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International Journal of Molecular... Jun 2023Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments... (Review)
Review
Though microscopy is most often intended as a technique for providing qualitative assessment of cellular and subcellular properties, when coupled with other instruments such as wavelength selectors, lasers, photoelectric devices and computers, it can perform a wide variety of quantitative measurements, which are demanding in establishing relationships between the properties and structures of biological material in all their spatial and temporal complexities. These combinations of instruments are a powerful approach to improve non-destructive investigations of cellular and subcellular properties (both physical and chemical) at a macromolecular scale resolution. Since many subcellular compartments in living cells are characterized by structurally organized molecules, this review deals with three advanced microscopy techniques well-suited for these kind of investigations, i.e., microspectrophotometry (MSP), super-resolution localization microscopy (SRLM) and holotomographic microscopy (HTM). These techniques can achieve an insight view into the role intracellular molecular organizations such as photoreceptive and photosynthetic structures and lipid bodies play in many cellular processes as well as their biophysical properties. Microspectrophotometry uses a set-up based on the combination of a wide-field microscope and a polychromator, which allows the measurement of spectroscopic features such as absorption spectra. Super resolution localization microscopy combines dedicated optics and sophisticated software algorithms to overcome the diffraction limit of light and allow the visualization of subcellular structures and dynamics in greater detail with respect to conventional optical microscopy. Holotomographic microscopy combines holography and tomography techniques into a single microscopy set-up, and allows 3D reconstruction by means of the phase separation of biomolecule condensates. This review is organized in sections, which for each technique describe some general aspects, a peculiar theoretical aspect, a specific experimental configuration and examples of applications (fish and algae photoreceptors, single labeled proteins and endocellular aggregates of lipids).
Topics: Animals; Microscopy, Fluorescence; Proteins; Optics and Photonics; Biophysics; Holography
PubMed: 37373120
DOI: 10.3390/ijms24129973 -
International Journal of Molecular... Apr 2023The review briefly describes various types of infrared (IR) and Raman spectroscopy methods. At the beginning of the review, the basic concepts of biological methods of... (Review)
Review
The review briefly describes various types of infrared (IR) and Raman spectroscopy methods. At the beginning of the review, the basic concepts of biological methods of environmental monitoring, namely bioanalytical and biomonitoring methods, are briefly considered. The main part of the review describes the basic principles and concepts of vibration spectroscopy and microspectrophotometry, in particular IR spectroscopy, mid- and near-IR spectroscopy, IR microspectroscopy, Raman spectroscopy, resonance Raman spectroscopy, Surface-enhanced Raman spectroscopy, and Raman microscopy. Examples of the use of various methods of vibration spectroscopy for the study of biological samples, especially in the context of environmental monitoring, are given. Based on the described results, the authors conclude that the near-IR spectroscopy-based methods are the most convenient for environmental studies, and the relevance of the use of IR and Raman spectroscopy in environmental monitoring will increase with time.
Topics: Vibration; Biological Monitoring; Spectrophotometry, Infrared; Spectrum Analysis, Raman; Spectroscopy, Near-Infrared; Spectroscopy, Fourier Transform Infrared
PubMed: 37108111
DOI: 10.3390/ijms24086947 -
Acta Physiologica (Oxford, England) Jul 2011Early in the last century August Krogh embarked on a series of seminal studies to understand the connection between tissue metabolism and mechanisms by which the... (Review)
Review
Early in the last century August Krogh embarked on a series of seminal studies to understand the connection between tissue metabolism and mechanisms by which the cardiovascular system supplied oxygen to meet those needs. Krogh recognized that oxygen was supplied from blood to the tissues by passive diffusion and that the most likely site for oxygen exchange was the capillary network. Studies of tissue oxygen consumption and diffusion coefficient, coupled with anatomical studies of capillarity in various tissues, led him to formulate a model of oxygen diffusion from a single capillary. Fifty years after the publication of this work, new methods were developed which allowed the direct measurement of oxygen in and around microvessels. These direct measurements have confirmed the predictions by Krogh and have led to extensions of his ideas resulting in our current understanding of oxygenation within the microcirculation. Developments during the last 40 years are reviewed, including studies of oxygen gradients in arterioles, capillaries, venules, microvessel wall and surrounding tissue. These measurements were made possible by the development and use of new methods to investigate oxygen in the microcirculation, so mention is made of oxygen microelectrodes, microspectrophotometry of haemoglobin and phosphorescence quenching microscopy. Our understanding of oxygen transport from the perspective of the microcirculation has gone from a consideration of oxygen gradients in capillaries and tissue to the realization that oxygen has the ability to diffuse from any microvessel to another location under the conditions that there exists a large enough PO(2) gradient and that the permeability for oxygen along the intervening pathway is sufficient.
Topics: Animals; Arterioles; Capillaries; Diffusion; Hemoglobins; Humans; Microcirculation; Microscopy; Muscle Contraction; Muscle, Skeletal; Oxygen; Oxygen Consumption; Partial Pressure; Sulfates; Venules
PubMed: 21281453
DOI: 10.1111/j.1748-1716.2010.02232.x -
Forensic Sciences Research 2021Signature examination is the most common examination performed by any document examiner. Determination of the authenticity of a handwritten signature on a questioned...
Signature examination is the most common examination performed by any document examiner. Determination of the authenticity of a handwritten signature on a questioned document is an important task for forensic document examiners in the forensic science field. As a result of continuous developments in technology, a signature stamp can now be created using a photosensitive seal to enable the reproduction of a handwritten signature. These stamps are commonly used in China and several other countries. In this study, 10 types of black photosensitive stamp-pad ink, 10 brands of fountain pen ink, 15 types of black gel ink and six types of black erasable gel ink found on the Chinese domestic market were collected and 10 photosensitive signature stamps were created using the signatures of 10 people. Microscopic analysis, infrared (IR) and fluorescence analyses and microspectrophotometry (MSP) techniques were used to examine the resulting photosensitive signature stamp impressions when applied to printing papers, writing papers and invoice papers. By comparing the printing and spectral characteristics of the photosensitive signature stamp impressions with those of the signatures executed using the fountain pens, gel pens and erasable gel pens, it was possible to determine whether each signature was written or stamped using a photosensitive signature stamp. To validate these results, a 96.7% absolute accuracy and a 99.3% detection rate were achieved over a total of 150 blind tests conducted by five forensic document examiners, thus demonstrating that a combination of the four analysis methods used in this work can provide a more scientific approach and improve the accuracy and the detection rate of the examination process.KEY POINTSA signature stamp is a photosensitive seal made in the style of a handwritten signature.Although microscopic analysis can usually provide better examination results, a comprehensive examination method that includes microscopic analysis and ink composition analysis is required to improve the accuracy and the detection rate of the examination process.This study collected and tested photosensitive stamp-pad inks, fountain pen inks, gel inks and erasable inks.Infrared and fluorescence analyses and microspectrophotometry were able to distinguish the photosensitive ink from both erasable ink and fountain pen ink. Supplemental data for this article are available online at https://doi.org/10.1080/20961790.2021.1898755.
PubMed: 34377575
DOI: 10.1080/20961790.2021.1898755 -
Current Opinion in Structural Biology Oct 2010Current efforts in structural biology aim to integrate structural information within the context of cellular organization and function. X-rays and infrared radiation... (Review)
Review
Current efforts in structural biology aim to integrate structural information within the context of cellular organization and function. X-rays and infrared radiation stand at opposite ends of the electromagnetic spectrum and act as complementary probes for achieving this goal. Intense and bright beams are produced by synchrotron radiation, and are efficiently used in the wavelength domain extending from hard X-rays to the far-infrared (or THz) regime. While X-ray crystallography provides exquisite details on atomic structure, Fourier transform infrared microspectroscopy (FTIRM) is emerging as a spectroscopic probe and imaging tool for correlating molecular structure to biochemical dynamics and function. In this manuscript, the role of synchrotron FTIRM in bridging the gap towards 'functional biology' is discussed based upon recent achievements, with a critical assessment of the contributions to biological and biomedical research.
Topics: Animals; Cells; Cells, Cultured; Humans; Microspectrophotometry; Molecular Imaging; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared
PubMed: 20739176
DOI: 10.1016/j.sbi.2010.07.007 -
Scientific Reports Jul 2023Dystrophic muscle is characterized by necrosis/regeneration cycles, inflammation, and fibro-adipogenic development. Conventional histological stainings provide essential...
Dystrophic muscle is characterized by necrosis/regeneration cycles, inflammation, and fibro-adipogenic development. Conventional histological stainings provide essential topographical data of this remodeling but may be limited to discriminate closely related pathophysiological contexts. They fail to mention microarchitecture changes linked to the nature and spatial distribution of tissue compartment components. We investigated whether label-free tissue autofluorescence revealed by Synchrotron deep ultraviolet (DUV) radiation could serve as an additional tool for monitoring dystrophic muscle remodeling. Using widefield microscopy with specific emission fluorescence filters and microspectroscopy defined by high spectral resolution, we analyzed samples from healthy dogs and two groups of dystrophic dogs: naïve (severely affected) and MuStem cell-transplanted (clinically stabilized) animals. Multivariate statistical analysis and machine learning approaches demonstrated that autofluorescence emitted at 420-480 nm by the Biceps femoris muscle effectively discriminates between healthy, dystrophic, and transplanted dog samples. Microspectroscopy showed that dystrophic dog muscle displays higher and lower autofluorescence due to collagen cross-linking and NADH respectively than that of healthy and transplanted dogs, defining biomarkers to evaluate the impact of cell transplantation. Our findings demonstrate that DUV radiation is a sensitive, label-free method to assess the histopathological status of dystrophic muscle using small amounts of tissue, with potential applications in regenerative medicine.
Topics: Animals; Dogs; Random Forest; Support Vector Machine; Muscular Dystrophies; Ultraviolet Rays; Microspectrophotometry; Microscopy; Stem Cell Transplantation; Male; Biopsy
PubMed: 37402811
DOI: 10.1038/s41598-023-37762-1 -
Journal of the Royal Society, Interface Feb 2017Buttercup ( spp.) flowers are exceptional because they feature a distinct gloss (mirror-like reflection) in addition to their matte-yellow coloration. We investigated...
Buttercup ( spp.) flowers are exceptional because they feature a distinct gloss (mirror-like reflection) in addition to their matte-yellow coloration. We investigated the optical properties of yellow petals of several and related species using (micro)spectrophotometry and anatomical methods. The contribution of different petal structures to the overall visual signal was quantified using a recently developed optical model. We show that the coloration of glossy buttercup flowers is due to a rare combination of structural and pigmentary coloration. A very flat, pigment-filled upper epidermis acts as a thin-film reflector yielding the gloss, and additionally serves as a filter for light backscattered by the strongly scattering starch and mesophyll layers, which yields the matte-yellow colour. We discuss the evolution of the gloss and its two likely functions: it provides a strong visual signal to insect pollinators and increases the reflection of sunlight to the centre of the flower in order to heat the reproductive organs.
Topics: Flowers; Models, Theoretical; Optics and Photonics; Pigmentation; Plant Epidermis; Ranunculus
PubMed: 28228540
DOI: 10.1098/rsif.2016.0933 -
Langmuir : the ACS Journal of Surfaces... Nov 2023Aspirin has been used for broad therapeutic treatment, including secondary prevention of cardiovascular disease associated with increased cholesterol levels. Aspirin and...
Aspirin has been used for broad therapeutic treatment, including secondary prevention of cardiovascular disease associated with increased cholesterol levels. Aspirin and other nonsteroidal anti-inflammatory drugs have been shown to interact with lipid membranes and change their biophysical properties. In this study, mixed lipid model bilayers made from 1-palmitoyl-2-oleoyl--glycero-3-phosphatidylcholine (POPC) or 1,2-dioleoyl--glycero-3-phosphatidylcholine (DOPC) comprising varying concentrations of cholesterol (10:1, 4:1, and 1:1 mole ratio of lipid:chol), prepared by the droplet interface bilayer method, were used to examine the effects of aspirin at various pH on transbilayer water permeability. The presence of aspirin increases the water permeability of POPC bilayers in a concentration-dependent manner, with a greater magnitude of increase at pH 3 compared to pH 7. In the presence of cholesterol, aspirin is similarly shown to increase water permeability; however, the extent of the increase depends on both the concentration of cholesterol and the pH, with the least pronounced enhancement in water permeability at high cholesterol levels at pH 7. A fusion of data from differential scanning calorimetry, confocal Raman microspectrophotometry, and interfacial tensiometric measurements demonstrates that aspirin can promote significant thermal, structural, and interfacial property perturbations in the mixed-lipid POPC or DOPC membranes containing cholesterol, indicating a disordering effect on the lipid membranes. Our findings suggest that aspirin fluidizes phosphocholine membranes in both cholesterol-free and cholesterol-enriched states and that the overall effect is greater when aspirin is in a neutral state. These results confer a deeper comprehension of the divergent effects of aspirin on biological membranes having heterogeneous compositions, under varying physiological pH and different cholesterol compositions, with implications for a better understanding of the gastrointestinal toxicity induced by the long term use of this important nonsteroidal anti-inflammatory molecule.
Topics: Aspirin; Phosphatidylcholines; Cholesterol; Lipid Bilayers; Water; Anti-Inflammatory Agents; Hydrogen-Ion Concentration
PubMed: 37939382
DOI: 10.1021/acs.langmuir.3c02242 -
Frontiers in Molecular Biosciences 2015The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the... (Review)
Review
The more than 100,000 protein structures determined by X-ray crystallography provide a wealth of information for the characterization of biological processes at the molecular level. However, several crystallographic "artifacts," including conformational selection, crystallization conditions and radiation damages, may affect the quality and the interpretation of the electron density maps, thus limiting the relevance of structure determinations. Moreover, for most of these structures, no functional data have been obtained in the crystalline state, thus posing serious questions on their validity in infereing protein mechanisms. In order to solve these issues, spectroscopic methods have been applied for the determination of equilibrium and kinetic properties of proteins in the crystalline state. These methods are UV-vis spectrophotometry, spectrofluorimetry, IR, EPR, Raman, and resonance Raman spectroscopy. Some of these approaches have been implemented with on-line instruments at X-ray synchrotron beamlines. Here, we provide an overview of investigations predominantly carried out in our laboratory by single crystal polarized absorption UV-vis microspectrophotometry, the most applied technique for the functional characterization of proteins in the crystalline state. Studies on hemoglobins, pyridoxal 5'-phosphate dependent enzymes and green fluorescent protein in the crystalline state have addressed key biological issues, leading to either straightforward structure-function correlations or limitations to structure-based mechanisms.
PubMed: 25988179
DOI: 10.3389/fmolb.2015.00012 -
The New Phytologist Mar 2021Hydrangea sepals exhibit a wide range of colors, from red, through purple, to blue; the purple color is a color mosaic. However, all of these colors are derived from the...
Hydrangea sepals exhibit a wide range of colors, from red, through purple, to blue; the purple color is a color mosaic. However, all of these colors are derived from the same components: simple anthocyanins, 3-O-glycosyldelphinidins, three co-pigment components, acylquinic acids and aluminum ions (Al ). We show the color mosaic is a result of graded differences in intravacuolar factors. In order to clarify the mechanisms of mosaic color, we performed single-cell analyses of vacuolar pH, and anthocyanin, co-pigment and Al content. From the sepals, a protoplast mixture of various colors was obtained. The cell color was evaluated by microspectrophotometry and vacuolar pH then was recorded by using a pH microelectrode. The organic and Al contents were quantified by micro-HPLC. We found that the bluer the cell, the greater the ratio of 5-O-acylquinic acids and Al to anthocyanins. Furthermore, reproducing experiments were conducted by mixing the components under various pH condition; all the colors could be reproduced in the various mixing conditions. Based on the above, we provide experimental evidence for cell color variation in hydrangea. Our study demonstrates the expression of phenotypic differences without any direct genomic control.
Topics: Aluminum; Anthocyanins; Color; Flowers; Hydrangea; Single-Cell Analysis
PubMed: 33220077
DOI: 10.1111/nph.17099