title: Automated multichannel fluorescent whole slide imaging and its application for cytometry.
abstract: Slide-based image cytometry (SBC) has several advantages over flow cytometry but it is not widely used because of its low throughput, complicated workflow, and high price. Fully automated microscopes became affordable with the advent of whole slide imaging (WSI) and they can be transformed into a cytometer. A MIRAX MIDI automated whole slide imager was used with metal-halide and light emitting diode (LED)-based fluorescent illumination, filter block changer, and a cooled monochrome charge coupled device camera. The MIRAX control software was further developed for fluorescent sample detection, autofocusing, multichannel digitization, and signal correction due to nonuniform illumination. Fluorescent calibration beads were used to verify the linearity of the system. The HistoQuant software package of the MIRAX viewer was used for image segmentation and quantitative analysis. The data was displayed by the histogram, scatter plot, and gallery functions of the same program. Fluorescent samples can be reliably detected, focused, and scanned. The measured integrated fluorescence showed linearity with exposure time and staining intensity. Automated fluorescent WSI with stable LED illumination and high-quality homogeneous fluorescent slides can be used conveniently for SBC.
authors.affiliation: 3DHISTECH, Ltd., Konkoly-Thege Miklós út 29-33, Building 18, Budapest H-1121, Hungary. viktor.varga@3dhistech.com
authors.forename: Viktor Sebestyén
authors.initials: VS
authors.lastname: Varga
authors.forename: Levente
authors.initials: L
authors.lastname: Ficsor
authors.forename: Viktor
authors.initials: V
authors.lastname: Kamarás
authors.forename: Viktor
authors.initials: V
authors.lastname: Jónás
authors.forename: Tibor
authors.initials: T
authors.lastname: Virág
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Tulassay
authors.forename: Béla
authors.initials: B
authors.lastname: Molnár
mesh_terms: D001331:Automation / Q000295:instrumentation*; D005453:Fluorescence; D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation*; D008863:Microspheres; D013997:Time Factors