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.
We propose a cancer diagnostics method using 3D reconstruction of fluorescence based optical imaging data. The
reconstruction of luminescence sources in biological tissue is investigated using data obtained from Monte Carlo
simulations as well as simulated data using the diffusion approximation. The absolute determination of the tumor
locations is dependent on the information gathered by the recorded data. To tackle the forward problem we used the
solution of the diffusion equation for a cube. The inverse problem is solved.
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.
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.
We propose a cancer diagnostics method using 3D reconstruction of fluorescence based optical
imaging data. The system was tested with analytical simulations. Phantom measurements will be
acquired and compared with the simulations.
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.
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.
Intensity modulation was introduced to total internal reflection fluorescence microscopy (TIRFM). This permits to
obtain information about the axial distribution of fluorescent dyes in close vicinity to the surface of adherent cells and
allows the application of (commercial) objective based TIRFM systems at a fixed angle of illumination. However, this
new method seems to be less suitable for quantitative measurements of cell-substrate topology as compared to variableangle
prism based TIRFM.
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.
One of the most promising clinical applications of 5-aminolevulinic acid (5-ALA) induced protoporphyrin IX (PP IX) is the fluorescence diagnosis and photodynamic treatment of malignant brain tumours. In order to obtain a deeper understanding of the cellular processes involved, U373-MG human glioblastoma cells were used as model system to study intracellular location, fluorescence properties and light-induced reactions of the photosensitizer. In comparison with ovarian cancer or breast cancer cell lines, glioblastoma cells accumulated PP IX only to a moderate extent, but were most effectively inactivated (highest photodynamic efficacy per photosensitizer molecule). In contrast to breast cancer cells, which showed a rather granular PP IX fluorescence pattern, PP IX fluorescence in U373-MG cells seemed to originate mainly from cellular membranes. In addition, photobleaching and lowering of fluorescence lifetimes during irradiation were comparably small for this cell line. Combining the results of fluorescence lifetime imaging microscopy (FLIM) and photobleaching measurements, we deduced that those cellular sites where PP IX fluorescence was most pronounced, contributed rather little to its photodynamic efficacy.
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.
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.
KEYWORDS: Luminescence, Energy transfer, Fluorescence spectroscopy, Microscopy, Fluorescence lifetime imaging, Molecules, Time resolved spectroscopy, Fluorescence resonance energy transfer, Absorption, Molecular energy transfer
An overview on some advanced techniques of fluorescence spectroscopy and fluorescence microscopy is given. These techniques include time-resolved fluorescence spectroscopy, energy transfer spectroscopy (FRET), total internal reflection fluorescence microscopy (TIRFM) and fluorescence lifetime imaging (FLIM). The principle of these methods is explained, and numerous applications are described.
An experimental setup for fluorescence lifetime imaging (FLIM) has been combined with total internal reflection fluorescence microscopy (TIRFM) in order to detect various membrane markers within living cells. The method is established using T47D human breast cancer cells transfected by a plasmid encoding for a membrane associated yellow fluorescent protein (EYFPmem). For further measurements the mitochondrial marker rhodamine 123 (R123) as well as the membrane marker laurdan are used. With increasing concentration R123 is accumulated outside the mitochondria, in particular within the plasma membrane, whereas mitochondrial fluorescence is quenched. Fluorescence lifetime of laurdan can be used to probe membrane dynamics, in particular the phase of membrane lipids. These lipids are in a rigid gel phase at temperatures around 24°C, whereas the gel phases and a liquid crystalline phase coexist at T ≥ 30°C. This phase pattern also depends on the age and the growth phase of the cells and may play a role in the uptake of pharmaceutical agents.
Fluorescence spectra and lifetime images of cultivated CHO cells incubated with the membrane marker laurdan are reported. The plasma membrane and intracellular membranes were distinguished using illumination of either the cell surface (by an evanescent wave) or of the whole cell. Parameters of membrane dynamics, probed by the generalized prolarization and the effective fluorescence lifetime of laurdan, were correlated with temperature, age and growth phase of the cells.
KEYWORDS: Human-machine interfaces, Virtual reality, Control systems, Navigation systems, 3D modeling, Visual process modeling, Cameras, 3D displays, Prototyping, Infrared radiation
In this paper we present our effort towards the goal of perceptual user interface for major interaction tasks, such as navigation/travel, selection/picking and personal data access, for virtual shopping. A set of 3-D navigation devices, vision-based pointing and personal access system are mainly discussed. The motivation and design principles behind these interfaces are also described. A prototype integration solution, which bring these devices together in virtual shopping environment, is given. These interfaces and interaction devices have been implemented and tested for evaluation.
A compact device for variable-angle total internal reflection flourescent microscopy was developed. A pulsed Nd:YAG laser operated at 355 nm was adapted using a multimode quartz fiber and collimating optics with a variable angle of incidence between 64 degrees and 72 degrees. Fluorescence spectra of BKEz-7 endothelial cells incubated with the membrane marker 6-dodecanoyl-2- dimethylamino-naphthalene were measured under TIR illumination as a function of the angle of incidence, incubation time and temperature. Emission bands around 440 nm and 490 nm were detected corresponding to laurdan locate within the gel phase or liquid crystalline phase of cellular lipids, respectively. The generalized polarization GP = (I440-I490)/(I440+I490) was used as a measure of intracellular temperature with a precision of ±1°C in the physiologically interesting range between 35°C and 38°C. Following pulsed laser excitation, the time delay between excitation and fluorescence detection was varied. A time gate at 10-15 ns after laser excitation revealed to be an optimum for spectral discrimination of the two emission bands. Fluorescence intensity IF of both bands decreased continuously when the angle of incidence Θ was increased. Between Θ = 68° and 72° the angular dependence corresponded to a fluorophore located within a thin layer (plasma membrane) at 150-200nm distance from the light reflecting surface. Between Θ=65° and 68° additional contributions from intracellular membranes were observed.
To establish optical in situ detection of mitochondrial malfunction,
nonradiative energy transfer from the coenzyme NADH to
the mitochondrial marker rhodamine 123 (R123) was examined. Dual
excitation of R123 via energy transfer from excited NADH molecules
as well as by direct absorption of light results in two fluorescence
signals whose ratio is a measure of mitochondrial NADH. A screening
system was developed in which these signals are detected simultaneously
using a time-gated (nanosecond) technique for energy transfer
measurements and a frequency selective technique for direct excitation
and fluorescence monitoring of R123. Optical and electronic
components of the apparatus are described, and results obtained from
cultivated endothelial cells are reported. The ratio of fluorescence
intensities excited in the near ultraviolet and blue–green spectral
ranges increased by a factor 1.5 or 1.35 after inhibition of the mitochondrial
respiratory chain by rotenone at cytotoxic or noncytotoxic
concentrations, respectively. Concomitantly the amount of mitochondrial
NADH increased. Excellent linearity between the number of
cells incubated with R123 and fluorescence intensity was found in
suspension.
KEYWORDS: Optical tweezers, Semiconductor lasers, Nd:YAG lasers, Luminescence, Absorption, Control systems, High power lasers, Microscopes, Diodes, Near infrared
To establish optical in situ detection of mitochondrial malfunction, non-radiative energy transfer from the coenzyme NADH to the mitochondrial marker rhodamine 123 (R123) was examined. Dual excitation of R123 via energy transfer from excited NADH molecules as well as by direct absorption of light results in two fluorescence signals whose ratio is a measure of mitochondrial NADH. An experimental setup was developed, where these signals are detected simultaneously using a time-gated technique for energy transfer measurements and a frequency selective technique for direct excitation and fluorescence monitoring of R123. Optical and electronic components of the apparatus are described, and preliminary result of cultivated endothelial cells are reported. Results are compared with those obtained from a previously established microscopic system and discussed in view of potential applications.
Cellular uptake of transparent Latex particles by J774A.1 mouse macrophages has been studied: First, single beads were kept within an optical light trap and located in close vicinity to individual cells. Uptake of the beads was visualized, and intrinsic fluorescence was detected in the spectral range of 420 - 530 nm. Second, time-gated fluorescence spectra of single cells were recorded at pre- selected times during one hour after cellular uptake. A rapid increase of autofluorescence and a subsequent decrease to the level of control cells within about 10 min. was measured within a time gate of 0 - 5 ns after the exciting laser pulses, and attributed to the 'free' coenzyme NAD(P)H. In contrast, fluorescence increase of NAD(P)H bound to proteins (measured within time gates of 5 - 10 ns or 10 - 15 ns) was less pronounced, and the subsequent decrease occurred within a longer period (about one hour).
To establish optical in situ detection of mitochondrial malfunction, non-radiative energy transfer from the coenzyme NADH to the mitochondrial marker rhodamine 123 (R123) was examined. Dual excitation of R123 via energy transfer from excited NADH molecules as well as by direct absorption of light results in two fluorescence signals whose ratio is a measure of mitochondrial NADH. These signals are detected simultaneously using a time-gated (nanosecond) technique for energy transfer measurements and a frequency selective technique for direct excitation and fluorescence monitoring of R123. Optical and electronic components of the experimental setup are described and compared with a previously established microscopic system.
KEYWORDS: Luminescence, Energy transfer, Spectroscopy, Rhodamine, Fluorescence spectroscopy, Control systems, Time resolved spectroscopy, Mode conditioning cables, Information operations, Mass attenuation coefficient
Energy transfer from NADH to the mitochondrial marker rhodamine 123 (R123) was used to probe mitochondrial malfunction of cultivated endothelial cells incubated with various inhibitors of specific enzyme complexes of the respiratory chain. Pronounced differences of 'energy transfer efficacy' of incubated cells as compared to controls were deduced from the ratio of fluorescence intensity and intracellular amount of the acceptor. A combination of cw and time-gated (nanosecond) fluorescence spectroscopy appeared to be an appropriate tool for probing mitochondrial malfunction in various kinds of diseases.
In comparison with optical tweezers using near infrared lasers, red emitting high power laser diodes ((lambda) equals 670 - 680 nm) may offer several advantages, such as easier adjustment, smaller beam diameter in the focal plane and low absorption of optical radiation. One-photon absorption by water or two-photon absorption by various cellular components may induce cell killing by heat or by photochemical reactions. To test cell viability, cultivated CHO cells were exposed to various light doses of this wavelength as well as to a moderate light dose of a conventional Nd:YAG laser. Loss of clonogenicity of about 50% as well as a reduction of cell growth were measured at 680 nm when using a maximum light dose of 2.4 GJ/cm2. In contrast, no cell damage was found at 670 nm as well as at 1064 nm when using a light dose of 340 MJ/cm2, which revealed to be sufficient for several applications.
Mitochondrial malfunction may be concomitant with changes of the redox states of the coenzymes nicotinamide adenine dinucleotide (NAD+/NADH), as well as flavin.mononucleotide or dinucleotide. The intrinsic fluorescence of these coenzymes was therefore proposed to be a measure of malfunction. Since mitochondrial fluorescence is strongly superposed by autofluorescence from various cytoplasmatic fluorophores, cultivated endothelial cells were incubated with the mitochondrial marker rhodamine 123 (R123), and after excitation of flavin molecules, energy transfer to R123 was investigated. Due to spectral overlap of flavin and R123 fluorescence, energy transfer flavin yields R123 could not be detected from their emission spectra. Therefore, the method of microscopic fluorescence excitation spectroscopy was established. When detecting R123 fluorescence, excitation maxima at 370 - 390 nm and 420-460 nm were assigned to flavins, whereas a pronounced excitation band at 465 - 490 nm was attributed to R123. Therefore, excitation at 475 nm reflected the intracellular concentration of R123, whereas excitation at 385 nm reflected flavin excitation with a subsequent energy transfer to R123 molecules. An enhanced energy transfer after inhibition of specific enzyme complexes of the respiratory chain is discussed in the present article.
A microscopic equipment is reported for examination of cellular autofluorescence and determination of energy transfer in vitro, which is proposed to be an appropriate tool to investigate mitochondrial malfunction. The method includes fluorescence microscopy combined with time-gated (nanosecond) fluorescence emission spectroscopy and is presently used to study mitochondrial metabolism of human myotube primary cultures Enzyme complexes of the respiratory chain, located at the inner mitochondrial membrane, were inhibited by various drugs, and fluorescence of the mitochondrial coenzyme nicotinamide adenine dinucleotide (NADH) as well as of the mitochondrial marker rhodamine 123 (R123) was examined. After inhibition of enzyme complex I (NADH-coenzyme Q reductase) by rotenone or enzyme complex III (coenzyme QH2-cytochrome c reductase) by antimycin a similar or increased NADH fluorescence was observed. In addition, energy transfer from excited states of NADH (energy donor) to R123 (energy acceptor) was deduced from a decrease of NADH fluorescence after coincubation with these inhibitors and R123. Application of microscopic energy transfer spectroscopy for diagnosis of congenital mitochondrial deficiencies is currently in preparation.
The application of total internal reflection fluorescence spectroscopy for probing fluorescence of protoporphyrin selectively in cell membranes is described. Penetration depths of the evanescent field were calculated for a wavelength of 543 nm. Penetration depths varied between 75 nm and 190 nm, depending on the incident angle of the light. In contrast to fluorescence spectra obtained by epi- illumination, spectra obtained by total internal reflection fluorescence spectroscopy were characterized by a very low autofluorescence background. This indicates that only protoporphyrin located in the plasma membrane or in close vicinity to the plasma membrane was excited. Furthermore total internal reflection fluorescence spectroscopy setup was used for the determination of photobleaching and polarized fluorescence measurements. Illumination of cells incubated with protoporphyrin resulted in a biexponential photobleaching with a rapidly and a slowly bleaching portion. During the whole period of light exposure a degree of polarization P equals minus 0.22 was determined.
Cytotoxicity, uptake, intracellular location and phototoxicity were compared for the naturally occurring porphyrins uroporphyrin III (UP), coproporphyrin III (CP) and protoporphyrin IX (PP), as well as for tetra-, tri-, di- and monosulphonated meso-tetraphenyl porphyrins (TPPSn with n equals 4, 3, 2a, 1) in cultivated endothelial cells from calf aorta (BKEz-7). In addition, the uptake of CP and PP in the chick chorioallantoic membrane (CAM) was studied after intravenous application. Hydrophilic porphyrins (UP, CP, TPPS4 and TPPS3) exhibit low cellular uptake as well as low cyto- and phototoxicity as compared with the more hydrophobic compounds. UP, CP, TPPS4 and TPPS3 were mainly located in lysosomes, whereas PP, TPPS2a and TPPS1 were preferentially observed in the cytoplasm -- supposed to be located in cellular membranes of organelles -- and exhibited pronounced light-induced reactions. A rapid uptake by the CAM vasculature (5 min. - 30 min. after application) and some delayed uptake by the CAM matrix (30 min. - 6 h with a maximum at 60 min.) were measured for CP and PP.
Phthalocyanines and m-TPPs were substituted with two or three t-butyl phenyl anchor groups to build up inclusion complexes with dimeric (beta) -cyclodextrins. These complexes proved to be stable enough to hinder complexation of the porphyrinoids with plasma lipoproteins and, so, offer an independent pathway to transfer porphyrinoids to photosensitize tumor tissue.
KEYWORDS: Luminescence, Energy transfer, Rhodamine, Molecules, Time resolved spectroscopy, Microscopy, Electron transport, Information operations, Fluorescence spectroscopy, Spectroscopes
A novel set-up for time-gated (nanosecond) detection of fluorescence spectra and images of microscopic samples was recently developed. The apparatus was now used to measure the fluorescence of mitochondrial coenzymes (NADH, flavins) and a marker molecule (rhodamine 123) in endothelial cells from calf aorta. In these cultivated cells the electron transport chain was inhibited at various sites of the inner mitochondrial membrane. It could be shown that the fluorescence intensity of the free coenzyme NADH depended on the site of inhibition. In addition, an increased energy transfer from mitochondrial coenzymes (NADH, flavins) to rhodamine 123 molecules was observed, if the inhibition occurred in complex I (NADH- coenzyme Q reductase) or complex III (coenzyme QH2-cytochrome c reductase) of the respiratory chain. The diagnostic potential of these findings are discussed.
Intracellular modifications of anionic photosensitizers during PDT were observed, dependent on the growth phase of the incubated cells. In addition to steady-state spectroscopy, time- resolved fluorescence spectra between different time gates were measured. Comparative results were obtained for the sensitizers meso-tetra(4-sulfonatophenyl)porphyrine (TPPS4) and aluminumsulfophthalocyanine (AlS2-3Pc). In both cases relocalization of the sensitizer could be observed exclusively for cells in the growing phase, whereas no relocalization was detected for cells in the stationary phase. Moreover, time-gated spectra were different for the two cell types. The formation of a fluorescent band around 615 nm during PDT for the sensitizer TPPS4 was observed mainly for cells in the growing phase. This species was correlated with a short fluorescence decay time (< 5 ns). Similar observations were done with AlS2-3Pc. A fluorescence band blue-shifted in the same order of magnitude (about 30 nm from the original band) and measured predominantly between an `early' time-gate (0-5 ns) was detected for cells in the growing phase.
The photosensitizers uroporphyrin III (UP III), coproporphyrin III (CP III), and protoporphyrin IX (PP IX) were examined with respect to their composition (by different molecular species), uptake and distribution in cells and tissues, intracellular pH value, cytotoxicity and formation of photoproducts. Whereas hydrophilic UP III and CP III were characterized by low cytotoxicity and preferential accumulation in lysosomes, hydrophobic PP IX was found to be rather cytotoxic and mainly localized in cellular membranes. Rapid uptake in tissues and some preferential location in tumor cells were detected for hydrophobic photosensitizers.
Based on novel time-resolving methods, the autofluorescence of saccharomyces, cultivated endothelial cells and biopsy specimens of human bladder was measured. The emission was found to be composed by the coenzymes NADH (free and protein-bound) and flavins with a concentration ratio of 100:1 between free NADH and flavin molecules. The fluorescence intensity of free NADH appeared to be a measure of the respiratory function. In addition, epithelial and connective tissues could be distinguished by the intensity ratio at 435 and 460 nm, which may be affected by the relation of bound and free NADH, but also by the extracellular fluorescence of elastin.
Intracellular modifications of sensitizers during PDT were observed with combined measurements of cw and time-gated microscopy and spectroscopy. Protoporphyrin when stimulated with 5-aminolevulinic acid (ALA) showed photobleaching after irradiation with blue light (1 - 10 J/cm2) in competition with the formation of photoproducts (i.e., photoprotoporphyrins). The anionic meso-tetra(4-sulfonatophenyl)porphyrin (TPPS4) was incubated in cells seeded at low cell density (isolated cells; 25 cells/mm2) or high cell density (confluent growing cells; 500 cells/mm2), respectively. A similar granular fluorescence pattern, cw and time-gated fluorescence spectra could be observed before light exposure in both cases. With increasing irradiation a redistribution from the cytoplasm to the nucleus in addition with a pronounced formation of a fluorescent band around 615 nm was dependent on the growth phase of the cells and could be detected mainly for isolated cells. This species was correlated with a short fluorescence decay time and detected with an `early' time gate between 0 - 5 ns, whereas the neutral TPPS4 molecule was detected within a `late' time-gate between 10 - 15 ns.
Athymic nude mice with subcutaneously xenotransplanted urothelial carcinoma received intravenously injections of the synthetic sensitizer meso-tetra (4-carboxyphenyl) porphyrin. Fluorescence at the tumors and the skin was excited using a Kr+ laser (407 nm) and was detected at 652 nm using a fiberoptical sensor in combination with an optical multichannel analyzer at different times after application of the sensitizer. Photodynamic treatment was carried out using an Ar+ laser pumped dye laser (650 nm; 100 mW/cm2; 100 J/cm2). The delay of tumor growth was determined in a follow-up period of four weeks and was correlated to the in vivo fluorescence measurements.
Various microscopic methods were established to measure the subcellular distribution and light-induced reactions of photosensitizing porphyrins. These methods include high resolution video microscopy, microspectrofluorometry, as well as time-resolved and time-gated fluorescence spectroscopy. By using these techniques, different components of hydrophylic and hydrophobic photosensitizers (e.g. monomers, aggregates, ionic species) were localized, and their intracellular reactions -- re-distribution, formation of photoproducts, pH-dependent spectral changes -- were studied.
Various microscopic techniques were used to study the dependency of photodynamically induced subcellular reactions on the metabolic state of cell cultures. TPPS4 and AlS2-3Pc were incubated in RR 1022 epithelial cells with varying cell density. To attain almost isolated cells (low cell density) or confluent growing cells (high cell density) 25 cells/mm2 or 500 cells/mm2 were seeded, respectively. Low cell density irradiation with blue light led to a change in the initial cytoplasmatic fluorescence pattern. For both sensitizers, TPPS4 as well as AlS2-3, a fluorescence relocalization and fluorescence intensity increase could be detected, moreover in the case of TPPS4 a fluorescence formation in the nucleus and nucleoli were detected. In contrast, for confluent growing cells no redistribution was observed.
Subcellular changes during photodynamic therapy (PDT) were probed simultaneously with two different microscopic techniques. These techniques include differential interference contrast (DIC) microscopy and fluorescence microscopy, in combination with video-contrast enhancement, averaging techniques, and digital image processing. The different cell organelles were identified by specific markers, such as rhodamine 123 and acridine orange. Subcellular morphological changes in fibroblastoid cells (3T3) and epithelial cells (RR 1022), incubated with the photosensitizer meso-tetra(4-sulfonatophenyl)porphyrin (TPPS4), were investigated during PDT treatment and correlated with the fluorescence pattern.
In order to study light-induced reactions during PDT, the fluorescence response of the photosensitizer meso-tetra(4-sulfonatophenyl)porphyrin (TPPS4) was observed in different cell systems and correlated with the sensitivity to photodynamic induced destructions. RR 1022 epithelial cells from the rat were grown on microscopic slides at a high and low cell density. Using video microscopy in combination with microspectrofluorometry we observed a different fluorescence behavior for high and low cell conditions during light exposure. A fluorescence relocalization from the cytoplasm to the nucleus and an intensity increase-- correlated with the formation of a new molecular species--could be detected only for low cell density. Moreover, cell cultures at a high density showed to be less sensitive to photodynamic destructions. In addition to cell culture-experiments, we observed the light-induced reactions of TPPS4 accumulated in multicellular tumor spheroids. For these measurements laser scanning microscopy was used. Fluorescence relocalization and intensity increase could be detected only for the peripheric parts of the spheroids. The different fluorescence response seems to reflect different metabolic and physiologic states of the cells.
Light-induced reactions of different hydrophilic meso-tetraphenyl porphyrins (TPPS4, TPPC4, T4MPyP) were investigated during PDT treatment. These measurements were carried out in vitro using micro-spectrofluorometry and were correlated with measurements in buffer solutions. In the case of the anionic TPPS4, before light exposure, a neutral free base and a protonated species could be detected simultaneously in the cells. During irradiation, the protonated species first disappeared. Due to the fact that a coexistence of a protonated and unprotonated species requires a pH value around 5, TPPS4 was at first located in the lysosomes (pH 5) and was released into the cytoplasm during irradiation as a result of lysosomal rupture. Further light exposure led to a drastic fluorescence formation in the nuclei, in particular the nucleoli of the cells, which was concomitant with a renewed observation of a fluorescence emission spectrum similar to that of the protonated species. In the case of the anionic TPPC4, a similar fluorescence increase was observed during irradiation. However, the formation of a protonated species played a less important role. The cationic T4MPyP again showed fluorescence increase during irradiation. Two Soret-bands separated by about 20 nm could be detected. The red-shifted band may be due to a special intercalation of T4MPyP in nucleic acids.
Present techniques and new perspectives of microscopic fluorescence spectroscopy in cellular diagnosis are outlined. Recent applications include the detection of mitochondrial respiratory deficiencies and the intracellular location and light-induced reactions of photosensitizing porphyrins.
Present techniques and new perspectives of microscopic fluorescence spectroscopy in cellular diagnosis are outlined. Recent applications include the detection of mitochondrial respiratory deficiencies and of the intracellular locatio and light-induced reactions of photosensitizing porphyrins.
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