Implementation of high quality and easy to use peltier cooled camera: A cost-conscious alternative for assessing isolated perfused mouse proximal tubule fluid absorption properties and apoptosis

Abstract

Introduction and aims: Fluorescein isothiocyanate (FITC) and 4’,6- diamidino-2-phenylindole (DAPI) are commonly used fluorophores to assess mouse proximal tubule fluid absorption properties and apoptosis, respectively. The aim of our study was to evaluate the performance of the SAC8.5 (SAC-imaging, Melbourne, Florida), a peltier-cooled black and white 8-bit CCD camera, with respect to fluorescence and transmission light imaging in living tissue as compared to a currently used photo multiplier (PMT) mounted on a Zeiss LSM 510 META laserscanning confocal microscope. Methods: FITC-inulin standard samples were dissolved in heated PBS and subsequently transferred to 1ml constant-bore capillary tubes (Drummond Scientific, Broomall, PA). Proximal tubules were isolated from the kidney through microdissection and were loaded with 300 nM DAPI (Molecular probes, Merelbeke, Belgium) for 5 minutes. Jurkat cells were fixated with 4% paraformaldehyde on poly L-cysteine coated coverslips and were loaded similarly. An equation describing the relationship between the fluorescence intensity and the FITC-concentration was obtained by fitting the experimental data using S-PLUS 2000 Professional Release 1 for Windows (Insightful, Seattle, WA, USA). All images were analyzed using Image J software (NIH, Bethesda, MD, USA). Results: Concerning quantitative FITC-inulin readings in 1 ml microcaps, the detected fluorescence signal was found to be linear with the FITC-inulin concentration (R2 =0.99, p<0.05). The SAC8.5 camera was able to detect FITC-inulin concentrations ranging from 0.1mg/ml to 2mg/ml. This means that a concentration as low as 5% of the maximum tested concentration could be measured. Besides this, the SAC8.5 camera was able to detect DAPI staining (excitation at 350 nm; emission at 460 nm) in isolated proximal tubules and Jurkat cells faster than the currently used PMT. In addition, the signal to noise ratio for the SAC8.5 camera was twice as large in comparison with the PMT. Furthermore, an interfacing with automated shutters to prevent rapid fluorophore bleaching was implemented with the user-friendly Astrovideo software (COOA, Portimão, Portugal). Conclusions:Rapid deterioration of the fluorescence signal due to bleaching and to degrading photochemical properties is one of the major drawbacks of pseudo-confocal detection of fluorescent stains. The currently implemented uncooled PMT detection requires about 15 sec for a 512 x 512 HxV image size. Even pixels that are not excited by the scanning mirrors in a confocal setup will bleach. By using a CCD or CMOS camera whole image spatial as well as temporal information can be collected much faster, so less deterioration will have taken place. In summary, we have demonstrated that the SAC8.5 camera is a high quality, easy to use and cost-conscious alternative for (quantitative) fluorescence measurements in living tissues.