Flow cytometry Laser-scanning

Unlike the conventional flow cytometry which scans the signal of individual bead, the Nanoplex Reader Raman Instrument acquires a total signal from the whole population of the beads within the laser spot (150 pm). Thus, increasing the portion of the beads that carry the specific signal in the laser spot area should improve the limit of detection of the assay. A multiple probe scheme is created to facilitate this characteristic (Fig. 19.5). In the assay, target DNA was first chopped into small... 

We have combined flow cytometry method, scanning force microscopy and confocal laser scanning microscopy for imaging structural features of leukemic cells from patients with acute lymphoblastic leukemia at different stage of the disease. 

Akagi et al. demonstrated the use of nanoparticles composed of amphiphilic poly (amino acid) derivatives as vaccine delivery and adjuvants [62, 102-104]. To evaluate the uptake of OVA encapsulated within y-PGA-Phe nanoparticles (OVA-NPs) by DCs, murine bone marrow-derived DCs were incubated with 250 nm-sized OVA-NPs for 30 min at 37 °C. The cells were then analyzed by flow cytometry (FCM) and confocal laser scanning microscopy (CLSM). OVA-NPs were efficiently taken up into DCs, whereas the uptake of OVA alone was barely detectable at the same concentration of OVA (Fig. 13). OVA-NPs were more efficiently taken up than OVA alone by DCs, and the uptake of OVA-NPs was inhibited at 4 °C. These results suggest that OVA-NPs were phagocytosed mainly via endocytosis by the DCs. In the case of OVA alone, an approximately 30-fold... 

In addition to flow cytometers, cytometric instrumentation has evolved to include Laser Scanning Cytometry (LSC). The LSC is a cross between a flow cytometer and an imaging cytometer. Data are equivalent to the flow cytometer but is slide-based. Endpoints include light scatter and fluorescence, but the instrument can also record position of individual cells on the slide so that cells of interest can be relocated and re-evaluated or photographed. LSC is valuable when cell morphology is important. 

Special emphasis is placed on the carbohydrate-mediated cell - target system interaction by describing hints and pitfalls of assays for cytoadhesion, specificity, cytoinvasion, and cytoevasion. In addition, basic considerations are presented to discriminate between active and passive uptake as well as to detect lysosomal accumulation. Finally, the pros and cons of two useful analytical techniques, namely, flow cytometry and confocal laser scanning microscopy, are described in detail. 

Keywords Cytoadhesion Cytoinvasion Lectin Confocal laser scanning microscopy Flow cytometry Uptake... 

Viability-based technologies Direct epifluorescent filter microscopy Membrane laser scanning Fluorescence cytometry Fluorescence flow cytometry... 

TAT-peptide modified liposomes were prepared and cellular association and intracellular distribution of (double) fluorescently labelled particles were assessed by flow cytometry and confocal laser scanning microscopy. 

Fluorescence detection confocal or laser scanning devices, CCD cameras, near-infrared imaging, surface plasmon resonance imaging, flow cytometry (for microspheres)... 

Fluorescent reporters allow detection in a single cell with epifluorescence and confocal laser scanning microscopy (Cormack et al., 1996 Tombolini et al., 1997 Phillips, 2001) or allow quantitative single-cell analysis with flow cytometry (Cormack et al., 1996 Valdivia and Ramakrishnan, 2000), enlarging their potential scope of use. 

Lemarchand K, Parthuisot N, Catala P, Lebaron P (2001) Comparative assessment of epifluores-cence microscopy, flow cytometry and solid phase cytometry in the enumeration of specific bacteria in water. Aquat Microb Ecol 25 301-309 Lepeuple AS, Delabre K, GUouppe S, IntertagUa L, de Roubin MR (2003) Laser scanning detection of FISH-labelled Escherichia coli from water samples. Water Sd Technol 47 123-129 Lisle JT, Hamilton MA, Willse AR, McFeters GA (2004) Comparison of fluorescence microscopy and sohd-phase cytometry methods for counting bacteria in water. Appl Environ Microbiol 70 5343-5348... 

As with flow cytometry, multiparameter apoptosis assays may also be performed by confocal laser scanning microscopy (CLSM). Using the approach similar to that detailed above for flow cytometry, we have examined NADPH content, mitochondrial membrane potential (CMX Rosamine fluorescence), and mitochondrial mass (Mitotracker Green), by CLSM. Figure 3 shows an example of a typical multiparameter assay performed by confocal microscopy. 

One advantage to examining apoptotic cells by laser-scanning cytometry is that subcellular structure may be examined. For instance, individual mitochondria may be examined for correlations among different parameters. Similarly, nuclear structure may also be examined in apoptotic cells. An example of the complimentarity of flow cytometry and CLSM is given in Fig. 4. TNF-a/ActD treated Hepa-1 cells were stained with CMX Rosamine... 

Fig. 4. Combined use of flow cytometry/cell sorting and confocal laser scanning microscopy. TNF-a/ActD treated Hepa-1 cells were stained with CMX Rosamine and then analyzed for mitochondrial membrane potential and NAD(P)H fluorescence (A). Cells in different regions of the cytogram were then sorted, and subsequently stained with the DNA fluorochrome Hoechst 33342. These cells were then examined by CLSM. (B), (C), and (D) show three-dimensional reconstructions of nuclei from cells sorted from healthy, early apoptotic, and late apoptotic populations. Whereas healthy cells show normal round nuclei, early and late apoptotic cells show progressive chromatin condensation/margination and nuclear fragmentation.

The laser scanning cytometer (LSC) is a microscope-based cytofluorometer that combines advantages of flow cytometry and image analysis and is finding wide applicability in many disciplines of biology and medicine (see reviews in refs. 8,9). LSC measures cell fluorescence rapidly and with similar accuracy as flow cytometer. However, since the xy coordi-... 

Porous silica supports such as MCM-41 have also been employed in the development of artificial hybrid pores due to their well-defined porous network and then-facile functionalization chemistry. For instance, Lopez and coworkers employed an MCM-41 support coated uniformly with a temperature-responsive poly(AI-isopropyl acrylamide) (PNIPAAm) polymer. At low temperatures, the polymer was hydrated and extended and the pores were closed no transport of fluorescein was observed when flow cytometry and confocal laser scanning microscopy were employed. In contrast, at high temperatures the polymer was hydrophobic and collapsed, allowing the transport of the dye (Figure 24). This system thus not only acts as a temperature sensor but also shows clear features of control as will become more important in the next section. 

In solid-phase cytometry (SPC), microorganisms are analysed by a combination of both epifluorescence microscopy and flow cytometry [139], Air is impacted on a water-soluble polymer film present in a standard Petri dish (polyvinyl alcohol plate). After filtration, viable cells are labeled. The filter is scanned by a laser in a solid-phase cytometer. Using different software, fluorescent particles are distinguished from microorganisms. 

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