scholarly journals Fast Optical Sectioning for Widefield Fluorescence Mesoscopy with the Mesolens based on HiLo Microscopy

2018 ◽  
Author(s):  
Jan Schniete ◽  
Aimee Franssen ◽  
John Dempster ◽  
Trevor Bushell ◽  
William Bradshaw Amos ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Computation Time ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Optical Sectioning ◽  
Full Field ◽  
Custom Software ◽  
Very Large Datasets ◽  
Acquisition Speed ◽  
Subcellular Resolution

ABSTRACTWe present here a fast optical sectioning method for optical mesoscopy based on HiLo microscopy, which makes possible imaging of specimens of up to 4.4 mm × 3 mm × 3 mm in volume in under 17 hours (estimated for a z-stack comprising 1000 images excluding computation time) with subcellular resolution throughout. Widefield epifluorescence imaging is performed with the Mesolens using a high pixel-number camera capable of sensor-shifting to generate a 259.5 Megapixel image, and we have developed custom software to perform HiLo processing of the very large datasets. Using this method, we obtain comparable sectioning strength to confocal laser scanning microscopy (CLSM), with sections as thin as 6.8±0.2 μm and raw acquisition speed of 1 minute per slice which is up to 30 times faster than CLSM on the full field of view (FOV) of the Mesolens of 4.4 mm with lateral resolution of 0.7 μm and axial resolution of 7 μm. We have applied this HiLo mesoscopy method to image fixed and fluorescently stained hippocampal neuronal specimens and a 5-day old zebrafish larva.

A technique in confocal laser microscopy for establishing biofilm coverage and thickness

1997 ◽  
Vol 36 (10) ◽  
pp. 117-124 ◽  
Author(s):  
G. Silyn-Roberts ◽  
G. Lewis
Keyword(s):  
Laser Scanning ◽  
Sewage Treatment ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rock Surface ◽  
Optical Sectioning ◽  
Image Analysis Technique ◽  
Rock Types ◽  
Analysis Technique ◽  
Biofilm Quantification

This study uses confocal laser scanning microscopy to determine the coverage and thickness of biofilms on rock types commonly used in wetland sewage treatment systems in New Zealand. Samples of scoria, greywacke and slag - with glass used as a comparison - were submerged in subsurface flow wetlands and examined after six weeks. An image analysis technique was used to quantitatively determine the coverage and thickness of each biofilm. The technique consisted of the biofilm quantification of each individual image obtained from the confocal optical sectioning. The results indicated that the biofilm coverage for the substrata types did not exceed 25%. However, there was a marked difference between the biofilm structures grown on the different substrata; that on glass formed thin spindly structures, and slag and scoria showed similar dense patches interspersed with open channel structures that followed the contours of the pocketed rock surface.

Confocal Laser Scanning Microscopy of Whole Mouse and Human Embryos: Apoptosis and Morphology.

1999 ◽  
Vol 5 (S2) ◽  
pp. 1084-1085
Author(s):  
Zucker Robert M. ◽  
Sulik Kathy ◽  
Owen T Price
Keyword(s):  
Sample Preparation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Human Embryos ◽  
Embryo Morphology ◽  
Optical Sectioning ◽  
Internal Structures ◽  
Opaque Surface ◽  
Made In

Two novel sample preparation procedures have been developed to visualize morphological structures in embryonic and fetal tissues. In the first, optimization of the sample preparation allows for the visualization of embryos with a thickness in excess of 500 microns. The morphology of the internal structures in these embryos is observed by optical sectioning. The general sample preparation procedures included paraformaldehyde fixation, methanol dehydration and clearing with either benzyl alcohol/benzyl benzoate or methyl salicylate. The dyes (i.e. LysoTracker Red, YoPro) are incorporated into living or fixed embryos to indicate a specific biological function or embryo morphology. Using these stained embryos, the detection of apoptosis and its subsequent quantification were made in addition to the observation of morphology.In the second technique, the confocal microscope has been adapted to produce images similar to those generated by a scanning electron microscope. For this application, the embryos are coated with a dilute solution of acridine orange resulting in opaque surface fluorescence.

Interspecies Uptake of Polymeric Microspheres

1998 ◽  
Vol 550 ◽  
Author(s):  
Chris Thanos ◽  
Maryellen Sandor ◽  
Yong Jong ◽  
Jules Jacob ◽  
Kay-Pong Yip ◽  
...  
Keyword(s):  
Light Microscopy ◽  
Laser Scanning ◽  
Polarized Light ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Optical Sectioning ◽  
Intestinal Tissue ◽  
Polymeric Microspheres ◽  
Particle Uptake

AbstractParticle uptake into intestinal tissue has seen increasing attention due to its implications in drug delivery. We attempted to observe a delivery system in vivo and examine uptake in different species. Microspheres were fabricated from polymers including polyanhydrides and delivered to an isolated loop of intestine in several species. The microspheres contained a dye either conjugated to a protein or incorporated freely and were used to qualitatively detect and locate the spheres in the villi of the length of the small intestine. Microspheres were dispersed, sized by a Coulter particle size analyzer, and characterized by confocal and cross-polarized light microscopy, FTIR and SEM. Coulter analysis revealed microspheres to be generally less than 5 microns in diameter. SEM typically showed homogeneous morphology among groups of microspheres. In vivo uptake experiments were performed in rodents, pigs, and ruminants using various microsphere formulations. Microspheres were delivered into the proximal end of the jejunum of anesthetized animals and allowed adequate transit time to be taken up. Animals were euthanized at various time points for explantation of tissue and sampling of blood. Excised samples were embedded inq polyvinyl alcohol, frozen, and cut into sections ranging between 7 and 14 μm in thickness. Our method of incorporating dyes allowed for simultaneous visualization by visible light microscopy and confocal laser scanning microscopy. Two-fluorochrome fluorescence of the microspheres and optical sectioning confirmed the presence of microspheres within intestinal tissue. The amount of uptake depended on the animal model, the duration of the experiment, and the composition of the microsphere. An assay for either the fluorescent dye, the protein attached to it, or the polymer encapsulating it may enable us to determine intracellular concentrations of mierospheres for the quantification of uptake.

Application of Confocal Laser Scanning Microscopy in Bone

1998 ◽  
Vol 02 (01) ◽  
pp. 65-71 ◽  
Author(s):  
H. Fujii ◽  
D. J. Wood ◽  
J. M. Papadimitriou ◽  
M. H. Zheng
Keyword(s):  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Bone Allograft ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser ◽  
Optical Sectioning ◽  
Sectioning Method ◽  
Plastic Embedding

The optical sectioning method of confocal laser scanning microscopy provides higher resolution than standard light microscope techniques. The use of optical rather than physical sections for detailed histological analyses of bone obviates the need for either decalcification or complex plastic embedding processes which are required as a routine for the preparation of thin microtome sections. In this study we have used confocal laser scanning microscopy for the morphological analyses of fresh unembedding human cortical bone, bone allograft and bone cement interfaces. Our results have indicated that such an approach has provided a relatively easy and rapid means for the assessment of the histology of normal and pathological bone.

scholarly journals Imaging Degraded Wood by Confocal Microscopy

1998 ◽  
Vol 6 (4) ◽  
pp. 14-15 ◽  
Author(s):  
Yoon Soo Kim ◽  
Adya Singh
Keyword(s):  
Laser Scanning ◽  
Science Studies ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Computer Assisted ◽  
Optical Sectioning ◽  
Archaeological Wood ◽  
Wide Range ◽  
High Resolution Images ◽  
Wood Science

The application of confocal laser scanning microscopy (CLSM) in the studies of biological materials is rapidly expanding because of the opportunity to produce sharp, high resolution images through optical sectioning and computer assisted 3-D reconstruction. At our institute CLSM is being used in a wide range of forestry and wood science studies.Recently we investigated the potential usefulness of CLSM in characterizing biologically degraded wood. The following are images produced from an archaeological wood which has been buried in a wet environment (rice field) for nearly 2,000 years in South Korea and is apparently degraded by bacteria. In an attempt to develop suitable techniques which can be used for routine examination of fragile degraded wood with CLSM, we have compared two different embedding methods for their suitability in preserving the integrity of cells. The embedding media are paraffin wax and LR White resin.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

Use of confocal laser scanning microscopy and a computer model to understand ink cavitation and filamentation

TAPPI Journal ◽  
2010 ◽  
Vol 9 (10) ◽  
pp. 7-15
Author(s):  
HANNA KOIVULA ◽  
DOUGLAS BOUSFIELD ◽  
MARTTI TOIVAKKA
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Print Quality ◽  
Length Scale ◽  
Offset Printing ◽  
Significant Difference ◽  
Printing Speed

In the offset printing process, ink film splitting has an important impact on formation of ink filaments. The filament size and its distribution influence the leveling of ink and hence affect ink setting and the print quality. However, ink filaments are difficult to image due to their short lifetime and fine length scale. Due to this difficulty, limited work has been reported on the parameters that influence filament size and methods to characterize it. We imaged ink filament remains and quantified some of their characteristics by changing printing speed, ink amount, and fountain solution type. Printed samples were prepared using a laboratory printability tester with varying ink levels and operating settings. Rhodamine B dye was incorporated into fountain solutions to aid in the detection of the filaments. The prints were then imaged with a confocal laser scanning microscope (CLSM) and images were further analyzed for their surface topography. Modeling of the pressure pulses in the printing nip was included to better understand the mechanism of filament formation and the origin of filament length scale. Printing speed and ink amount changed the size distribution of the observed filament remains. There was no significant difference between fountain solutions with or without isopropyl alcohol on the observed patterns of the filament remains.

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