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Automated four-color analysis of leukocytes by scanning fluorescence microscopy using quantum dots

: Bocsi, József; Lenz, Dominik; Mittag, Anja; Varga, Viktor Sebestyén; Molnar, Béla; Tulassay, Zsolt; Sack, Ulrich; Tárnok, Attila


Cytometry. Part A 69 (2006), Nr.3, S.131-134
ISSN: 1552-4922
ISSN: 0196-4763
Fraunhofer IZI ()
SBC; SFM; bleaching; fluorescence mounting medium; QD605; QD655

Scanning fluorescence microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al., Cytometry A 2004, 61:1–8).
We developed a four-color staining protocol (DNA, CD3, CD4, and CD8) for the lymphocyte phenotyping by SFM.
Organic (Alexa488, FITC, PE-Alexa610, CyChrom, APC) and inorganic (quantum dot (QD) 605 or 655) fluorochromes were used and compared in different combinations. Measurements were performed in suspension by flow cytometer (FCM) and on slide by SFM.
Both QDs were detectable by the appropriate Axioplan-2 and FCM filters and the AxioCam BW-camera. CD4/CD8 ratios were highly correlated (P = 0.01) between the SFM and FCM.
Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs. © 2006 International Society for Analytical Cytology
Flow cytometer (FCM) and Laser scanning cytometer (LSC,1) are standard instruments for quantitative multicolor phenotyping of leukocytes; spectral imaging confocal microscopy is optimal for morphological cytometric analysis (2). The Scanning fluorescence microscope (SFM) is a slide-based, software-controlled new technique for automated motorized microscopes equipped with a scientific digital camera. It can also measure multiple fluorescent labeling and is appropriate for multichannel cytometric analysis (3, 4). Current development of digital cameras and computer systems promise broad spreading of SFM technique.
In fluorescence microscopy, fluorescence emission is stimulated by high intensity UV or visible light. Light absorption induces an excited state of the dye (usually singlet-state), leading to fluorescence. However, the excited state of the dye may undergo chemical reactions such as photo-oxidation, leading to its destruction as evidenced by fading or bleaching of the fluorescence and subsequent decrease in signal intensity during measurement.
High-content analysis of biological specimens requires multicolor labeling for polychromatic (1) or hyperchromatic cytometry (5). The hyperchromatic analysis needs spectrally combinable labels with stable long-lasting fluorescence. Conventional organic fluorochromes bleach more or less rapidly in buffer during illumination (6). This is overcome by using antifading solutions as mounting media (7, 8).
Fluorescent semiconductor nanocrystals (quantum dots, QDs) offer new ways in multiparametric characterization of biological samples. In comparison with organic fluorophores, the QDs have unique optical properties such as, size (5–20 nm diameter, similar size as a typical protein), and composition tunable fluorescence emission from visible to infrared wavelength. In high absorption from UV across a wide range of the spectrum, narrow and symmetric emission spectra, intensive brightness, and photostability are the characteristics (9, 10). QDs are produced with appropriate mean emission for the well-proved fluorescence systems and filter combinations. This should enable combination of QDs in multicolor staining protocols with the other “classical” fluorochromes. QDs have recently been successfully covalently linked to reactive molecules such as peptides, antibodies, nucleic acids, or small-molecule ligands (9, 10). The development of high-sensitivity and high-specificity probes that lack the intrinsic limitations of organic dyes and fluorescent proteins is of considerable interest in many areas of biomedicine, ranging from molecular and cellular biology to molecular imaging and diagnostics.
The T-helper/T-cytotoxic (CD4+/CD8+) lymphocytes were selected as a model system. We developed a four-color fluorescence labeling method to demonstrate applicability of QDs in immunophenotyping by slide-based cytometry (SBC).