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Conventional photothermal (PT) and photoacousic (PA) imaging, spectroscopy, and cytometry are preferentially based on positive PT/PA effects, when signals are above background. Here, we introduce PT/PA technique based on detection of negative signals below background. Among various new applications, we propose label-free in vivo flow cytometry of circulating clots. No method has been developed for the early detection of clots of different compositions as a source of thromboembolism including ischemia at strokes and myocardial infarction. When a low-absorbing, platelet-rich clot passes a laser-irradiated vessel volume, a transient decrease in local absorption results in an ultrasharp negative PA hole in blood background. Using this phenomenon alone or in combination with positive contrasts, we demonstrated identification of white, red, and mixed clots on a mouse model of myocardial infarction and human blood. The concentration and size of clots were measured with threshold down to few clots in the entire circulation with size as low as 20 μm. This multiparameter diagnostic platform using portable personal high-speed flow cytometer with negative dynamic contrast mode has potential to real-time defining risk factors for cardiovascular diseases, and for prognosis and prevention of stroke or use clot count as a marker of therapy efficacy. Possibility for label-free detection of platelets, leukocytes, tumor cells or targeting themby negative PA probes (e.g., nonabsorbing beads or bubbles) is also highlighted., (Copyright © 2011 International Society for Advancement of Cytometry.)

Flow cytometric assays for quantifying polymorphonuclear leucocyte (PMN) function involve peripheral blood fractionation methods. However, most are of limited use for conditions like pediatrics and thalassemia, as they may require large blood volumes or complicated by red blood cell contamination. In whole blood assay, 500-mul aliquots of normal or thalassemic whole blood were incubated with bis-carboxyethyl-carboxyfluorescein pentaacetoxymethylester (BCECF-AM)-labeled Candida albicans cells and phycoerythrin-conjugated anti-CD13 monoclonal antibody for PMNs. Phagocytosis was quantitated by gating on CD13 label PMNs and determining the frequency of phagocytosed yeast using flow cytometry. The killing activity was quantitated by estimating the number of viable fluorescence retaining yeast cells liberated following lysis of PMNs. In normal control or thalassemia samples, nonphagocytosed yeast and PMNs with or without phagocytosed yeast were readily distinguished by two-color dot plot, permitting phagocytosis estimates. Viable and nonviable yeast killed by whole blood PMNs were distinguished by side scatter and fluorescence, permitting killing estimates. The patterns seen using whole blood was similar to that seen with fractionated PMNs. Data from thalassemia patients showed similar opsonophagocytosis but slightly decreased intracellular killing of yeast cells when compared with normal subjects. This assay provides an alternative method to assess PMN function in small blood samples, which could be especially useful in conditions like thalassemia and pediatric patients., ((c) 2007 International Society for Analytical Cytology)

Background: Copper(II) is a heavy metal whose levels have increased in some marine ecosystems to polluting levels. Dinoflagellates, an important phytoplankton group, are at the base of aquatic food chains and bioaccumulation of copper by these microorganisms can result in complex ecosystem alterations, so we investigated how copper disturbs those cells., Methods: Cytotoxic effects of sublethal and lethal copper concentrations ranging from 4.2 nM (control condition) to 3.13 microM estimated labile copper were studied in batch cultures of Amphidinium carterae. Cell morphology, motility, autofluorescence, and fluorescein diacetate (FDA)-dependent fluorescence generation were evaluated by flow cytometry (FCM) and microscopy., Results: Exposure of A. carterae to toxic levels of copper impaired cell mobility, delayed cell proliferation, led to increased green autofluorescence, and at 3.13 microM labile copper also induced encystment and death. Chlorophyll fluorescence, however, was not affected. Kinetic FCM assay of FDA-dependent fluorescence generation showed a dose-dependent enhancement of fluorescein fluorescence immediately after copper addition and in cultures with sustained exposure to this toxicant., Conclusions: Our data suggest that copper toxicity occurs quickly at the membrane level in relation to oxidative stress generation. Based on fluorescence kinetic studies, the Na(+)/H(+) antiporter seemed to be affected by copper, thereby affecting intracellular pH., (Copyright 2001 Wiley-Liss, Inc.)

Xenotransplantation requires monitoring of complex cellular interactions in vitro. A tool to monitor cell proliferation in detail would be instrumental in understanding these cellular interactions in heterogeneous xenogeneic lymphocyte cultures and in patients after xenotransplantation. To accomplish this, we used a fluorescent cell proliferation marker, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE), in combination with flow cytometry. CFSE, a green fluorescent molecule, binds covalently to intracellular macromolecules. Each cell division reduces the fluorescent intensity per cell by half and shows a characteristic multipeak pattern in flow cytometric analysis. For this study, human lymphocytes were labeled with CFSE and cultured in the presence of irradiated porcine lymphocytes. Cell proliferation was detected in CFSE-labeled lymphocytes in both a single and a multiparameter flow cytometry setting. Concurrently, tritiated ((3)H) thymidine incorporation, a common method to measure gross cell proliferation, was assessed. The kinetics of CFSE-labeled cell proliferation correlated with (3)H-thymidine incorporation in that both methods showed a lag phase for days 1-3 and a log phase for days 4-7. Multiparameter flow cytometric monitoring of mixed lymphocyte cultures allowed phenotyping and assessment of viability of proliferating populations in heterogeneous xenogeneic stimulated human lymphocyte cultures and complemented the classical (3)H-thymidine incorporation assay. The use of this technique will allow a wide array of immunologic parameters to be measured in a heterogeneous xenogeneic mixed lymphocyte culture. The information gained from these assays is essential to understanding the biological significance of xenogeneic cellular interaction and for monitoring the immune status of the xenotransplanted patient., (Copyright 2000 Wiley-Liss, Inc.)

The sensitivity of six fluorophores to glutathione (GSH) was evaluated in living rat cortical neuronal/glial mixed cultures during the first 23 days in vitro (DIV). Four of the dyes require glutathione-S-transferase (GST) to form a fluorescent conjugate, potentially conferring specificity for GSH: these included t-butoxycarbonyl-Leu-Met-7-amino-4-chloromethylcoumarin (CMAC), 7-amino-4-chloromethylcoumarin (CMAC-blue), monochlorobimane (MCB), and 5-chloromethylfluorescein diacetate (CMFDA). The final two dyes examined, 2,3-naphthalenedicarboxaldehyde (NDA) and o-phthaldehyde (OPD), do not require GST for adduct formation with GSH. To examine the specificity of the dyes for GSH, cultures grown less than 6 DIV were pretreated with diethyl maleate or DL-buthionine-(S, R)-sulfoximine to deplete endogenous GSH. This resulted in a substantial loss of staining by CMAC, CMAC-blue, and MCB and partial loss of staining by OPD, indicating specificity for GSH, while staining by CMFDA or NDA was not altered, indicating a lack of specificity for GSH. Neurons experienced a dramatic decline in GSH levels relative to astrocytes between 5-6 DIV, as shown by a loss of neuronal staining with CMAC, CMAC-blue and MCB. This decrease in staining was not due to a decrease in GST activity, as neurons stained with the GST-insensitive OPD also exhibited a decline in GSH-sensitive staining. Immunolabeling experiments demonstrated that CMAC staining co-localized with GFAP-positive astrocytes, but not with MAP-2-positive neurons, in 18 DIV cultures. Finally, CMAC was exploited as a specific morphological marker of astrocytes in cultures aged >5 DIV. CMAC staining was employed to monitor astrocyte proliferation and to resolve astrocytes in living mixed cultures co-loaded with the Ca(2+)-sensitive dye, calcium green 5N-AM. GLIA 30:329-341, 2000. Published 2000 Wiley-Liss, Inc.

Background and Objectives: The aim of the study was to evaluate the feasibility of retinal thermal damage assessment in a rabbit eye model by using laser-induced release of liposome-encapsulated dye., Study Design/materials and Methods: After anesthesia, thermosensitive liposomes (DiStearoyl Phosphatidyl Choline: DSPC) loaded with 5,6-carboxyfluorescein were injected intravenously to pigmented rabbits. Retinal photocoagulations were performed with a 810nm diode laser (P=100-400 mW, laser spot=500 microm, 1s) (OcuLight, IRIS Medical Instruments, Mountain View, CA). Fluorescence measurements in the area of the laser exposures were then realized with a digitized angiograph (CF-60UVi, Canon-Europe, The Netherlands; OcuLab, Life Science Resources, UK)., Results: Fluorescent spots were observed for power ranging from 100 +/- 5 mW to 400 +/- 5 mW. The fluorescence intensity increased linearly with the power and reached a plateau at 280 +/- 5 mW. The fluorescence intensity was correlated to the maximum temperature at the center of the laser spot with a linear increase from 42 +/- 3 degrees C to 65 +/- 3 degrees C. These results are in agreement with our two previous studies with DSPC liposomes for temperature measurements in a tissue model and then in a vascular model., Conclusion: This preliminary study demonstrates the possibility of a laser-induced release of liposome-encapsulated dye for a quantification of diode laser induced thermal damage in ophthalmology. Such a method could be useful for a real-time monitoring of laser photocoagulation for conditions such as choroidal neovascular membranes when a precise thermal damage is required near the foveolar area.

We report the development of a new fluorescence-activated cell sorter (FACS)-based reporter gene system utilizing the enzymatic activity of the E. coli beta-glucuronidase (gus) gene. When loaded with the Gus substrate fluorescein-di-beta-D-glucuronide (FDGlcu), individual mammalian cells expressing and translating gus mRNA liberate sufficient levels of intracellular fluorescein for quantitative analysis by flow cytometry. This assay can be used to FACS sort viable cells based on Gus enzymatic activity, and the efficacy of the assay can be measured independently by using a fluorometric lysate assay. Furthermore, both the beta-glucuronidase and the previously described E. coli beta-galactosidase enzymes have high specificities for their cognate substrates, allowing each reporter gene to be measured by FACS independently.

A four-colour flow cytometry technique is described for the determination of the number and phenotype of conjugate-forming mononuclear effector cells from human blood. The discriminatory power of previously described techniques was improved by labelling the effector cells with antibodies that simultaneously identified natural killer (NK), T, and myeloid cells and by labelling the tumour target cells with fluorescein octadecyl ester (FOE), so that cell conjugates could be differentiated from nonbinding effector cells more effectively than by the use of light scatter. The simultaneous characterisation of the three classes of conjugate-forming cells within a heterogeneous human peripheral blood mononuclear cell (PBMC) population made it possible to investigate conjugate formation by each subpopulation without purification, or semipurification (e.g., by monocyte removal), of the subpopulations. The binding of PBMCs to K562 and RPMI 1788 targets was examined. Binding for all three PBMC subpopulations was optimal at 37 degrees C and was negligible at 0 degree C (except for some T cell binding to RPMI 1788 cells). At 37 degrees C, maximum binding was essentially achieved by 10 min. Sodium azide inhibited the majority of the conjugation by NK and T cells, and that inhibition could be removed by washing the cells prior to conjugation, whereas azide had a negligible effect on the binding by monocytes. It appears that effective conjugation by human peripheral blood NK and T lymphocytes requires the operation of an energy-dependent process, differentiating it from conjugation by monocytes.

We report on 1) the development of a flow cytometry-based technique for detecting beta-galactosidase in differentiating cultures of Bacillus subtilis and 2) the application of this technique in the study of early developmental gene expression. The problems associated with generating detectable signals (despite the small size of B. subtilis cells) have been overcome using the fluorogenic substrate 5-octanolyaminofluorescein di-beta-D-galactopyranoside (C8-FDG). Additionally, to control for background fluorescence during the staining process, we included a control population in the C8-FDG staining mixture that consists of cells devoid of the lacZ gene prestained with another dye, PKH26. The distinct emission spectra of C8-fluorescein and PKH26 allow nonspecific C8-FDG staining in this control population to be monitored using two-color analysis. This technique has been applied in the study of developmental gene expression in sporulating cultures of B. subtilis, and it has been found that such cultures are heterogeneous, comprising two cell populations. One population is induced for expression of early sporulation genes, which is determined using lacZ fusions, whereas the other remains uninduced. These results have allowed us to understand better the patterns of gene expression exhibited by wild-type and mutant cultures early during the development process of spore formation.

Environmentally sensitive molecules have many potential cellular applications. We have investigated the utility of a pH sensitive ligand for the formyl peptide receptor, CHO-Met-Leu-Phe-Phe-Lys (SNAFL)-OH (SNAFL-seminaphtho-fluorescein), because in previous studies (Fay et al.: Biochemistry 30:5066-5075, 1991) protonation has been used to explain the quenching when the fluoresceinated formyl pentapeptide ligand binds to this receptor. Moreover, acidification in intracellular compartments is a general mechanism occurring in cells during processing of ligand-receptor complexes. Because the protonated form of SNAFL is excited at 488 nm with emission at 530 nm and the unprotonated form is excited at 568 nm with emission at 650 nm, the ratio of protonated and unprotonated forms can be examined by multiparameter flow cytometry. We found that the receptor-bound ligand is sensitive to both the extracellular and intracellular pH. There is a small increase in the pKa of the ligand upon binding to the receptor consistent with protonation in the binding pocket. Once internalized, spectral changes in the probe consistent with acidification and ligand dissociation from the receptor are observed.

Previous studies have identified unique cell surface antigens which are associated with the specific binding of lymphocytes to high endothelial venules (HEV). Evidence is presented in this paper which demonstrates that uptake of the fluorescent dye calcein by lymphocytes represents an additional marker for the lymph node homing subpopulation of lymphocytes. Calcein exhibits a characteristic ability to label lymphocytes differentially into two distinct populations, based on fluorescence intensity, that does not occur with three other structurally related, fluorescein-based dyes. In vivo lymphocyte migration studies revealed that cells displaying the "dull" fluorescence phenotype, although entering all lymphoid organs examined, preferentially homed to the lymph nodes, particularly the popliteal lymph node (PLN). By contrast, lymphocytes displaying the "bright" phenotype were essentially excluded from entering lymphoid organs, where entry is HEV dependent, but were observed entering spleen, where entry is HEV independent. Furthermore, a high proportion (76.5%) of lymphocytes displaying the dull fluorescence phenotype expressed the PLN homing receptor MEL-14. Based on these observations it is suggested that calcein uptake may be a marker for general membrane properties, such as fluidity and plasticity, essential for the passage of lymphocytes through HEV.

Detailed studies have been performed on various spectroscopic properties such as time dependence and excitation wavelength dependence of the fluorescence anisotropy for fluorescein molecules introduced into rat thymus lymphocytes. Experimental results have been found to be well interpreted in terms of the coexistence of two types of dye molecules, i.e., free and bound molecules. The fluorescence spectrum of only the bound molecules has been obtained from the difference in the time-resolved spectra of fluorescence with two polarization directions. The time gate has been set at a sufficiently late time after the excitation, so that the polarization memories of the free molecules are lost. The spectrum thus determined agrees very well with that calculated from the spectral data in the stationary condition. From the above results, we come to the conclusion that the main factors which determine the fluorescence anisotropy inside the cell are the fraction and the anisotropy of the bound dye molecules. Finally, we discuss how these factors are related to biological quantities.