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.
A device for selective plane illumination microscopy (SPIM) of three-dimensional multicellular spheroids, in culture medium under stationary or microfluidic conditions, is described. Cell spheroids are located in a micro-capillary and a light sheet, for illumination, is generated in an optical setup adapted to a conventional inverse microscope. Layers of the sample, of about 10 μm or less in diameter, are, thus, illuminated selectively and imaged by high resolution fluorescence microscopy. SPIM is operated at low light exposure even if a larger number of layers is imaged and is easily combined with laser scanning microscopy. Chinese hamster ovary cells expressing a membrane-associated green fluorescent protein are used for preliminary tests, and the uptake of the fluorescent marker, acridine orange via a microfluidic system, is visualized to demonstrate its potential in cancer research such as for the detection of cellular responses to anticancer drugs.
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.
A heating device is presented, which can be attached on top of a standard microtiter plate, permitting individual and fast
heating of the specimen by resistor elements without direct contact. All wells of the microtiter plate remain accessible
from above and beyond for sensory (e.g. optical) measurements, independently from the bottom geometry of the wells.
By various circuit configurations it is possible to realize either homogenous heating all over the plate or heating of
individual arrays or clusters of wells, e.g. for maintaining defined temperature gradients. Main applications include
quantitative measurements of temperature dependent parameters in the area of diagnostics, serology, cell culture- and
immunological research.
The heating device is validated by various referencing temperature kinetics and infrared images, as well as by selective
investigations of cell membranes upon total internal reflection of a laser beam. For this purpose, particularly with regard
to potential pharmaceutical applications, fluorescence decay kinetics of cancer cells incubated with temperature-dependent
dyes, e.g. 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan), is determined.
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.
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 novel tomographic screening reader for 3-dimensional cell cultures is described. The method is based on structured
illumination and permits imaging with high axial resolution and 3-D reconstruction of single cells or clusters.
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.
A setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living
cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within
the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by an evanescent
electromagnetic field. Two different screening procedures for the detection of fluorescence arising from the plasma
membrane of living cells by High Throughput Screening (HTS) and High Content Screening (HCS), are distinguished. In
the first case a rapid measurement of large sample numbers based on fluorescence intensity, and in the second case a
high content of information from a single sample based on the parameters fluorescence lifetime (Fluorescence Lifetime
Screening, FLiS) and fluorescence anisotropy (Fluorescence Lifetime Polarization Screening, FLiPS) is achieved. Both
screening systems were validated using cultivated cells incubated with different fluorescent markers (e. g. NBD-cholesterol)
as well as stably transfected cells expressing a fluorescent membrane-associating protein. In addition,
particularly with regard of potential pharmaceutical applications, the kinetics of the intracellular translocation of a
fluorescent protein kinase c fusion protein upon stimulation of the cells was determined. Further, a caspase sensor based
on Förster Resonance Energy Transfer (FRET) between fluorescent proteins was tested. Enhanced cyan fluorescent
protein (ECFP) anchored to the inner leaflet of the plasma membrane of living cells transfers its excitation energy via a
spacer (DEVD) to an enhanced yellow fluorescent protein (EYFP). Upon apoptosis DEVD is cleaved, and energy
transfer is disrupted, as proven by changes in fluorescence intensity and decay times.
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.
A high content screening (HCS) system for fluorescence measurements at surfaces, in particular the plasma membrane of living cells, is described. The method is based on multiple total internal reflections (TIRs) of an incident laser beam within the glass bottom of a microtiter plate such that up to 96 individual samples could be illuminated by an evanescent electromagnetic field. Fluorescence lifetimes and time-resolved fluorescence anisotropies of these samples were assessed. While fluorescence lifetime represents a general measure for the interaction of a marker molecule with its microenvironment, the rotational diffusion time corresponds to the relaxation time of a molecule from a position with a defined orientation into a position with an arbitrary orientation. Thus, time-resolved fluorescence anisotropy reflects the viscosity of the microenvironment, i.e., membrane fluidity in the case of living cells. For all measurements in this study, either human glioblastoma cells incubated with the fluorescent membrane marker NBD-cholesterol or human breast cancer cells expressing a membrane-associating fluorescent protein were used.
A novel setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate (cell substrate), such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. Main prerequisites are an appropriate thickness and a high transmission of the glass bottom, which is attached to the 96-well cell culture plate by a noncytotoxic adhesive. Glass rods of rectangular cross sections are optically coupled to this bottom for TIR illumination. Fluorescence arising from the plasma membrane of living cells is detected simultaneously from all samples using an integrating charge-coupled device (CCD) camera. The TIR fluorescence reader is validated using cultivated cells incubated with different fluorescent markers, as well as stably transfected cells expressing a fluorescent membrane-associating protein. In addition, particularly with regard to potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells is determined.
A novel setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of
living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR)
within the bottom of a microtiter plate, such that up to 96 individual samples are illuminated simultaneously by an
evanescent electromagnetic field. In general, two different screening procedures (1) High Throughput Screening (HTS)
and (2) High Content Screening (HCS) are distinguished, where in the first case a rapid measurement of large sample
numbers, and in the second case a high information content from a single sample is desired. In particular, a HCS system
for the parameters fluorescence lifetime (Fluorescence Lifetime Screening, FLiS) and fluorescence anisotropy
(Fluorescence Lifetime Polarization Screening, FLiPS) has been established and integrated into an existing HTS-system.
A novel setup for fluorescence measurements of surfaces of biological samples, in particular cell membranes, is described. The method is based on multiple total internal reflections (TIR) of a laser beam at the surface of a multi-well plate, such that 96 individual samples are excited simultaneously. Main prerequisites are an appropriate thickness and high transmission of the glass bottom, a non-cytotoxic adhesive, and appropriate glass rods for TIR illumination. Fluorescence from the cell surface is detected simultaneously using an integrating CCD camera and appropriate optical filters. For validation of the system, cells incubated with the fluorescence marker NBD as well as transfected cells expressing a fluorescent membrane protein are used. In addition, intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation is examined. The method appears well suitable for high throughput screening (HTS), since neither washing of the samples nor any readjustment of the equipment after changing of individual plates are necessary.
A novel setup for fluorescence measurements of surfaces of biological samples, in particular cell membranes, is described. The method is based on multiple total internal reflection (TIR) of a laser beam on the surface of a multi-well plate, such that 96 individual samples are excited simultaneously. Main prerequisites are an appropriate thickness and high transmission of the glass bottom, a non-cytotoxic adhesive, and appropriate glass rods for TIR illumination. Fluorescence from cell surface is detected simultaneously using an integrating CCD camera and appropriate optical filters. For validation of the system, transfected cells expressing a fluorescent membrane protein are used. In addition, intracellular translocation of green fluorescent protein kinase c upon stimulation is examined. The method appears well suitable for high throughput screening (HTS), since neither washing of the samples nor any re-adjustment of the equipment after changing of individual plates are necessary.
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