Pentaferometer: a solid Sagnac interferometer.

A solid two-beam Sagnac-type interferometer is described that is especially adapted for use with a microscope for Fourier excitation and emission fluorescence spectroscopy. Its advantages are its compactness and stability, and because it is an integral optical element, the need for eliminating vibration and for shielding from air currents is greatly reduced.

Interferometric measurement of fluorescence excitation spectra.

A Michelson interferometer has been adapted as an excitation source for fluorescence spectroscopy. A moving fringe pattern was generated by linear displacement of the movable mirror of the Michelson interferometer coupled to a xenon-arc lamp. This spectrally modulated source was monitored by a reference photomultiplier and used for exciting a Rhodamine B solution.

Microfluorometric study of oxygen dependence of (1"-pyrene butyl)-2-rhodamine ester probe in mitochondria of living cells.

The access to oxygen concentration is of importance in various organelles of living cells, especially in mitochondria. A new probe, (1"-pyrene butyl)-2-rhodamine ester, was designed to target this organelle. We present here the properties of the probe in such an environment.

Microspectrofluorometry and fluorescence imaging in the study of human cytopathology.

The study of energy pools and dynamics of specific pathways in living cells by microspectrofluorometry and fluorescence imaging produces spectral and topographic images characterizing structural and functional changes associated with cytopathology. Microspectro-fluorometry and fluorescence imaging have been applied, together with organelle morphometry to a number of cells mimicking certain cytopathologies, including melanoma cells, long-term malignant cells, and gene-defective cells. These investigations of cellular pathology indicate that there is a convergence of various physiopathological processes.

Multiprobe fluorescence imaging and microspectrofluorimetry of cell transformation and differentiation: implications in terms of applied biochemistry and biotechnology.

The dichotomy of cellular transformation versus differentiation does not preclude the hypothesis of a unified underlying mechanism that can switch either way as a result of growth factors, cell-membrane receptors, secondary messengers, integrating switch kinases and/or nuclear receptors. Its study for biopharmaceutical and biotechnological applications requires a methodology capable of dealing with such pleiotropy. In the multiprobe-multiparameter approach, one must remain wary of cumulative toxic effects and misinterpretations.

Imaging of cells by autofluorescence: a new tool in the probing of biopharmaceutical effects at the intracellular level.

The success of biopharmaceuticals relies on the ability to have reliable probes to interpret their mechanisms of action in situ at the intracellular level in terms of cell organelles and microcompartments. One of the most effective probes is the endogenous coenzyme NAD(P)H and its fluorescence transients obtained by the microinjection or perfusion of metabolic intermediates and modifiers, in the presence of drugs and inhibitors. The approach in fluorescence microtopography and microspectrofluorimetry is based on the premise that natural cell fluorescence (autofluorescence) holds a decisively greater potential in unravelling intracellular physiopathological processes than extrinsic fluorescence or artificial pseudocolouring.

An in situ study of beta-glucosidase activity in normal and Gaucher fibroblasts with fluorogenic probes.

Beta-glucosidase activity was evaluated in situ by means of fluorogenic probes in normal human fibroblasts and fibroblasts from homozygous carriers of the Gaucher trait. Probe internalization, targeting to lysosomes and post-cleavage probe retention were the primary concerns. Internalization and targeting were attempted by in situ photosensitized labilization of lysosomal membranes, lysosomotropic detergents and the use of low density lipid (LDL) or the receptor ligand apolipoprotein E (ApoE).

Direct connection between myelinosomes, endoplasmic reticulum and nuclear envelope in mouse hepatocytes grown with the amphiphilic drug, quinacrine.

Mouse hepatocytes grown in 4 microM quinacrine had numerous myelinosomes which were directly connected to expanded cisternae of the rough endoplasmic reticulum (RER). The cisternae of the RER either subtended the electron transparent space of the myelinosome, expanded to form the outer membrane of the myelinosome or penetrated into it. Material of low electron density was frequently seen within the area where the cisternae penetrated into the electron transparent space of the myelinosome.

Is rhodamine 123 a photosensitizer?

Because of conflicting reports on the photoxicity of rhodamine 123 (Rh 123), we have undertaken a study of Rh 123 photosensitization in several in vitro systems. First, Rh 123 is not a photodynamic agent and does not react with singlet oxygen. Second, when bound to cytochrome c (Cyt c), Rh 123 photosensitizes ferro Cyt c but not ferri Cyt c degradation by an oxygen-independent process.

Bioregulatory mechanisms at the level of cell organelle interactions: microspectrofluorometric in situ studies.

The spatiotemporal analysis of bioregulatory mechanisms at the level of intracellular multienzyme complexes and organelle interactions is made possible by the availability of endogenous and exogenous fluorescence probes, the development of microspectrofluorometers allowing one- and two-dimensional scans of intracellular fluorescence reactions, and the use of micromanipulatory techniques enabling the rapid alteration of metabolic states. Absorbed photons are not only a tool for quantitative evaluation of metabolic processes, they can also trigger alterations of cell membranes and functions as mediated by photosensitizer drugs. In the hierarchy of intracellular organization different levels of complexity are accessible to study, such as the regulation of multienzyme complexes and the interaction of organelle complexes.

Interaction of tetrapyrrolic rings with rhodamine 110 and 123 and with rhodamine 110 derivatives bearing a peptidic side chain.

Rhodamines 110 and 123, and rhodamine 110 linked via peptide bonds to Arg, Cbz-Arg and Cbz-Ile-Pr-Arg interact with free base porphyrins or cytochrome C. Rhodamines 110 and 123 essentially form 1:1 complexes while the other derivatives form 2:1 complexes. The possible biological implications of these results are discussed.

A microspectrofluorometric study of the effect of anthralin, an antipsoriatic drug, on cellular structures and metabolism.

The microspectrofluorometric approach has been used to investigate in single living cells in culture fundamental questions raised by the use of anthralin, a potent antipsoriatic drug. This method allows fluorescence determinations on the intracellular fate of the drug as well as the recognition of structural and metabolic alterations induced by the drug. In the absence of demonstrable adduct formation with DNA, the antipsoriatic, i.

Microspectrofluorometry of fluorescent photoproducts in photosensitized cells. Relationship to cellular quiescence and senescence in culture.

Microspectrofluorometry of L and WI-38 cells reveals chemical/structural changes due to quiescence or senescence, i.e., lipid peroxidation, spontaneous or photosensitized by hematoporphyrin.

The differential effects of dimethylnitrosamine and ethionine on mitochondrial and extramitochondrial dehydrogenases in single intact cells.

In studying the bioenergetics of living cells, the microfluorometric analysis of coenzyme (NAD(P)H) responses to microinjected respiratory and glycolytic substrates enables, in principle, a search for qualitative/quantitative differences in normal versus carcinogen-treated (short-term, long-term) and malignant cells. Responses are compared in L-cells, same adapted to hypertonic media (i.e.

Metabolic control and compartmentation in single living cells.

Microspectrofluorometry of cell coenzymes (NAD(P)H, flavins) in conjunction with sequential microinjections into the same cell of metabolites and modifiers, reveals aspects of the regulatory mechanisms of transient redox changes of mitochondrial and extramitochondrial nicotinamide adenine dinucleotides. The injection of ADP in the course of an NAD(P)H transient produced by glycolytic (e.g.

Microspectrofluorometry of carcinogens in living cells.

A microspectrofluorometric approach has been used to follow the changes undergone by the carcinogen benzo(a)pyrene in malignant L cells, inducible Buffalo rat liver (BRL) cells and oncogenic mouse embryo C3H/10 T 1/2, clone 8 (CCL 226) cells. Since it is known that benzo(a)pyrene (BP) is converted metabolically to at least 40 metabolites, including phenols, epoxides, quinones, dihydrodiols, diol epoxides, and water-soluble conjugates, the interpretation of blue- and red-spectral shifts in fluorescence emission observed in BP-treated cells, compared to the original BP emission, undoubtedly presents considerable difficulties, but a certain number of facts clearly emerge. The sequence of blue-red shifts expressive of intracellular interactions and detoxification of the carcinogen is accelerated in the induced BRL compared to non-induced, and it is also generally accelerated in the malignant and inducible lines compared to the oncogenic line.