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Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification
Citation:
ZUCKER, R. M., R. G. ELLIS-HUTCHINGS, AND J. M. ROGERS. Confocal Microscopy and Flow Cytometry System Performance: Assessment of QA Parameters that affect data Quanitification. Presented at Microscopy and Microanalysis 2008 Meeting, Albuquerque, NM, August 03 - 07, 2008.
Impact/Purpose:
Presentation @ Microscopy & Microanalysis 2008 Meeting
Description:
Flow and image cytometers can provide useful quantitative fluorescence data. We have devised QA tests to be used on both a flow cytometer and a confocal microscope to assure that the data is accurate, reproducible and precise. Flow Cytometry: We have provided two simple performance criteria to determine if a flow cytometer is aligned correctly and properly functioning. The system can be assessed for alignment and functionality by measuring the coefficient of variation (CV), peak channels and hisogram distributions, from uniform single intensity beads. The CV of the bead population is a measure of the alignment of the system and the overall functionality. A low CV indicates good alignment while high CV indicates either an alignment problem or a fluidic problem. Increasing he flow rate should increase the CV but the histogram distribution should stay relatively symmetrical. Deviations from a symmetrical pattern indicate a system that is not aligned optimally. The 2nd test monitors the cleanliness of the system and the amount of background light scatter. Failure to obtain good values in both of these tests will compromise the systems ability to detect dim fluorescence from background and will affect the precision of the measurement. It is important that some of these bead intensities are similar to the intensity of the samples measured. The CV test used to check flow cell blockage and alignment and the 2nd sensitivity test will be used to evaluate the machine contamination and ability to detect low level fluorescence. A machine that is set up correctly will pass the CV test and the sensitivity test. These values should be measured daily prior to using the machine. When the machine is not aligned correctly the sensitivity data will be affected. Confocal Microscopy: The emergence of confocal laser scanning microscopy (CLSM) as a technique capable of optically generating serial sections of whole-mount tissue and then reassembling the computer-stored images as a virtual 3-dimentional structure offers a viable alternative to traditional sectioning approaches. However, the imaging of such whole-mounts presents technical problems of its own. One of the major problems with using a confocal microscope to image whole organs and embryos is the penetration of laser light into tissues. By optimizing the sample preparation technique, better quality images can be derived. In addition, the confocal microscope acquisition variables must be optimized (i.e. objective lens, averaging, pinhole size, bleaching, PMT voltage, laser excitation source, and spectral registration).as it will affect the accuracy of the data. The CLSM has enormous potential in many biological fields. One of the goals in using a CLSM is to quantify fluorescence. The accuracy of these measurements demands that the system be in correct alignment with stable laser power and spectral restorations. This evaluation can not be made using a histological slide to create a “pretty picture” which appears to be the most common method to check the performance of a CLSM system. We have developed testing procedures to replace this subjective approach with objective measurements of field illumination, lens function, total laser power, laser stability, dichhroic reflectance, axial resolution, galvanometer scanning stability, overall machine stability, and system noise. We have developeda additional tests to measue spectral performance to serve as guidelines for investigators to assess both the performance of their instruments as well as the quality of their data. Recently we have applied this technique to observe glomeruli in kidneys. By staining with YoPro-1, we have been able to visualize and count glomeruli using Imaris software in thick kidney tissue (600 microns) derived from a postnatal day 22 rat. Currently we are evaluating the data from rats treated with chemicals or undernourished during pregnancy to establish what advantages this technique offers over the exhaustive stereology method, the current standard for glomeruli quantification. The understanding of the spectra derived from the tissue has been extremely useful in optimizing our staining protocols. We have continued to incrementally improve the tissue staining and preparation techniques to achieve better quality 3D images. The YoPro-1 spectra had a maximum peak around 525 nm while the kidney tubule background had increased fluorescence intensity at longer wavelengths. The spectra derived from these different confocal spectral machines all showed that YoPro-1 concentrated in glomeruli in the 500-530 range yielding a higher intensity and stained the background with a higher intensity in the 550-650 nm range. We have also evaluated various hardware parameters which affect our image acquisition. These include the type of objectives, numerical aperture of objectives, confocal microscope vendors, zoom factor, pinhole size, averaging, intensity compensation, z step size, PMT voltage and laser power. Quality assurance of the confocal microscope is necessary for this quantification to be successful and these factors will be discussed in the presentation. Confocal spectoadci equipment was evaluated using the MIDL lamp for accuracy. Spectroscopic imaging of kidney features was conducted and comparisons made using a PARISS spectrograph, Leica SP1, Nikon C1Si and a Zeiss Meta 510. We have continued to incrementally improve the YoPro staining of glomeruli tissue and sample preparations techniques to achieve better quality 3D images.