Wolfgang S. L. Strauss

Variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM): realization and application of a compact illumination device

A novel compact illumination device in variable‐angle total internal reflection fluorescence microscopy (VA‐TIRFM) is described. This device replaces the standard condensor of an upright microscope. Light from different laser sources is delivered via a monomode fibre and focused onto identical parts of a sample under variable angles of total internal reflection. Thus, fluorophores in close proximity to a cell–substrate interface are excited by an evanescent wave with variable penetration depth, and localized with high (nanometre) axial resolution. In addition to quantitative measurements in solution, fluorescence markers of the cytoplasm and the plasma membrane, i.e. calcein and laurdan, were examined using cultivated endothelial cells. Distances between the glass substrate and the plasma membrane were determined using the mathematical algorithm of a four‐layer model, as well as a Gaussian‐shaped intensity profile of the illumination spot on the samples. Distances between 0 and 30 nm in focal contacts and between 100 and 300 nm in other parts of the cell were thus determined. In addition to measurements of cell–substrate topology, the illumination device appears appropriate for numerous applications in which high axial resolution is required, e.g. experiments on endocytosis or exocytosis, as well as measurements of ion concentrations proximal to the plasma membrane. The compact illumination device is also suitable for combining TIRFM with further innovative techniques, e.g. time‐resolved fluorescence spectroscopy, fluorescence lifetime imaging (FLIM) or fluorescence resonance energy transfer (FRET).

Fluorescence formation during photodynamic therapy in the nucleus of cells incubated with cationic and anionic water-soluble photosensitizers

The variations of fluorescence during light exposure of the cationic sensitizers methylene blue (MB) and meso-tetra(4N-methylpyridyl)porphyrin (T4MPyP) as well as the anionic meso-tetra(4-sulphonatophenyl)porphyrin (TPPS4) were measured at different intracellular sites using video-intensified microscopy in combination with microspectrofluorometry. Before light exposure the sensitizers were localized in distinct parts of the cytoplasm, especially in fluorescent organelles. During irradiation a drastic fluorescence formation and increase in the cytoplasm and nucleus, which was most pronounced in the nucleoli, could be observed for the cationic sensitizers as well as TPPS4. In the case of MB the increase in fluorescence was concomitant with a spectral shift in the emission spectra. For TPPS4 and T4MPyP the formation of a second species with a Soret band shifted towards longer wavelengths was observed and correlated with the fluorescence increase in the nucleoli. Cell deformations also took place.

Laser-assisted optoporation of single cells

The plasma membrane of Chinese hamster ovary cells was made permeable using the focused beam of an argon ion laser (488 nm) and phenol red as a light absorbing dye. Small circular dark spots on the cell surface appeared immediately after laser irradiation and disappeared within about 5 min. They were related to transient changes in membrane properties, which could be visualized using the fluorescent marker laurdan, and were probably due to a local increase in temperature. According to a colony forming assay, cell viability was maintained by using light doses up to 2.5 MJ/cm(2) applied for 1 s. In addition to measurements of the efflux of the cytoplasmic marker calcein, cell transfection using a green fluorescent protein (GFP) coding plasmid was studied: brightly fluorescent GFP with an emission maximum around 510 nm was observed within part of the cells after 24 h. The transfection rates after laser irradiation were around 30% for younger subcultures and less than 10% for aging cells. This may be due to age dependent changes in the phase transition of membrane lipids from gel phase to liquid crystalline phase. High transfection rates, visual control and universality towards various cell lines are possibly the main advantages of laser-assisted optoporation in comparison with presently existing methods of cell transfection.

Autofluorescence Lifetime Imaging of Cultivated Cells Using a UV Picosecond Laser Diode

Lifetime images of autofluorescence of cultivated endothelial cells were recorded using a novel picosecond laser diode in the near ultraviolet range (375 nm). In contrast to existing picosecond light sources this wavelength permits efficient excitation of the free and protein bound coenzyme NADH with fluorescence lifetimes of 0.4–0.5 ns and 2.0–2.5 ns, respectively. The effective fluorescence lifetime τeff (depending on both lifetimes) was homogenously distributed over the cells with some shortening in the perinuclear region, possibly close to mitochondria. A slight decrease of τeff was observed after inhibition of the mitochondrial respiratory chain, whereas a slight increase was observed after inhibition of the glycolytic pathway, thus indicating variations of the ratio of free and protein bound NADH. Although present applications are still limited by their low pulse energy (≤5 pJ), uv picosecond laser diodes have a large potential in high resolution fluorescence microscopy and fluorescence lifetime endoscopy.

Light exposure and cell viability in fluorescence microscopy

Test systems for measuring cell viability in optical microscopy (based on colony formation ability or lysosomal integrity) were established and applied to native cells as well as to cells incubated with fluorescence markers or transfected with genes encoding for fluorescent proteins. Human glioblastoma and Chinese hamster ovary cells were irradiated by various light doses, and maximum doses where at least 90% of the cells survived were determined. These tolerable light doses were in the range between 25 J cm−2 and about 300 J cm−2 for native cells (corresponding to about 250−3000 s of solar irradiance and depending on the wavelength as well as on the mode of illumination, e.g. epi‐ or total internal reflection illumination) and decreased to values between 50 J cm−2 and less than 1 J cm−2 upon application of fluorescent markers, fluorescent proteins or photosensitizers. In high‐resolution wide field or laser scanning microscopy of single cells, typically 10−20 individual cell layers needed for reconstruction of a 3D image could be recorded with tolerable dose values. Tolerable light doses were also maintained in fluorescence microscopy of larger 3D samples, e.g. cell spheroids exposed to structured illumination, but may be exceeded in super‐resolution microscopy based on single molecule detection.

Light Dose is a Limiting Factor to Maintain Cell Viability in Fluorescence Microscopy and Single Molecule Detection

A test system for cell viability based on colony formation has been established and applied to high resolution fluorescence microscopy and single molecule detection. Living cells were irradiated either by epi-illumination or by total internal reflection (TIR) of a laser beam, and light doses where at least 90% of irradiated cells survived were determined. These light doses were in the range of a few J/cm2 up to about 200 J/cm2 depending on the wavelength of illumination as well as on the presence or absence of a fluorescent dye (e.g., the membrane marker laurdan). In general, cells were less sensitive to TIR than to epi-illumination. However, comparably high light doses needed for repetitive excitation of single molecules limit the application of super-resolution microscopy to living cells.

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.

Cell viability in optical tweezers: high power red laser diode versus Nd:YAG laser

Viability of cultivated Chinese hamster ovary cells in optical tweezers was measured after exposure to various light doses of red high power laser diodes (lambda = 670-680 nm) and a Nd:yttrium-aluminum-garnet laser (lambda = 1064 nm). When using a radiant exposure of 2.4 GJ/cm2, a reduction of colony formation up to a factor 2 (670-680 nm) or 1.6 (1064 nm) as well as a delay of cell growth were detected in comparison with nonirradiated controls. In contrast, no cell damage was found at an exposure of 340 MJ/cm2 for both wavelengths, and virtually no lethal damage at 1 GJ/cm2 applied at 1064 nm. Cell viabilities were correlated with fluorescence excitation spectra and with literature data of wavelength dependent cloning efficiencies. Fluorescence excitation maxima of the coenzymes NAD(P)H and flavins were detected at 365 and 450 nm, respectively. This is half of the wavelengths of the maxima of cell inactivation, suggesting that two-photon absorption by these coenzymes may contribute to cellular damage. Two-photon excitation of NAD(P)H and flavins may also affect cell viability after exposure to 670-680 nm, whereas one-photon excitation of water molecules seems to limit cell viability at 1064 nm.

Intracellular fluorescence behaviour of meso-tetra(4-sulphonatophenyl)porphyrin during photodynamic treatment at various growth phases of cultured cells

Meso-tetra(4-sulphonatophenyl)porphyrin (TPPS4) taken up by cells is mainly localized in lysosomes as previously shown by fluorescence microscopical and fluorescence spectroscopical investigations. In the present study the intracellular fluorescence behaviour and the intracellular amount of this dye at various growth periods of cells were examined. For cells irradiated in the growth phase a relocalization of TPPS4 from the lysosomes into the cytoplasm and finally into the nucleus was observed. In contrast, for cells irradiated in the stationary phase no redistribution could be detected and therefore no evidence for severe damage of the lysosomal membranes and subsequently for the release of lytical enzymes is given. In both cases lethal damage of the cells was achieved as examined using the trypan blue exclusion test. This indicates that damage of the lysosomes is less important in the photodynamic inactivation of cells sensitized by TPPS4.

Relation between intracellular location and photodynamic efficacy of 5-aminolevulinic acid-induced protoporphyrin IXin vitro. Comparison between human glioblastoma cells and other cancer cell lines

A promising clinical application of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PP IX) is fluorescence detection and photodynamic treatment of residual tumour tissue during surgical resection of high grade malignant glioma. U373 MG human glioblastoma cells were used as a model system to study the relation between intracellular location and photodynamic efficacy of 5-ALA-induced PP IX in more detail. Therefore, ultra-sensitive fluorescence microscopy, using either optical excitation of whole cells or selective excitation of the plasma membrane by an evanescent electromagnetic field, was combined with quantitative measurements of intracellular porphyrin amount and phototoxicity. Glioblastoma cells accumulated PP IX to a moderate extent as compared to T47D breast cancer cells (high accumulation) or OV2774 ovarian cancer cells (low accumulation). Although photodynamic inactivation of the different cell lines (decreasing in the order T47D > U373 MG > OV2774) seemed to be directly related to PP IX accumulation, examination of the data in more detail revealed that photodynamic efficacy per photosensitizer molecule (PE) was about two times higher in glioblastoma and ovarian cancer cells as compared to breast cancer cells. The different photodynamic efficacy of PP IX was related to the different intracellular location. In contrast to breast cancer cells where PP IX fluorescence was localized in small granules, PP IX fluorescence in glioblastoma cells and ovarian cancer cells originated mainly from cellular membranes. Thus, the intracellular location of PP IX in a predominantly lipophilic environment, characterized by a comparably high photostability (probed by photobleaching and photoproduct formation) and a lower degree of porphyrin aggregation (probed previously by fluorescence decay kinetics), seems to be the key factor for high photodynamic efficacy of 5-ALA-induced PP IX. In the case of OV2774 ovarian cancer cells, however, a low PP IX accumulation limited cell inactivation upon irradiation, whereas the results obtained for glioblastoma cells are encouraging to develop PDT to an additional therapeutic option for the treatment of tumour margins in patients who underwent fluorescence-guided resection of high grade malignant glioma after 5-ALA administration.