Scanning fluorescent microscopy is an alternative for quantitative fluorescent cell analysis

Abstract

Background Nasal polyps in patients with cystic fibrosis (CF) are believed to be phenotypically different than polyps affecting non‐CF patients. The objective of this study was to characterize the expression of inflammatory cytokines within nasal polyps from CF and non‐CF, aspirin‐tolerant patients. “Regulated on activation, normal T‐cell expressed and secreted” (RANTES) is a chemotactic cytokine involved in the recruitment and activation of eosinophils. Multiple molecular studies of non‐CF polyps have established that RANTES may play an important role in nasal polyposis. Our study suggests RANTES may be upregulated in CF polyps relative to non‐CF polyps. Methods Nasal polyps were prospectively obtained from CF and non‐CF, aspirin‐tolerant patients. The Quantibody™ Human Cytokine Array I from RayBiotech, Inc., was used to identify differences in cytokine expression between protein extracts of two polyp groups. Four CF polyp extracts and 4 non‐CF, aspirin‐tolerant polyp extracts were each incubated on identical antibody subarrays, each containing 20 human cytokines in quadruplicate. Western blot analysis confirmed expression of RANTES. Results The protein microarray suggests a greater than 4‐fold upregulation of RANTES in CF polyps relative to non‐CF polyps. Western blot analysis confirmed expression of RANTES in CF polyps. Conclusion Chemokines such as RANTES are responsible for the activation of inflammatory cells within the lamina propria of nasal polyps. We have demonstrated that RANTES may be an important cytokine associated with CF polyps. © 2011 ARS‐AAOA, LLC.

Quantitative histology by multicolor slide‐based cytometry

“…To this end the section could be mosaicked into detail images that then are analyzed by slide-based cytometry algorithms as in the LSC. This could be done by present instruments such as the scanning fluorescence microscope (6).…”

“…High-end flow cytometers equipped with two or more lasers permit assessment of nine (or even more) markers on each cell (2). Slide-based methods such as laser scanning cytometry (LSC) (3), laser scanning microscopy (LSM) (4,5), and scanning fluorescent microscopy (6) offer the opportunity to analyze fluorochrome-labeled specimens. These methods are applied mainly to single cells from blood, tissues, or cell cultures (7)(8)(9), and are widely used in research and clinical diagnosis.…”

Scanning fluorescent microscopy analysis is applicable for absolute and relative cell frequency determinations

“…In our study, we performed stoichiometric quantification of the fluorescence, which was based on the calibration procedure described in our previous work (20), using digital slide, compensation images, and standardization beads. Further results in this field have also been published by other groups (28).…”

“…Based on this digital slide, virtual microscopy software simulates the basic functions of the microscope (magnification and stage movement). This technique was modified in order to perform SFM and to support cytometric evaluation of fluorescent calibration beads (20). SFM technology proved to be linear and stoichiometric on fluorescent calibration beads using Hg arc lamp excitation inhomogeneity-compensated fluorescent images, and has shown its feasibility for cytometric measurement.…”

Automated multichannel fluorescent whole slide imaging and its application for cytometry

“…Since that time different systems were developed to push the boundaries of image cytometry. The slide-based systems include laser scanning cytometry (3,(12)(13)(14)(15), standard (16)(17)(18)(19), enhanced wide-field (6,(20)(21)(22)(23) and confocal fluorescent microscopes (4,5,24). FCM can also be extended with imaging capabilities (25)(26)(27).…”