PubMed Health⌕ Search
Home/Archive/PubMed

PubMed · 7924689

Fluorescence array detector for large-field quantitative fluorescence cytometry.

K D Wittrup·R J Westerman·R Desai
View original source ↗Export metadata ↓

Abstract

A prototypic fluorescence array detector (FAD) has been designed and constructed which is capable of quantifying single-cell fluorescence emissions from a statistically significant population of cell-sized objects (over 10(3)) on a solid substrate. The system is comprised of a cryogenically cooled CCD, 50 mW air-cooled argon ion laser, and optics that image a large (1 x 1 cm) field at 1:1 (no magnification). The CCD is effectively treated as a two-dimensional array of 2.7 x 10(5) independent 20 x 20 microns photodetectors, with each cell-sized object imaged across only a few CCD pixels. Algorithms have been developed for focusing, image segmentation, shading correction, and noise rejection; performance data for the FAD with fluorescent calibration beads are presented. The FAD is a simple alternative to microscope-based imaging cytometry, allowing large-field imaging without a scanning stage.

Explore related subjects

Flow CytometryImage Processing, Computer-AssistedJournal ArticleResearch Support, U.S. Gov't, Non-P.H.S.

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

K D Wittrup, R J Westerman, R Desai. 1994-07-01. Fluorescence array detector for large-field quantitative fluorescence cytometry.. https://doi.org/10.1002/cyto.990160304

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related citations

PubMed1998

Direct visualization of antigen-specific T cells: HTLV-1 Tax11-19- specific CD8(+) T cells are activated in peripheral blood and accumulate in cerebrospinal fluid from HAM/TSP patients.

Human T lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropic spastic paraparesis is a demyelinating inflammatory neurologic disease associated with HTLV-1 infection. HTLV-1 Tax11-19-specific cytotoxic T cells have been isolated from HLA-A2-positive patients. We have used a peptide-loaded soluble HLA-A2-Ig complex to directly visualize HTLV-1 Tax11-19-specific T cells from peripheral blood and cerebrospinal fluid without in vitro stimulation. Five of six HTLV-1-associated myelopathy/tropic spastic paraparesis patients carried a significant number (up to 13.87%) of CD8(+) lymphocytes specific for the HTLV-1 Tax11-19 peptide in their peripheral blood, which were not found in healthy controls. Simultaneous comparison of peripheral blood and cerebrospinal fluid from one patient revealed 2.5-fold more Tax11-19-specific T cells in the cerebrospinal fluid (23.7% vs. 9.4% in peripheral blood lymphocyte). Tax11-19-specific T cells were seen consistently over a 9-yr time course in one patient as far as 19 yrs after the onset of clinical symptoms. Further analysis of HTLV-1 Tax11-19-specific CD8(+) T lymphocytes in HAM/TSP patients showed different expression patterns of activation markers, intracellular TNF-alpha and gamma-interferon depending on the severity of the disease. Thus, visualization of antigen-specific T cells demonstrates that HTLV-1 Tax11-19-specific CD8(+) T cells are activated, persist during the chronic phase of the disease, and accumulate in cerebrospinal fluid, showing their pivotal role in the pathogenesis of this neurologic disease.

Flow Cytometry↗
PubMed1998

CXCR4 sequences involved in coreceptor determination of human immunodeficiency virus type-1 tropism. Unmasking of activity with M-tropic Env glycoproteins.

The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4 and one of a cadre of chemokine receptors triggers conformational changes in the HIV-1 envelope (Env) glycoprotein that lead to membrane fusion. The coreceptor activity of the second extracellular loop of CXCR4, which is restricted to dual tropic and T-tropic strains, was insensitive to the removal of charged residues either singly or in combinations by alanine scanning mutagenesis or to the conversion of acidic residues to lysine. Conversion of Asp-187 to a neutral residue exclusively unmasked activity with M-tropic Env in fusion and infection experiments. Insertion of the D187V mutation into chimeras containing extracellular loop 2 of CXCR4 in a CXCR2 framework also resulted in the acquisition of M-tropic coreceptor activity. The independence of CXCR4 coreceptor activity from charged residues and the extension of its repertoire by removing Asp-187 suggest that this interaction is not electrostatic and that coreceptors have the potential to be utilized by a spectrum of Env, which may be masked by charged amino acids in extracellular domains. These findings indicate that the primary structural determinants of coreceptors that program reactivity with M-, dual, and T-tropic Env are surprisingly subtle and that relatively insignificant changes in CXCR4 can dramatically alter utilization by Env of varying tropism.

Flow Cytometry↗
PubMed1998

Analysis of intracellular Th1 cytokine secretion data using parametric methodology.

Flow cytometric methods are widely used for analyzing cytokine release from immune cell populations. However, difficulties are frequently encountered in the analysis of flow cytometric data from intracellular cytokine staining because the cytokine positive and cytokine negative histograms overlap considerably. This study compares models containing Gaussian, Giddings, Haarhoff-van der Linde (HVL) and Weibull distributions for fitting flow cytometric intracellular cytokine histogram peaks. The results show that flow cytometric data for the Th1 cytokines, interferon-gamma and interleukin-2, in peripheral blood are well described by a model consisting of the sum of two log-normal distributions but the other distributions tested also showed satisfactory fits. The model-based approach may potentially eliminate the need to use markers derived from isotype control staining. The results obtained using peak fitting were also compared to the widely practised 99% division line or marker method. The percent positive calculated using the 99% division line or marker methods correlates poorly with the percent area under the cytokine-positive peak. However, when the model is used to calculate a cytokine-positive percentage analogous to that determined by the 99% division line or marker method, the two methods correlate well.

Flow Cytometry↗