major role in its movement and division. It's also helps organise the content of cells and is the base for intracellular transport. Important components of the cytoskeleton are microtubules, which are hollow cylindrical beams (25 nm in diameter) that assemble from protein building blocks called tubulin. Deficiencies in microtubules are related to many diseases including cancer and Alzheimer. Given their important role, microtubules are heavily investigated in many laboratories. One way to study microtubules is to isolate them from cells and image them using light microscopy. Over the years a number of imaging techniques have been used. These techniques have a number of drawbacks which are addressed by ongoing efforts which this work is a part of. Here, we present a method based on light interference that produce high quality images of microtubules. The technique is cheap and easy to implement making it accessible to a wide base of researchers.
Topics: Animals; Cattle; Fluorescence; Light; Microscopy, Fluorescence; Microscopy, Interference; Microtubules
PubMed: 30044498
DOI: 10.1111/jmi.12744 -
Analytical Biochemistry May 1992We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not...
We describe a new fluorescence imaging methodology in which the image contrast is derived from the fluorescence lifetime at each point in a two-dimensional image and not the local concentration and/or intensity of the fluorophore. In the present apparatus, lifetime images are created from a series of images obtained with a gain-modulated image intensifier. The frequency of gain modulation is at the light-modulation frequency (or a harmonic thereof), resulting in homodyne phase-sensitive images. These stationary phase-sensitive images are collected using a slow-scan CCD camera. A series of such images, obtained with various phase shifts of the gain-modulation signal, is used to determine the phase angle and/or modulation of the emission at each pixel, which is in essence the phase or modulation lifetime image. An advantage of this method is that pixel-to-pixel scanning is not required to obtain the images, as the information from all pixels is obtained at the same time. The method has been experimentally verified by creating lifetime images of standard fluorophores with known lifetimes, ranging from 1 to 10 ns. As an example of biochemical imaging we created life-time images of Yt-base when quenched by acrylamide, as a model for a fluorophore in distinct environments that affect its decay time. Additionally, we describe a faster imaging procedure that allows images in which a specific decay time is suppressed to be calculated, allowing rapid visualization of unique features and/or regions with distinct decay times. The concepts and methodologies of fluorescence lifetime imaging (FLIM) have numerous potential applications in the biosciences. Fluorescence lifetimes are known to be sensitive to numerous chemical and physical factors such as pH, oxygen, temperature, cations, polarity, and binding to macromolecules. Hence the FLIM method allows chemical or physical imaging of macroscopic and microscopic samples.
Topics: Algorithms; Fluorescence; Methods
PubMed: 1519759
DOI: 10.1016/0003-2697(92)90112-k -
Biosensors Feb 2023The combination of different imaging modalities into single imaging platforms has a strong potential in biomedical sciences as it permits the analysis of complementary...
The combination of different imaging modalities into single imaging platforms has a strong potential in biomedical sciences as it permits the analysis of complementary properties of the target sample. Here, we report on an extremely simple, cost-effective, and compact microscope platform for achieving simultaneous fluorescence and quantitative phase imaging modes with the capability of working in a single snapshot. It is based on the use of a single illumination wavelength to both excite the sample's fluorescence and provide coherent illumination for phase imaging. After passing the microscope layout, the two imaging paths are separated using a bandpass filter, and the two imaging modes are simultaneously obtained using two digital cameras. We first present calibration and analysis of both fluorescence and phase imaging modalities working independently and, later on, experimental validation for the proposed common-path dual-mode imaging platform considering static (resolution test targets, fluorescent micro-beads, and water-suspended lab-made cultures) as well as dynamic (flowing fluorescent beads, human sperm cells, and live specimens from lab-made cultures) samples.
Topics: Male; Humans; Microscopy; Semen; Holography; Lighting; Calibration
PubMed: 36832019
DOI: 10.3390/bios13020253 -
The Journal of Biophysical and... Jul 1956In this paper, a procedure for correlating electron microscope and light microscope cytochemical studies using immediately adjacent serial thin and thick sections has...
In this paper, a procedure for correlating electron microscope and light microscope cytochemical studies using immediately adjacent serial thin and thick sections has been described and discussed. This technique, combined with the Feulgen reaction for DNA, has been of particular value in framing and answering both general and specific questions about the nucleus. The results may be summarized as follows:- Apparent nuclear homogeneity in the electron microscope is not due to loss of DNA as evidenced by positive Feulgen reactions in such nuclei. Arrangement of Feulgen-positive material in chromosomes, heterochromatin, perinuclear and perinucleolar chromatin, etc., is similar to that customarily observed in the light microscope but this is not necessarily reflected in a cursory survey of the electron image. Careful comparison of light and electron images shows that fine differences in structure are associated with chromatin localization. Primary spermatocyte prophase chromosomes of crayfish have been positively identified by their Feulgen-positive nature. Core-like axial structures in such chromosomes have been observed (9) and are described further. A remarkable feature of spermiogenesis in the crayfish is an elaboration of the nuclear envelope of the spermatid accompanying the formation of what becomes a mass of convoluted membranes in the sperm. In the spermatid, perinuclear chromatin follows outpocketings of the nuclear envelope into the cytoplasm. In the early sperm, on the other hand, although the nuclear envelope is continuous with the system of convoluted membranes, the chromatin is distinct from it and is retained in the nucleus proper by some mechanism independent of the nuclear envelope. None of the above observations was apparent from the electron microscope images alone; they were possible only by virtue of the correlated cytochemical and electron microscope study of adjacent sections. The successful use of other cytochemical tests, such as the PAS reaction for certain carbohydrates, in such correlated studies is also described.
Topics: Cell Nucleus; Chromatin; Chromosomes; Cytoplasm; DNA; Electrons; Heterochromatin; Light; Male; Microscopy; Microscopy, Electron; Nuclear Envelope; Spermatogenesis; Spermatozoa
PubMed: 13357576
DOI: 10.1083/jcb.2.4.397 -
Lab on a Chip Jun 2010Despite the rapid progress in optical imaging, most of the advanced microscopy modalities still require complex and costly set-ups that unfortunately limit their use...
Despite the rapid progress in optical imaging, most of the advanced microscopy modalities still require complex and costly set-ups that unfortunately limit their use beyond well equipped laboratories. In the meantime, microscopy in resource-limited settings has requirements significantly different from those encountered in advanced laboratories, and such imaging devices should be cost-effective, compact, light-weight and appropriately accurate and simple to be usable by minimally trained personnel. Furthermore, these portable microscopes should ideally be digitally integrated as part of a telemedicine network that connects various mobile health-care providers to a central laboratory or hospital. Toward this end, here we demonstrate a lensless on-chip microscope weighing approximately 46 grams with dimensions smaller than 4.2 cm x 4.2 cm x 5.8 cm that achieves sub-cellular resolution over a large field of view of approximately 24 mm(2). This compact and light-weight microscope is based on digital in-line holography and does not need any lenses, bulky optical/mechanical components or coherent sources such as lasers. Instead, it utilizes a simple light-emitting-diode (LED) and a compact opto-electronic sensor-array to record lensless holograms of the objects, which then permits rapid digital reconstruction of regular transmission or differential interference contrast (DIC) images of the objects. Because this lensless incoherent holographic microscope has orders-of-magnitude improved light collection efficiency and is very robust to mechanical misalignments it may offer a cost-effective tool especially for telemedicine applications involving various global health problems in resource limited settings.
Topics: Equipment Design; Equipment Failure Analysis; Holography; Lenses; Lighting; Microfluidics; Microscopy; Miniaturization; Telemedicine
PubMed: 20401422
DOI: 10.1039/c000453g -
The Surgeon : Journal of the Royal... Dec 2023High quality surgical lighting is central to successful performance in the operating room and therefore to both patient care and treatment. This article discusses the... (Review)
Review
High quality surgical lighting is central to successful performance in the operating room and therefore to both patient care and treatment. This article discusses the origins of surgical lighting from the 1800s to today, with a focus on the four main forms. Their uses, advantages, and disadvantages are evaluated in an effort to identify the improvements required to improve today's current state of surgical lighting. Whilst these four mainstream types have served well for the past thirty years, the literature exposes opportunities for improvement and can be used to guide the pathway to transition from manual conventional methods to a more automated lighting (AL) approach. The concept of AL has been proposed using established and known technical approaches such as artificial intelligence (AI), 3D sensor tracking algorithms and thermal imaging. Whilst AL seems incredibly promising, further focused research must be undertaken to maximise its' effectiveness and allow for successful integration of this new technology into operating rooms today.
Topics: Humans; Lighting; Artificial Intelligence; Operating Rooms; Algorithms
PubMed: 37328393
DOI: 10.1016/j.surge.2023.05.004 -
Optics Express Oct 2014Light field microscopy has been proposed as a new high-speed volumetric computational imaging method that enables reconstruction of 3-D volumes from captured projections...
Light field microscopy has been proposed as a new high-speed volumetric computational imaging method that enables reconstruction of 3-D volumes from captured projections of the 4-D light field. Recently, a detailed physical optics model of the light field microscope has been derived, which led to the development of a deconvolution algorithm that reconstructs 3-D volumes with high spatial resolution. However, the spatial resolution of the reconstructions has been shown to be non-uniform across depth, with some z planes showing high resolution and others, particularly at the center of the imaged volume, showing very low resolution. In this paper, we enhance the performance of the light field microscope using wavefront coding techniques. By including phase masks in the optical path of the microscope we are able to address this non-uniform resolution limitation. We have also found that superior control over the performance of the light field microscope can be achieved by using two phase masks rather than one, placed at the objective's back focal plane and at the microscope's native image plane. We present an extended optical model for our wavefront coded light field microscope and develop a performance metric based on Fisher information, which we use to choose adequate phase masks parameters. We validate our approach using both simulated data and experimental resolution measurements of a USAF 1951 resolution target; and demonstrate the utility for biological applications with in vivo volumetric calcium imaging of larval zebrafish brain.
Topics: Algorithms; Equipment Design; Imaging, Three-Dimensional; Light; Microscopy, Fluorescence; Models, Theoretical
PubMed: 25322056
DOI: 10.1364/OE.22.024817 -
Nature Communications Nov 2021A limitation of standard brightfield microscopy is its low contrast images, especially for thin specimens of weak absorption, and biological species with refractive...
A limitation of standard brightfield microscopy is its low contrast images, especially for thin specimens of weak absorption, and biological species with refractive indices very close in value to that of their surroundings. We demonstrate, using a planar photonic chip with tailored angular transmission as the sample substrate, a standard brightfield microscopy can provide both darkfield and total internal reflection (TIR) microscopy images with one experimental configuration. The image contrast is enhanced without altering the specimens and the microscope configurations. This planar chip consists of several multilayer sections with designed photonic band gaps and a central region with dielectric nanoparticles, which does not require top-down nanofabrication and can be fabricated in a larger scale. The photonic chip eliminates the need for a bulky condenser or special objective to realize darkfield or TIR illumination. Thus, it can work as a miniaturized high-contrast-imaging device for the developments of versatile and compact microscopes.
Topics: Equipment Design; Image Enhancement; Microscopy; Nanoparticles; Photons; Surface Plasmon Resonance
PubMed: 34824261
DOI: 10.1038/s41467-021-27231-6 -
Analytical Chemistry Feb 2019Advancement of discrete frequency infrared (DFIR) spectroscopic microscopes in image quality and data throughput are critical to their use for analytical measurements....
Advancement of discrete frequency infrared (DFIR) spectroscopic microscopes in image quality and data throughput are critical to their use for analytical measurements. Here, we report the development and characterization of a point scanning instrument with minimal aberrations and capable of diffraction-limited performance across all fingerprint region wavelengths over arbitrarily large samples. The performance of this system is compared to commercial state of the art Fourier transform infrared (FT-IR) imaging systems. We show that for large samples or smaller set of discrete frequencies, point scanning far exceeds (∼10-100 fold) comparable data acquired with FT-IR instruments. Further we show improvements in image quality using refractive lenses that show significantly improved contrast across the spatial frequency bandwidth. Finally, we introduce the ability to image two tunable frequencies simultaneously using a single detector by means of demodulation to further speed up data acquisition and reduce the impact of scattering. Together, the advancements provide significantly better spectral quality and spatial fidelity than current state of the art imaging systems while promising to make spectral scanning even faster.
Topics: Color; Equipment Design; Software; Spectrophotometry, Infrared
PubMed: 30605317
DOI: 10.1021/acs.analchem.8b04749 -
BioTechniques Nov 2001Chemiluminescence has become a standard tool in biomedical research. Chemiluminescent probes are used for immunoassays, nucleic acid identification, reporter gene... (Review)
Review
Chemiluminescence has become a standard tool in biomedical research. Chemiluminescent probes are used for immunoassays, nucleic acid identification, reporter gene assays, measuring enzyme activity, and the detection of ions and small molecules such as Ca2+, ATP, NO, O2- and H2O2. Along with the development of new chemiluminescent probes, significant progress has been made in techniques to measure chemiluminescence. Ultra-sensitive photometers or luminometers have become widely available and can be obtained with automatic injectors and microplate readers. In addition, imaging photon detectors have been developed that allow the imaging of chemiluminescence from gels, blots, and microplates. Imaging photon detectors have also been attached to microscopes and allow imaging of chemiluminescent probes and reporter genes in cells and tissues. Specific methods of photon collection, storage, and analysis have been developed for microscopic imaging of chemiluminescence. Two of these methods are discussed in detail. The first is a method of data storage that allows days of continuous imaging without creating oversized files. The second is a method for calibrating photon imaging microscopes using a low-light standard. Such calibration will be helpful for comparing the performance of various photon imaging systems and for comparing data obtained in different laboratories.
Topics: Animals; Calibration; Humans; Information Storage and Retrieval; Luminescent Measurements; Microscopy; Photons
PubMed: 11730016
DOI: 10.2144/01315rv01