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Results for “Fluorescence Microscopy”

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PubMed2004

Concepts for nanoscale resolution in fluorescence microscopy.

Spatio-temporal visualization of cellular structures by fluorescence microscopy has become indispensable in biology. However, the resolution of conventional fluorescence microscopy is limited by diffraction to about 180 nm in the focal plane and to about 500 nm along the optic axis. Recently, concepts have emerged that overcome the diffraction resolution barrier fundamentally. Formed on the basis of reversible saturable optical transitions, these concepts might eventually allow us to investigate hitherto inaccessible details within live cells.

Animals↗
PubMed1975

[Fluorescent microscopy in the diagnosis of Dictyocaulus infection in sheep].

Fluorescence microscopy was employed to study Dictyocaulus infection in 15 experimentally infected lambs each given 2000 D. filaria larvae. Studied were also the sera of 10 control lambs and of 20 sheep and goats infected with other helminths. Antibodies were established after the 14th day in 53 per cent of the lambs, the 20th day in 86.6 per cent, and the 27th day in all animals infected with the parasite. Antigens were found mainly in the wall of the digestive and reproductive organs, and in the cuticle of the larvae. As a method of early diagnosis fluorescence microscopy is simple, precise, and readily applicable in establishing Dictyocaulus infections.

Animals↗
PubMed1980

Studies on structural changes of F-actin and myosin in living, intact and damaged muscle fibres by means of polarized ultraviolet fluorescence microscopy.

By means of polarized ultraviolet fluorescence microscopy the structural changes of F-actin and myosin were discovered at the changing a functional state of a living muscle fibre and during spreading degeneration (Zenker's necrosis). The character of conformational changes of F-actin and myosin at activation, contraction, contracture and rigor is similar, but the number of changed macromolecules depends on a fibre state. At fibre local damage in its morphological intact parts there was found an alternation of zones, reflecting two states unusual for a fibre. During spreading degeneration these states transform into irreversible contracture and then into rigor. Similar changes were observed in muscle fibres obtained from denervated muscles.

Actins↗
PubMed1989

[Status and developmental trends in the use of fluorescence microscopy studies in microbiology].

There are many applications for fluorescence microscopy in the field of microbiology for diagnostic and scientific purposes. Autofluorescence as well as secondary fluorescence induced by staining of specimen with fluorochromes or with fluorochrome labeled antibodies are used for detection and differentiation of microorganisms. Small demands for object preparation and short test times are advantages for screening tests. During the last time considerable progresses in methods and technical equipments are noticeable and offer numerous new applications for fluorescence microscopy. New microscopes show clearly increased intensity of excitation. This requires a selection of object slides and cover glasses with lowest autofluorescence, blocking of fading by addition of PPD to embedding medium and a better adaptation of filters for excitation and fluorescence to the characteristics of fluorochromes. Multiple fluorochroming and combined application of fluorescence and alternative contrast techniques such as phase contrast are of rising importance in practice.

Bacteria↗
PubMed2006

To see or not to see: lateral organization of biological membranes and fluorescence microscopy.

In the last few years several experimental strategies based on epi-, confocal and two photon excitation fluorescence microscopy techniques have been employed to study the lateral structure of membranes using giant vesicles as model systems. This review article discusses the methodological aspects of the aforementioned experimental approaches, particularly stressing the information obtained by the use of UV excited fluorescent probes using two-photon excitation fluorescence microscopy. Additionally, the advantages of utilizing visual information, to correlate the lateral structure of compositionally simple membranes with complex situations, i.e., biological membranes, will be addressed.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2001

Cellular screening assays using fluorescence microscopy.

The recent development of automated fluorescence imaging systems has enabled fluorescence microscopy to be used for the purposes of compound screening. This information-rich technique has found various applications, including screening for the effect of kinase inhibitors on the cytoskeleton, agonist-stimulated receptor internalization, and protein phosphorylation and acetylation. The discovery of novel fluorescent proteins and new fluorescent dyes will find many applications in multichannel fluorescence imaging assays.

Animals↗
PubMed2003

Fluorescence microscopy: a powerful technique to detect low GUS activity in vascular tissues.

We have previously shown that the Eucalyptus gunnii EgCAD2 promoter was preferentially expressed in vascular tissues in different transgenic plants (poplar, tobacco, Arabidopsis and grapevine). In order to delineate the cis elements governing this vascular expression pattern, promoter deletion analysis was performed allowing us to identify the proximal region [-340/-124] as essential for vascular cambium/xylem-specific expression. In plants transformed with the smallest promoter region [-124/+117], the GUS activity was difficult to detect using conventional bright field microscopy. To overcome this problem, we used fluorescence microscopy, enabling us to show that the [-124/+117] region contained cis-elements driving activity in phloem fibres but not in secondary xylem. The technical improvement of the histochemical detection of GUS activity using fluorescence microscopy enables accurate investigation of low GUS activity in phenol-rich tissues.

Eucalyptus↗
PubMed2000

A comparison of optical geometries for combined flash photolysis and total internal reflection fluorescence microscopy.

Total internal reflection fluorescence (TIRF) microscopy, used in conjunction with flash photolysis, provides a useful way of investigating the kinetics of macromolecular interactions. We compare different TIRF optical geometries to establish an optimal combination. Excitation light was introduced via four different arrangements: (1) a prism positioned on the microscope optical axis, (2) an offset prism with propagation through a silica slide trans to the objective lens, (3) an offset prism with propagation through a silica coverslip cis to a water-immersion objective lens and (4) a prismless arrangement using a high NA oil-immersion objective lens. Photolysis was achieved using a Xe flash lamp and a customised silica condenser lens. Single myosin molecules labelled with a Cy3 fluorophore were used as a test sample. Although the offset trans prism gave the best signal-to-background ratio, a customised thin rhombic prism incorporated, on axis, into the flash condenser assembly was almost as good and was more practical for scanning multiple fields. An oil-immersion lens gave the brightest image for sample depths < 30 micrometer but above this limit, a water-immersion lens was better. The prismless arrangement may offer advantages in other situations but it is important to check the actual numerical aperture of the objective lens.

Animals↗
PubMed2001

Multi-photon fluorescence microscopy--the response of plant cells to high intensity illumination.

Multi-photon fluorescence microscopy has been cited for its advantage in increased depth penetration due to low linear absorption and scattering coefficient of biological specimen in the near infrared (NIR) range. Because of the need of high peak power for efficiently exciting two-photon fluorescence, the relationship between cell damage and peak power has become an interesting and much debated topic in the applications of multi-photon fluorescence microscopy. It is conceivable that at high illumination intensity, non-linear photochemical processes have impacts on cell physiology and viability in ways much different from low illumination in the linear domain. In this article, we discuss some of the issues in two-photon fluorescence microscopy, including the degree of transparency of the specimen, a comparison of single- and two-photon excited fluorescence spectra, and the cell damage under high intensity illumination, using plant cells as a model.

Absorption↗
PubMed2004

Laser-assisted fluorescence microscopy for measuring cell membrane dynamics.

Membranes of living cells are characterized by laser-assisted fluorescence microscopy, in particular a combination of microspectrofluorometry, total internal reflection fluorescence microscopy (TIRFM), fluorescence lifetime imaging (FLIM) and Forster resonance energy transfer (FRET) spectroscopy. The generalized polarization (GP, characterizing a spectral shift which depends on the phase of membrane lipids) as well as the effective fluorescence lifetime (tau(eff)) of the membrane marker laurdan were revealed to be appropriate parameters for membrane stiffness and fluidity. GP decreased with temperature, but increased during cell growth and was always higher for the plasma membrane than for intracellular membranes. Microdomains of different fluorescence lifetimes tau(eff) were observed at temperatures above 30 degree C and disappeared during cell aging. Non-radiative energy transfer was used to detect laurdan selectively in close proximity to a molecular acceptor (DiI) and may present a possibility for measuring membrane dynamics in specific microenvironments.

Animals↗