CONFOCAL MICROSCOPY APPLIED TO PALEONTOLOGICAL SPECIMENS

2016 ◽  
Vol 22 ◽  
pp. 39-55 ◽  
Author(s):  
Alexander D. Ball ◽  
Tomasz Goral ◽  
Seyit A. Kamanli
Keyword(s):  
Confocal Microscopy ◽  
Laser Scanning ◽  
Fluorescent Dyes ◽  
Three Dimensional ◽  
Sample Surface ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Image Stack ◽  
Selective Staining ◽  
Wide Range

AbstractConfocal laser scanning microscopy is a well-established optical technique allowing for three-dimensional (3-D) visualization of fluorescent specimens with a resolution close to the diffraction limit of light. Thanks to the availability of a wide range of fluorescent dyes and selective staining using antibodies, the technique is commonly used in life sciences as a powerful tool for studying different biological processes, often at the level of single molecules. However, this type of approach is often not applicable for specimens that are preserved in historical slide collections, embedded in amber, or are fossilized, and cannot be exposed to any form of selective staining or other form of destructive treatment. This usually narrows the number of microscopic techniques that can be used to study such specimens to traditional light microscopy or scanning electron microscopy. However, these techniques have their own limitations and cannot fully reveal 3-D structures within such barely accessible samples. Can confocal microscopy be of any help? The answer is positive, and it is due to the fact that many paleontological specimens exhibit a strong inherent autofluorescence that can serve as an excellent source of emitted photons for confocal microscopy visualizations either through reconstruction of the induced autoflourescent signal from the sample, or through reconstruction of the reflected signal from the sample surface. Here, we describe the workflow and methodology involved in acquiring confocal data from a sample and reprocessing the resulting image stack using the image-processing program imageJ before reconstructing the data using the open-source 3-D rendering program, Drishti. This approach opens new possibilities for using confocal microscopy in a nondestructive manner for visualizing complex paleontological material that has never previously been considered as suitable for this type of microscopic technique.

scholarly journals Hydrophobic analogues of rhodamine B and rhodamine 101: potent fluorescent probes of mitochondria in living C. elegans

2012 ◽  
Vol 8 ◽  
pp. 2156-2165 ◽  
Author(s):  
Laurie F Mottram ◽  
Safiyyah Forbes ◽  
Brian D Ackley ◽  
Blake R Peterson
Keyword(s):  
Fluorescent Probes ◽  
Rhodamine B ◽  
Laser Scanning ◽  
Fluorescent Dyes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
C Elegans ◽  
Wide Range ◽  
Rhodamine 101

Mitochondria undergo dynamic fusion and fission events that affect the structure and function of these critical energy-producing cellular organelles. Defects in these dynamic processes have been implicated in a wide range of human diseases including ischemia, neurodegeneration, metabolic disease, and cancer. To provide new tools for imaging of mitochondria in vivo, we synthesized novel hydrophobic analogues of the red fluorescent dyes rhodamine B and rhodamine 101 that replace the carboxylate with a methyl group. Compared to the parent compounds, methyl analogues termed HRB and HR101 exhibit slightly red-shifted absorbance and emission spectra (5–9 nm), modest reductions in molar extinction coefficent and quantum yield, and enhanced partitioning into octanol compared with aqueous buffer of 10-fold or more. Comparison of living C. elegans (nematode roundworm) animals treated with the classic fluorescent mitochondrial stains rhodamine 123, rhodamine 6G, and rhodamine B, as well as the structurally related fluorophores rhodamine 101, and basic violet 11, revealed that HRB and HR101 are the most potent mitochondrial probes, enabling imaging of mitochondrial motility, fusion, and fission in the germline and other tissues by confocal laser scanning microscopy after treatment for 2 h at concentrations as low as 100 picomolar. Because transgenes are poorly expressed in the germline of these animals, these small molecules represent superior tools for labeling dynamic mitochondria in this tissue compared with the expression of mitochondria-targeted fluorescent proteins. The high bioavailabilty of these novel fluorescent probes may facilitate the identification of agents and factors that affect diverse aspects of mitochondrial biology in vivo.

scholarly journals One-step preparation of antimicrobial silicone materials based on PDMS and salicylic acid: insights from spatially and temporally resolved techniques

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Luca Barbieri ◽  
Ioritz Sorzabal Bellido ◽  
Alison J. Beckett ◽  
Ian A. Prior ◽  
Jo Fothergill ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Laser Scanning ◽  
Pathogenic Bacteria ◽  
Pharmaceutical Products ◽  
Laser Scanning Microscopy ◽  
Wound Dressings ◽  
Confocal Laser ◽  
Vis Spectroscopy ◽  
Wide Range ◽  
One Step

AbstractIn this work, we introduce a one-step strategy that is suitable for continuous flow manufacturing of antimicrobial PDMS materials. The process is based on the intrinsic capacity of PDMS to react to certain organic solvents, which enables the incorporation of antimicrobial actives such as salicylic acid (SA), which has been approved for use in humans within pharmaceutical products. By combining different spectroscopic and imaging techniques, we show that the surface properties of PDMS remain unaffected while high doses of the SA are loaded inside the PDMS matrix. The SA can be subsequently released under physiological conditions, delivering a strong antibacterial activity. Furthermore, encapsulation of SA inside the PDMS matrix ensured a diffusion-controlled release that was tracked by spatially resolved Raman spectroscopy, Attenuated Total Reflectance IR (ATR-IR), and UV-Vis spectroscopy. The biological activity of the new material was evaluated directly at the surface and in the planktonic state against model pathogenic bacteria, combining confocal laser scanning microscopy, electron microscopy, and cell viability assays. The results showed complete planktonic inhibition for clinically relevant strains of Staphylococcus aureus and Escherichia coli, and a reduction of up to 4 orders of magnitude for viable sessile cells, demonstrating the efficacy of these surfaces in preventing the initial stages of biofilm formation. Our approach adds a new option to existing strategies for the antimicrobial functionalisation of a wide range of products such as catheters, wound dressings and in-dwelling medical devices based on PDMS.

Behaviour of nucleolar proteins in nuclei lacking ribosomal genes. A study by confocal laser scanning microscopy

1991 ◽  
Vol 98 (1) ◽  
pp. 99-105
Author(s):  
D. Hernandez-Verdun ◽  
M. Robert-Nicoud ◽  
G. Geraud ◽  
C. Masson
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Specific Marker ◽  
Nucleolar Proteins ◽  
Fibrillar Component

The behaviour of nucleolar proteins in cycling PtK1 cells and in micronuclei with or without NORs was investigated by immunofluorescence using antibodies from autoimmune sera and confocal laser scanning microscopy. These antibodies were shown by electron microscopy to recognize antigens confined to only one of the three basic nucleolar components: fibrillar centres (FC), dense fibrillar component (DFC) and granular component (GC). Serial optical sections allowed us to determine the three-dimensional organization of these components in the nucleolus of cycling cells. Furthermore, clear differences were found in the distribution of the various antigens in micronucleated cells. Three patterns could be observed: (1) the FC antigens were found mainly in the nucleoli, but also in varying amounts in the dots; (2) surprisingly, the DFC antigens were found to accumulate preferentially in the dots; (3) the GC-specific marker stained intensively the nucleoli as well the dots. The results are interpreted with regard to possible mechanisms for targeting nucleolar proteins to the site of nucleolar formation.

scholarly journals Astrogliosis in an Experimental Model of Hypovitaminosis B12: A Cellular Basis of Neurological Disorders due to Cobalamin Deficiency

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2261
Author(s):  
Zuzanna Rzepka ◽  
Jakub Rok ◽  
Justyna Kowalska ◽  
Klaudia Banach ◽  
Justyna Magdalena Hermanowicz ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Cobalamin Deficiency ◽  
Confocal Laser ◽  
In Vivo Model ◽  
Cellular Expression ◽  
Wide Range ◽  
Vitro Model

Cobalamin deficiency affects human physiology with sequelae ranging from mild fatigue to severe neuropsychiatric abnormalities. The cellular and molecular aspects of the nervous system disorders associated with hypovitaminosis B12 remain largely unknown. Growing evidence indicates that astrogliosis is an underlying component of a wide range of neuropathologies. Previously, we developed an in vitro model of cobalamin deficiency in normal human astrocytes (NHA) by culturing the cells with c-lactam of hydroxycobalamin (c-lactam OH-Cbl). We revealed a non-apoptotic activation of caspases (3/7, 8, 9) in cobalamin-deficient NHA, which may suggest astrogliosis. The aim of the current study was to experimentally verify this hypothesis. We indicated an increase in the cellular expression of two astrogliosis markers: glial fibrillary acidic protein and vimentin in cobalamin-deficient NHA using Western blot analysis and immunocytochemistry with confocal laser scanning microscopy. In the next step of the study, we revealed c-lactam OH-Cbl as a potential non-toxic vitamin B12 antagonist in an in vivo model using zebrafish embryos. We believe that the presented results will contribute to a better understanding of the cellular mechanism underlying neurologic pathology due to cobalamin deficiency and will serve as a foundation for further studies.

Confocal Laser Scanning Microscopy of Hamster Cerebellum using FM4-64 as Intracellular Staining

1998 ◽  
Vol 4 (S2) ◽  
pp. 1126-1127
Author(s):  
O. Castejón ◽  
P. Sims
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Granular Layer ◽  
Fluorescent Dyes ◽  
Laser Scanning Microscopy ◽  
Climbing Fibers ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Fluorescence Signal

Confocal laser scanning microscopy is an excellent method to study nerve cell morphology and the three-dimensional distribution and interrelationship of dentrites and axons in the central nervous system. The cerebellum has been taking as a model of a gray center.The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum,5 mm thick, were incubated in 10,30 and 100 μm solutions of FM4-64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances .They exhibited high fluorescence signal at the level of myelin sheath.Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fibers bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies.

scholarly journals Software-Based Three-Dimensional Deconvolution Microscopy of Cytoskeletal Proteins in Cultured Fibroblast Using Open-Source Software and Open Hardware

2019 ◽  
Vol 5 (12) ◽  
pp. 88
Author(s):  
Kazuo Katoh
Keyword(s):  
Open Source ◽  
Open Source Software ◽  
Laser Scanning ◽  
Low Cost ◽  
Three Dimensional ◽  
Cytoskeletal Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Actual Measurement ◽  
Open Hardware

As conventional fluorescence microscopy and confocal laser scanning microscopy generally produce images with blurring at the upper and lower planes along the z-axis due to non-focal plane image information, the observation of biological images requires “deconvolution.” Therefore, a microscope system’s individual blur function (point spread function) is determined theoretically or by actual measurement of microbeads and processed mathematically to reduce noise and eliminate blurring as much as possible. Here the author describes the use of open-source software and open hardware design to build a deconvolution microscope at low cost, using readily available software and hardware. The advantage of this method is its cost-effectiveness and ability to construct a microscope system using commercially available optical components and open-source software. Although this system does not utilize expensive equipment, such as confocal and total internal reflection fluorescence microscopes, decent images can be obtained even without previous experience in electronics and optics.

scholarly journals Applicability of Confocal Raman Microscopy to Observe Microstructural Modifications of Cream Cheeses as Influenced by Freezing

Foods ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 679 ◽  
Author(s):  
Marcello Alinovi ◽  
Germano Mucchetti ◽  
Ulf Andersen ◽  
Tijs A. M. Rovers ◽  
Betina Mikkelsen ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Dyes ◽  
Raman Microscopy ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Confocal Raman Microscopy ◽  
Freezing And Thawing ◽  
High Moisture ◽  
Cream Cheese ◽  
Confocal Raman

Confocal Raman microscopy is a promising technique to derive information about microstructure, with minimal sample disruption. Raman emission bands are highly specific to molecular structure and with Raman spectroscopy it is thus possible to observe different classes of molecules in situ, in complex food matrices, without employing fluorescent dyes. In this work confocal Raman microscopy was employed to observe microstructural changes occurring after freezing and thawing in high-moisture cheeses, and the observations were compared to those obtained with confocal laser scanning microscopy. Two commercially available cream cheese products were imaged with both microscopy techniques. The lower resolution (1 µm/pixel) of confocal Raman microscopy prevented the observation of particles smaller than 1 µm that may be part of the structure (e.g., sugars). With confocal Raman microscopy it was possible to identify and map the large water domains formed during freezing and thawing in high-moisture cream cheese. The results were supported also by low resolution NMR analysis. NMR and Raman microscopy are complementary techniques that can be employed to distinguish between the two different commercial formulations, and different destabilization levels.

Sign in / Sign up

Export Citation Format

Share Document