In view of a pharmacological test system with living cells we examined Förster Resonance Energy Transfer (FRET) between fluorescent proteins upon Total Internal Reflection (TIR) of a laser beam. We measured in particular HeLa cells expressing membrane associated EGFR-CFP and Grb2-YFP prior to and subsequent to stimulation with the epidermal growth factor EGF. In addition, HeLa cells expressing a membrane associated Green Fluorescent Protein (HeLa hFR-GPI-GFP) were incubated with the energy acceptor Nile Red for further validation. Measurements range from fluorescence microscopy to Fluorescence Lifetime Imaging (FLIM) of a larger number of samples in a TIR reader.
A miniaturized setup for sample rotation on a microscope stage has been developed, combined with light sheet, confocal or structured illumination microscopy and applied to living cells as well as to small organisms. This setup permits axial tomography with improved visualization of single cells or small cell clusters as well as an enhanced effective 3D resolution upon sample rotation.
Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z-stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.
Non-radiative cell membrane associated energy transfer (FRET) from a cyan (ECFP) to a yellow (EYFP) fluorescent protein is used for detection of apoptosis in multi-cellular tumor spheroids. Low light exposure in light sheet fluorescence microscopy is combined with the detection of spectral changes and prolonged fluorescence lifetimes of the donor ECFP upon apoptosis. In view of future label-free detection preliminary experiments of light scattering microscopy with high angular resolution are reported which may give additional information on morphological changes.
A new concept of three-dimensional imaging of tumor cell spheroids by light sheet-based fluorescence microscopy and nanosecond ratio imaging is described. Due to its low light dose and alternative excitation by two laser wavelengths (391 and 470 nm), this method maintains cell viability and permits recording of real-time kinetics. A genetically encoded sensor permits measurement of the redox state of glutathione and visualization of the impact of oxygen radicals. The pharmaceutically relevant system is tested upon addition of an oxidizing agent (H 2 O 2 ), as well as upon addition of the apoptosis-inducing agent staurosporine.
The uptake and intracellular distribution of the cytostatic drug doxorubicin is visualized in 2D and 3D systems of
human breast cancer cells and fibroblasts by fluorescence microscopy and spectroscopy. Fluorescence lifetime imaging
(FLIM) and scattering experiments with high angular resolution are suggested to probe apoptotic reactions. A light
scattering microscope as well as a light sheet module for 3D fluorescence microscopy have been developed and are
used for this purpose.
Autofluorescence spectra, images, and decay kinetics of U251-MG glioblastoma cells prior and subsequent to activation of tumor suppressor genes are compared. While phase contrast images and fluorescence intensity patterns of tumor (control) cells and less malignant cells are similar, differences can be deduced from autofluorescence spectra and decay kinetics. In particular, upon near UV excitation, the fluorescence ratio of the free and protein-bound coenzyme nicotinamid adenine dinucleotide depends on the state of malignancy and reflects different cytoplasmic (including lysosomal) and mitochondrial contributions. While larger numbers of fluorescence spectra are evaluated by principal component analysis, a multivariate data analysis method, additional information on cell metabolism is obtained from spectral imaging and fluorescence lifetime imaging microscopy.
A novel setup for fluorescence imaging of 3-dimensional cell cultures is described. The method is based on structured
illumination by various light sources and detection of images in individual cellular planes.
Autofluorescence and Raman spectra, images and decay kinetics of U251-MG glioblastoma cells prior and after
activation of tumor suppressor genes are compared. While phase contrast images and fluorescence patterns of the tumor
(control) cells and the less malignant cells are similar, differences can be deduced from autofluorescence spectra and
decay times. In particular, upon excitation around 375nm, the fluorescence ratio of the protein bound and the free
coenzyme NADH depends on the state of malignancy. Slight differences are also observed in Raman spectra of these cell
lines, in particular at wave numbers around 970 cm-1. While larger numbers of fluorescence and Raman spectra are
evaluated by the method of multivariate data analysis, additional information is obtained from spectral images and
fluorescence lifetime images (FLIM).
A microfluidic system was developed and combined with optical tweezers for single cell sorting. This system consists of
a glass chip of 300 μm thickness with an etched crosswise channel structure, a silicon layer for sealing and a PMMA
substrate for tubular coupling. Selected cells are trapped and moved in perpendicular direction to the main flow for
recovery in special reservoirs and further evaluation (e.g. by polymerase chain reaction, PCR). In addition, maximum
light doses and exposure times for maintaining cell viability were determined.
Methods of wide-field fluorescence microscopy for measuring membrane dynamics of living cells are described, including spectral imaging as well as anisotropy imaging of the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene (laurdan). Plasma membranes are selected by illumination with an evanescent electromagnetic field and distinguished from intracellular membranes assessed by whole-cell illumination. While fluorescence spectra of laurdan appeared red-shifted with decreasing membrane stiffness, fluorescence anisotropy and rotational correlation times were reduced with increasing membrane fluidity. Membrane stiffness was found to increase with decreasing temperature and increasing amounts of cholesterol and was always higher for the plasma membrane than for intracellular membranes. These effects may have some clinical relevance in the research of drug resistance or cell aging.
A membrane associated caspase sensor based on Förster Resonance Energy Transfer (FRET) between enhanced cyan
fluorescent protein (Mem-ECFP) and yellow fluorescent protein (EYFP) is reported. Upon apoptosis a caspase sensitive
amino acid peptide linker (DEVD) between these proteins is cleaved, and pronounced changes of fluorescence spectra
and lifetimes are observed. Membrane selective detection of fluorescent proteins in cultivated HeLa cervix carcinoma
cells is achieved by total internal reflection fluorescence microscopy (TIRFM) with high sensitivity and resolution.
Light dose plays an important role for maintaining viability in optical microscopy of living cells. Therefore, a colony
forming assay was established, and non-phototoxic light doses were determined for glioblastoma cells. These doses
ranged from a few 1 J/cm2 or even less for cells incubated with fluorescence markers or photosensitizers up to about
100 J/cm2 for non-incubated cells. Microscopic methods were adapted to those light doses, and often wide field methods
appeared to be more appropriate than laser scanning methods.
A fluorescence reader for the detection of Förster resonance energy transfer (FRET) on surfaces of living cells is described. The method is based on multiple total internal reflections (TIR) of an incident laser beam within a glass slide, such that individual samples on top of the glass slide are illuminated simultaneously by an evanescent electromagnetic field. Enhanced cyan fluorescent protein (ECFP) anchored to the inner leaflet of the plasma membrane is optically excited and transfers its excitation energy via the peptide linker Asp-Glu-Val-Asp (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved, and energy transfer is disrupted, as proven by an increase of fluorescence intensity as well as of fluorescence lifetime of the donor ECFP. Due to selective excitation of membrane-associated fluorophores, intracellular fluorescence and background luminescence from the surrounding medium are eliminated. Therefore, this test system appears to be a sensitive device for the detection of apoptosis and more generally for drug screening or in vitro diagnosis on a nanometer scale.
Intracellular accumulation and location of photosensitizers, e.g. 5-ALA induced protoporphyrin IX, are crucial parameters for the efficiency of photodynamic therapy (PDT). Fluorescence microscopy has proved to be a powerful technique to assess these parameters, however, even at low light doses around or below 1 J/cm² cells may be irreversibly damaged. Therefore, prior to microscopic experiments non-phototoxic light doses were determined, and experimental conditions of laser scanning (LSM) and wide field microscopy were adapted to these doses. Wide field images appeared more brilliant than LSM images, thus demonstrating some advantage of simultaneous over sequential detection. In addition, human glioblastoma cells appeared less sensitive towards illumination by an evanescent electromagnetic field than towards epi-illumination, since only their plasma membranes and adjacent parts were exposed to light.
Methods of wide field fluorescence microscopy for measuring membrane dynamics in living cells are described. These
methods are based on laser pulse excitation of the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene
(laurdan) whose emission spectra, fluorescence decay kinetics and anisotropies are sensitive to membrane stiffness and
fluidity. Plasma membranes are selected by illumination with an evanescent electromagnetic field and distinguished from
intracellular membranes assessed by whole cell illumination. While fluorescence spectra of laurdan appeared red-shifted
with decreasing membrane stiffness, fluorescence anisotropy and rotational relaxation times were reduced with
increasing membrane fluidity. Membrane stiffness was found to increase with decreasing temperature and increasing
amounts of cholesterol. In addition, membrane stiffness of the plasma membrane was always higher than that of
intracellular membranes. These effects may have some influence on pathogenesis of certain diseases, uptake of
pharmaceutical agents or cell aging. Present experiments are limited to fluorescence microscopy with total internal
reflection (TIR) or epi-illumination, but corresponding methods can also be used for screening of larger cell collectives,
e.g. in microtiter plates.
Cholesterol content is an important factor for membrane dynamics of living cells. With well defined protocols of depletion and enrichment the impact of cholesterol on membrane dynamics was examined by fluorescence microscopy. In addition, the intracellular cholesterol
content was determined with biochemical methods. Changes of cholesterol amounts in cell membranes have previously been related to specific disease and may have some influence on the uptake of pharmaceutical agents. A combination of conventional and total internal reflection fluorescence microscopy was applied to the fluorescence marker laurdan, a polarity-sensitive probe, whose electronic excitation energy is different in polar and non-polar environment. Once incorporated into cell membranes, the fluorescence of laurdan shows a spectral shift towards longer wavelength
when its molecules get into contact with adjacent water molecules, e.g. when a phase transition from the tightly packed gel phase to the liquid crystalline phase of membrane lipids occurs. The generalized polarization (GP, characterizing this spectral shift) as well as the
fluorescence lifetime (τ) of laurdan revealed to be appropriate measures for membrane stiffness and fluidity. GP generally decreased with increasing temperature and was always higher for the plasma membrane than for intracellular membranes. Enrichment of cholesterol
caused a pronounced increase, whereas depletion of cholesterol caused a decrease of GP. In addition, pronounced changes of the fluorescence lifetime pattern occurred in the subnanosecond range. GP, and τ were determined as integral values of single cells or small cell collectives and were also displayed as microscopic images.
Membrane dynamics has a large impact on cellular uptake and release of various metabolites or pharmaceutical agents.
For a deeper understanding of the cellular processes involved, we used U373-MG human glioblastoma cells as a model
system. As conventional microscopy does not permit to investigate individual layers in living cells, we used structured
illumination techniques and total internal reflection fluorescence microscopy (TIRFM) to analyse the plasma membrane
and intracellular membranes of living cells selectively. Optical image sections provide a high resolution and the
possibility of 3D reconstruction.
Membranes of living cells were characterized by the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene
(laurdan). Due to its spectral and kinetic properties this fluorescence marker appears appropriate for measuring
membrane stiffness and fluidity. After excitation with linearly polarized laser pulses, membrane fluidity of human
glioblastoma cells was determined by measurements of steady-state and time-resolved fluorescence anisotropy r(t),
since with increasing viscosity of the environment, the rotation of an excited molecule is impeded. The corresponding
time constant &tgr;r of molecular relaxation decreased with temperature and increased with the amount of cholesterol. In
addition, fluorescence anisotropy r(t) values of the plasma membrane were larger than the values of intracellular
membranes for all temperatures in the range of 16°C≤T≤41°C.
Total internal reflection fluorescence microscopy (TIRFM) and non-radiative energy transfer (FRET) measurements
have been combined in order to examine co-localization of the amyloid precursor protein (APP) and the ?-site APPcleaving
enzyme (BACE) in human glioblastoma cells. So far, these proteins have been co-localized within whole cells
(depending on the intracellular amount of cholesterol) and in some cases also within their plasma membranes. This
supports the present hypothesis of localization within lipid domains on the cell surface and co-internalization via
endocytosis.
Total internal reflection fluorescence microscopy (TIRFM) is used to measure non-radiative energy transfer between membrane associated proteins in living cells. Measurements are concentrated on focal contacts and their associated proteins focal adhesion kinase (FAK) and Paxillin (Pax) which play major roles with respect to cell migration, growth, and survival. These proteins are visualized after fusion with variants of green fluorescent protein (ECFP and EYFP), and an intermolecular energy transfer ECFP -> EYFP is deduced from fluorescence spectra as well as from fluorescence decay kinetics of single cells.
Membrane dynamics of human glioblastoma cells were investigated using the intercalating fluorescence marker 6-dodecanoyl-2-dimethylamino naphthalene (laurdan). In particular its generalized polarization (GP), which describes a spectral shift depending on the phase of membrane lipids, was used as a measure of membrane stiffness, whereas its fluorescence lifetime τ and its rotational diffusion time tr were used to characterize membrane fluidity. Upon excitation with linearly polarized pulsed laser light the parallel and perpendicular components of fluorescence from the sample were measured simultaneously using an imaging device with polarization sensitivity. So far, membrane dynamics depended on temperature and cell age as well as the on intracellular amount of cholesterol. In addition, the plasma membrane (assessed by illumination with an evanescent electromagnetic wave) appeared to be stiffer than intracellular membranes (assessed by epiillumination of the cells).
Membranes of living cells are characterized by laser-assisted fluorescence microscopy, in particular a combination of microspectrofluorometry, total internal reflection fluorescence microscopy (TIRFM) and fluorescence decay kinetics. The generalized polarization (GP, characterizing a spectral shift which depends on the phase of membrane lipids), the time constant of fluorescence anisotropy (τr) as well as the fluorescence lifetime (τ) of the membrane marker laurdan revealed to be appropriate measures for membrane stiffness and fluidity. GP decreased with increasing temperature and was always higher for the plasma membrane than for intracellular membranes. The latter effect was correlated with the intracellular content of cholesterol, which could be modified using defined protocols of depletion or enrichment. Concomitant with generalized polarization the fluorescence lifetime τ increased with the content of cholesterol. Changes of cholesterol amounts in cell membranes have previously been related to specific diseases and may have some influence on the uptake of pharmaceutical agents.
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