Phase modulation nanoscopy: a simple approach to enhanced optical resolution

2015 ◽  
Vol 177 ◽  
pp. 507-515 ◽  
Author(s):  
Robert Pal
Keyword(s):  
Phase Modulation ◽  
Laser Scanning ◽  
Optical Resolution ◽  
Super Resolution ◽  
Laser Scanning Microscopy ◽  
Optical Diffraction ◽  
Optical Modulator ◽  
Confocal Laser ◽  
Electro Optical Modulator ◽  
Experimental Resolution

A new modular super-resolution technique called Phase Modulation Nanoscopy (PhMoNa) has been developed in order to break the optical diffraction barrier in Confocal Laser Scanning Microscopy (LSCM). This technique is based on using spatially modulated illumination intensity, whilst harnessing the fluorophore's non-linear emission response. It allows experimental resolution in both lateral and axial domains to be improved by at least a factor of 2. The work is in its initial phase, but by using a custom built Electro Optical Modulator (EOM) in conjunction with functionalised Ln(iii) complexes as probes, a sub-diffraction resolution of ∼60 nm was achieved of selected cellular organelles in long term live cell imaging experiments.

scholarly journals Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Nazar Oleksiievets ◽  
Yelena Sargsyan ◽  
Jan Christoph Thiele ◽  
Nikolaos Mougios ◽  
Shama Sograte-Idrissi ◽  
...  
Keyword(s):  
Fluorescence Lifetime ◽  
Laser Scanning ◽  
Super Resolution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wide Field ◽  
Fluid Exchange ◽  
Multiplexed Imaging ◽  
Bio Imaging ◽  
A Cell

AbstractDNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

scholarly journals Confocal Laser-Scanning Fluorescence-Lifetime Single-Molecule Localisation Microscopy

2020 ◽  
Author(s):  
Jan Christoph Thiele ◽  
Dominic Helmerich ◽  
Nazar Oleksiievets ◽  
Roman Tsukanov ◽  
Eugenia Butkevich ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Spectral Properties ◽  
Laser Scanning ◽  
Super Resolution ◽  
Stimulated Emission ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Localisation Microscopy

AbstractFluorescence lifetime imaging microscopy (FLIM) is an important technique that adds another dimension to the intensity and colour information of conventional microscopy. In particular, it allows for multiplexing fluorescent labels that have otherwise similar spectral properties. Currently, the only super-resolution technique that is capable of recording super-resolved images with lifetime information is STimulated Emission Depletion (STED) microscopy. In contrast, all Single-Molecule Localisation Microscopy (SMLM) techniques that employ wide-field cameras completely lack the lifetime dimension. Here, we combine Fluorescence-Lifetime Confocal Laser-Scanning Microscopy (FL-CLSM) with SMLM for realising single-molecule localisation-based fluorescence-lifetime super-resolution imaging (FL-SMLM). Besides yielding images with a spatial resolution much beyond the diffraction limit, it determines the fluorescence lifetime of all localised molecules. We validate our technique by applying it to direct STochastic Optical Reconstruction Microscopy (dSTORM) and Points Accumulation for Imaging in Nanoscale Topography (PAINT) imaging of fixed cells, and we demonstrate its multiplexing capability on samples with two different labels that differ only by fluorescence lifetime but not by their spectral properties.

Mammalian Apoptosis in Whole Neonatal Ovaries, Embryos and Fetal Limbs Using Confocal Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 598-599
Author(s):  
Robert M Zucker
Keyword(s):  
Limb Development ◽  
Laser Scanning ◽  
Optical Resolution ◽  
Confocal Microscope ◽  
Laser Scanning Microscopy ◽  
Dimensional Structure ◽  
Confocal Laser ◽  
Depth Of Penetration ◽  
Technical Problems ◽  
Sample Technique

The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimensional structure offers a viable alternative to traditional sectioning approaches. However, the imaging of such whole-mounts presents technical problems of its own. One of the major problems with using a confocal microscope to image whole organs and embryos is the depth of penetration of the laser light into the tissue. We have optimized the confocal microscope performance and developed a sample technique that increases the optical resolution of the system.CLSM has been used to study cellular death (apoptosis) during GD 8-12 normal rat/mouse embryonic development, neonatal ovarian development (PD10-45) and fetal limb development (GD 11-15). LysoTracker Red (LT) was incorporated into the tissue prior to laser excitation. It is aldehyde fixable stain that concentrates into acidic structures or into cells that have high lysosomal activity.

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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