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EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: APPLICATIONS FOR IMAGING MORPHOLOGY AND DEATH IN EMBRYOS AND REPRODUCTIVE TISSUE/ORGANS
Citation:
Zucker, R M. EVALUATION OF CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: APPLICATIONS FOR IMAGING MORPHOLOGY AND DEATH IN EMBRYOS AND REPRODUCTIVE TISSUE/ORGANS. Presented at Microscopy & Microanalysis, Long Beach, CA, August 04 - 09, 2001.
Description:
The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. It is remarkable that procedures to test the performance of these machines are not done routinely by most investigators and thus many of the machines in the field are working at levels of sub optimal performance. When machines are checked, it is usually a subjective assessment of performance made by primarily evaluating the system using a specific test slide provided by each user's laboratory. We have devised test methods on the Leica TCS-SP and TCS-4D systems to ensure our machine was working correctly and delivering the correct performance. Tests measuring field illumination, lens clarity, laser power and stability, dichroic functionality, spectral registration, axial resolution, overall machine stability, and system noise were derived or perfected in order to determine the CLSM system performance. It is anticipated this type of data will help in setting performance standards for proper functioning of confocal microscopes and eliminate the subjectivity in evaluating the CLSM. These tests will also serve as a guide for other investigators to insure that their machines are working correctly and providing data that is accurate with the necessary resolution, sensitivity and precision.
The coefficient of variation (CV) is defined as the standard deviation ( ) of the fluorescent intensity of a population of beads or pixels expressed as a proportion or percentage of the mean ( ) intensity (CV= / ). The field of flow cytometry has historically used the CV of a population of bead intensities to determine if the flow cytometer is aligned correctly and performing properly. In confocal microscopy 100,000 pixels are measured from within one homogeneous Spherotech 10 micron bead which is analogous to measuring 10,000 intensity beads using a flow cytometer. In both cases a distribution of intensities, yielding a CV of intensities is measured.
This bead CV analysis provides a systematic method of reducing noise and increasing image clarity. The CV of pixel intensities is dependent on a number of machine variables that include frame averaging, photo multiplier tube (PMT) voltage, and laser stability and laser power. The relationship between averaging, laser power, pinhole and PMT is explained in statistical terms to provide the rational for image quality on a confocal microscope. If image quality cannot be achieved by having a low PMT setting, then frame averaging will be necessary to reduce the CV and improve image quality. This data may be used not only to compare whether one's system is functioning correctly, but it may also be used to compare different confocal microscopy systems, lasers and components for total system performance and sensitivity. This type of data will be useful in the future to test the performance of many types of fluorescence optical systems. The ability to apply estimates of image noise (CV) may be a better way to assess the confocal system performance compared to the subjective histological/biological slide that is normally used.
We have previously demonstrated that cell death (apoptosis) can be visualized in growth day (GD) 8-9 whole embryos treated in vitro with hydroxurea or other toxicants. This work was has been extended to measure apoptosis in neonatal ovaries and GD 14 limbs. Millimeter pieces of ovarian tissue can be visualized in three dimensions to examine oocytes and follicles contained within the tissue. By combining LysoTracker Red (LT) stain with morphological criteria, attretic and regressing follicles were revealed in ovarian tissue. LT stained the dying granulosa cell layer in attretic follicles to a greater extent than in healthy antral follicles as measured by relative fluorescent intensity.
Our ultimate research objective was to try to quantify fluorescence from a CLSM. To achieve this goal it is essential to have a stable machine with known parameters. This visualization technique was also applied to GD 14 developing rat limbs treated with 5- flurouracil (5-FU). The cells contained in the proliferation zone were damaged and accumulated a greater amount of LT as measured by fluorescent intensity. Attempts to quantify the concentration of LT in 5-FU treated tissues and correlate that value to apoptosis were made. Due to many of the machine and sample handling variables that occur in this experiment the data obtained is naturally a controversial topic that will be discussed in terms of cell death in normal GD 10 rat embryos somites and GD 14 developing limbs treated in vivo with 5-FU.
In contrast to the LT technique in which depth of laser penetration is essential to observe morphological structures, a technique has been developed to observe the surface of a 3-D structure with a CLSM. By over staining the surface of an embryo with a reflecting dye (i.e. acridine orange) and not clearing the tissue, the confocal microscope can be used as a Pseudo scanning electron microscope for low power organisms or tissues. This has the advantage of using aldehyde or ethanol fixatives and not preparing the tissue for conventional SEM.