title: Simplified confocal microscope for counting particles at low concentrations.
abstract: We describe a compact scanning confocal fluorescence microscope capable of detecting particles concentrations less than 100 particles∕ml in ~15 min. The system mechanically moves a cuvette containing ~3 ml of sample. A relatively large confocal volume is observed within the cuvette using a 1 mm pinhole in front of a detection PMT. Due to the motion of the sample, particles traverse the confocal volume quickly, and analysis by pattern recognition qualifies spikes in the emission intensity data and counts them as events. We show linearity of detection as a function of concentration and also characterize statistical behavior of the instrument. We calculate a detection sensitivity of the system using 3 μm fluorescent microspheres to be 5 particles/ml. Furthermore, to demonstrate biological application, we performed a dilution series to quantify stained E. coli and yeast cells. We counted E. coli cells at a concentration as low as 30 cells∕ml in 10 min/sample.
authors.affiliation: Diagnostics Research, Abbott Diagnostics Division, Abbott Park, Illinois 60064, USA.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Skinner
authors.forename: Kerry M
authors.initials: KM
authors.lastname: Swift
authors.forename: Qiaoqiao
authors.initials: Q
authors.lastname: Ruan
authors.forename: Sergio
authors.initials: S
authors.lastname: Perfetto
authors.forename: Enrico
authors.initials: E
authors.lastname: Gratton
authors.forename: Sergey Y
authors.initials: SY
authors.lastname: Tetin
mesh_terms: D004926:Escherichia coli / Q000166:cytology; D005434:Flow Cytometry; D018613:Microscopy, Confocal / Q000295:instrumentation*; D008863:Microspheres; D012441:Saccharomyces cerevisiae / Q000166:cytology; D013997:Time Factors
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