Intravital research on hydrophobic interactions in protein fibrils using a fluorescence polarization method

Fluorescent probe of acridine orange (AO) is known to be bound exclusively by materials of thick protofibril muscle fibres. The dependence of fluorescence anisotropy of AO in frog muscle fibres on the temperature has been studied. It has been found that the fluorophore orientation decreases with temperature, i.e. here the adsorbent cold denaturation takes place. This phenomenon was demonstrated for living, skinned and glycerinated muscle fibres. It shows that hydrophobic interactions contribute much to stabilization of the myosin structure. After contraction of glycerinated muscle fibres at the action of ATP, the AO fluorescence anisotropy grows independently of the temperature which is indicative of structural rearrangements in materials of thick protofibrils upon contraction.     

Intravital imaging

Until recently, the idea of observing life deep within the tissues of a living mouse, at a resolution sufficient to pick out cellular behaviors and molecular signals underlying them, remained a much-coveted dream. Now, a new era of intravital fluorescence microscopy has dawned. In this Primer, we review the technologies that made this revolution possible and demonstrate how intravital imaging is beginning to provide quantitative and dynamic insights into cell biology, immunology, tumor biology, and neurobiology.     

High-Resolution Intravital Microscopy

Cellular communication constitutes a fundamental mechanism of life, for instance by permitting transfer of information through synapses in the nervous system and by leading to activation of cells during the course of immune responses. Monitoring cell-cell interactions within living adult organisms is crucial in order to draw conclusions on their behavior with respect to the fate of cells, tissues and organs. Until now, there is no technology available that enables dynamic imaging deep within the tissue of living adult organisms at sub-cellular resolution, i.e. detection at the level of few protein molecules. Here we present a novel approach called multi-beam striped-illumination which applies for the first time the principle and advantages of structured-illumination, spatial modulation of the excitation pattern, to laser-scanning-microscopy. We use this approach in two-photon-microscopy - the most adequate optical deep-tissue imaging-technique. As compared to standard two-photon-microscopy, it achieves significant contrast enhancement and up to 3-fold improved axial resolution (optical sectioning) while photobleaching, photodamage and acquisition speed are similar. Its imaging depth is comparable to multifocal two-photon-microscopy and only slightly less than in standard single-beam two-photon-microscopy. Precisely, our studies within mouse lymph nodes demonstrated 216% improved axial and 23% improved lateral resolutions at a depth of 80 µm below the surface. Thus, we are for the first time able to visualize the dynamic interactions between B cells and immune complex deposits on follicular dendritic cells within germinal centers (GCs) of live mice. These interactions play a decisive role in the process of clonal selection, leading to affinity maturation of the humoral immune response. This novel high-resolution intravital microscopy method has a huge potential for numerous applications in neurosciences, immunology, cancer research and developmental biology. Moreover, our striped-illumination approach is able to improve the resolution of any laser-scanning-microscope, including confocal microscopes, by simply choosing an appropriate detector.     

A fluorescence study on the mobility of surface antigens of untreated tumor cells and of tumor cells undergoing cell-mediated lysis

Heterologous (rabbit) antibodies were raised against murine P-815 mastocytoma cells of DBA/2 origin. Antisera and IgG preparations were highly cytotoxic, whereas Fab fragments thereof lost all activity. Fab fragments also showed a much lower avidity than IgG, both for tumor and normal DBA/2 and C57 spleen cells as measured by the release of iodinated Fab and IgG. Both preparations bound specifically to P-815 cells since they were capable of inhibiting T cell-mediated target cell lysis. The binding of IgG and monovalent Fab fragments was studied by fluorescence. Rhodamine-coupled IgG bound homogeneously in the cold and quickly formed patches upon warming but did not form caps even after prolonged incubation at 37 degrees C. Rhodamine-coupled Fab fragments also bound homogeneously. Their distribution was unaltered after incubation at 37 degrees C even when tumor cells formed uropod-like tails. Fab fragments, however, could be induced to cap with a second and third antibody layer. P-815 cells labeled with rhodamine-coupled Fab fragments were incubated with cytolytic T cells (CTL). The conjugates formed between CTL and fluorescent target cells were observed. No gross redistribution of surface antigens on target cells was observed even at late stages of the lytic process. CTL, therefore, do not seem to operate via a redistribution of surface antigens.     

Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis

Accurate quantification of disseminated tumor cells in hematological samples is of fundamental importance in clinical oncology. However, even highly standardized protocols allow only a rough estimation of the total analyzed cell number, as sample processing may have adverse effects on the number of cells available for analysis. The fluorescence-based microscopic scanning system (MetaCyte) detects, counts, captures, and relocates immunolabeled tumor cells in hematopoietic samples. We report on a cell-counting approach that has been implemented into the scanning system to precisely quantify the number of cells per slide. The cell-counting function, which was designed to determine the number of all nucleated (DAPI-stained) cells on the slide, allows an accurate counting of the tumor cells and the total number of cells analyzed in the given microscopic sample. The reliability of the cell-counting approach was demonstrated by the analysis of DAPI-stained images with 18-1,363 nucleated cells. A good correlation (r(2) = 0.965) between the manually and automatically gained results was observed. The counting accuracy could even be optimized after implementing a correction factor. To prove or disprove an interslide variation, routine bone marrow cytospin preparations from neuroblastoma patients were immunostained for GD2/FITC and counterstained with DAPI. Automatic cell counting of cytospin preparations from the same patients showed significant differences in the total cell number (up to 67% cell loss during preparation, with a maximum interslide difference of 4.7 x 10(5) mononuclear cells). We conclude that determination of the tumor cell content in hematopoietic samples is only reliable when it is performed together with accurate cell counting.     

Estradiol decreases retention of rhodamine 123 fluorescence in GH4C1 pituitary tumor cells

Rhodamine 123 is a lipophilic cationic fluorescent dye that localizes in mitochondria. We found that 17 beta-estradiol changes the ability of GH4C1 cells, clonal rat pituitary tumor cells, to retain rhodamine 123. Cells incubated with 10 micrograms/ml rhodamine 123 for 30 min at 37 C took up about equal amounts of rhodamine 123, as determined by fluorescence microscopy, regardless of whether they had been treated with estradiol. After three 5-min washes at 37 C, cells treated with 1 nM estradiol for 7 days before incubation with rhodamine 123 had lost more fluorescence than untreated cells. We further characterized the effect by flow cytometry. The difference in fluorescence between control and treated cells ranged from 50- to 500-fold. The effect of estradiol was maximal at 10(-10) M and took a week to develop fully. The effect is specific for estradiol, because estradiol and diethylstilbestrol reduced retention of rhodamine 123 fluorescence at 10(-10) M, but the same concentrations of dihydrotestosterone, progesterone, dexamethasone, and cholesterol did not. To test if the effect on rhodamine 123 fluorescence was caused by activation of the multidrug resistance transport system, we examined the effect of estradiol on the retention of daunomycin, a known substrate of the transport system. Estradiol treatment caused a 3-fold decrease in daunomycin fluorescence. We isolated clones resistant to estradiol-induced loss of rhodamine 123 fluorescence by flow cytometry and found that two clones still showed an estradiol-induced decrease in daunomycin fluorescence equivalent to that of the parent line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Terbium fluorescence studies of cisplatin binding to GH3/B6 pituitary tumor cells

Terbium (Tb3+) fluorescence was used to investigate the interaction of cisplatin with GH3/B6 pituitary tumor cells. The binding of cisplatin to GH3/B6 cells quenched the fluorescence intensity of bound Tb3+. The IC50 for cisplatin inhibition of Tb3+-GH3/B6 fluorescence was determined to be 190 microM. Cisplatin was found to non-competitively inhibit the cellular binding of Tb3+, causing a dramatic decrease in the maximum number of high-affinity Tb3+ binding sites (by 33%), without markedly affecting their binding affinity. The half-life for the cellular binding of cisplatin was calculated to be 2.7 min. It was suggested that the plasma membrane of GH3/B6 cells contain a specific protein receptor for binding cisplatin.     

Intravital Fluorescence Excitation in Whole-Animal Optical Imaging

Whole-animal fluorescence imaging with recombinant or fluorescently-tagged pathogens or cells enables real-time analysis of disease progression and treatment response in live animals. Tissue absorption limits penetration of fluorescence excitation light, particularly in the visible wavelength range, resulting in reduced sensitivity to deep targets. Here, we demonstrate the use of an optical fiber bundle to deliver light into the mouse lung to excite fluorescent bacteria, circumventing tissue absorption of excitation light in whole-animal imaging. We present the use of this technology to improve detection of recombinant reporter strains of tdTomato-expressing Mycobacterium bovis BCG (Bacillus Calmette Guerin) bacteria in the mouse lung. A microendoscope was integrated into a whole-animal fluorescence imager to enable intravital excitation in the mouse lung with whole-animal detection. Using this technique, the threshold of detection was measured as 10³ colony forming units (CFU) during pulmonary infection. In comparison, the threshold of detection for whole-animal fluorescence imaging using standard epi-illumination was greater than 10⁶ CFU.     

Simultaneous fluorescence immunophenotyping and interphase cytogenetics: a contribution to the characterization of tumor cells.

In immunocytochemical studies, the phenotypic evaluation of tumor cells is often complicated by accompanying normal cells, representing the original tissue or infiltrating leukocytes. This holds particularly true for tissues with a great morphological and immunophenotypical variability, such as bone marrow. A method that identifies mitotic tumor cells by chromosomal aberrations and permits the subsequent immunophenotypical analysis was a first progress, demonstrated by Teerenhovi et al. However, the results are usually hampered by the low number of analyzable mitoses. We demonstrate here a method that simultaneously combines immunophenotyping and in situ hybridization with centromere-specific probes. Using our method, numerically aberrant tumor cells can be identified by interphase cytogenetics and subsequently analyzed immunophenotypically. Since all interphase cells can be analyzed, we are not limited by the number and banding quality of analyzable mitoses.     

Vitamin B6 fluorescence in cells]

The band of cell fluorescence with the maximum of 395-400 nm is registered. This band is exposed on the phone of the tryptophane by wavelength excitation lambdaex=250-260 nm, and in pure scape by lambdaex=310-326 nm. Pyridoxin - substrate of vitamin B6 has identical parameters of spectra excitation and emission of neutral (pH 7) and acid (pH 2) solutions. After temperature damage of cells the intensity of this band increases.     

Modulation of fluorescence in Heliobacterium modesticaldum cells

In what appears to be a common theme for all phototrophs, heliobacteria exhibit complex modulations of fluorescence yield when illuminated with actinic light and probed on a time scale of μs to minutes. The fluorescence yield from cells of Heliobacterium modesticaldum remained nearly constant for the first 10–100 ms of illumination and then rose to a maximum level with one or two inflections over the course of many seconds. Fluorescence then declined to a steady-state value within about one minute. In this analysis, the origins of the fluorescence induction in whole cells of heliobacteria are investigated by treating cells with a combination of electron accepters, donors, and inhibitors of the photosynthetic electron transport, as well as varying the temperature. We conclude that fluorescence modulation in H. modesticaldum results from acceptor-side limitation in the reaction center (RC), possibly due to charge recombination between P₈₀₀ ⁺ and A₀ ^?.     

Apoptosis resistance in tumor cells

Various antitumor agents induce apoptotic cell death in tumor cells. Since the apoptosis program in tumor cells plays a critical role in the chemotherapy-induced tumor cell killing, it is suggested that the defect in the signaling pathway of apoptosis could cause a new form of multidrug resistance in tumor cells. This article describes the recent findings concerning the mechanisms of chemotherapy-induced apoptosis and discusses the implication of apoptosis resistance in cancer chemotherapy. This revised version was published online in August 2006 with corrections to the Cover Date.     

Intracytoplasmic desmosomes in tumor cells

The occurrence of intracytoplasmic desmosomes in some normal and tumor cells was suggested to develop by cell fusion by previous investigators. In this study, similar structures were found in tumor biopsies and tumor cells in culture. It is suggested that these structures develop by membrane incorporation and dissolution in cells with active membrane turnover.