Confocal laser scanning microscopy (CLSM) is a technique that is capable of generating serial sections of whole-mount tissue and then reassembling the computer acquired images as a virtual 3-dimensional structure. In many ways CLSM offers an alternative to traditional sectioning approaches. However, the imaging of whole-mount tissues presents technical limitations of its own. One of the major problems using CLSM to image whole organs and embryos is the penetration of laser light into the tissue.. High quality morphological images begin by optimizing the sample preparation technique (1-2). Additional factors include evaluating CLSM performance by optimizing the acquisition variables (i.e. objective lens, averaging, pinhole size, bleaching, PMT voltage, laser excitation source, and spectral registration) of the confocal microscope. (3-4) Confocal microscopy has been used by our laboratory to study cell death and morphology in embryos, ovaries, eyes, ears, and limbs (1-2). The technique has revealed structural morphology and visualized areas of cell death by the uptake of the LysoTracker dye into phagolysomes. LysoTracker Red (LT) is fixable by paraformaldehyde and concentrates in acidic compartments of cells. In whole tissues, this accumulation indicates regions of high lysosomal activity and phagocytosis. LT staining is an indicator of apoptotic cell death and correlates with other standard apoptotic assays (1-2). LT staining revealed cell death regions in mammalian limbs, neonatal ovaries, fetuses and embryos. The mammalian samples were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MeOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). The use of BABB matches the refractive index of the tissue to that of the suspending medium. BABB helps increase the penetration of laser light during CLSM by reducing the amount of light scattering artifacts and allows for the visualization of morphology in thick tissue. Following this treatment, the tissues were nearly transparent. This sample preparation procedure, combined with the optimization of CLSM instrument factors, allowed for the detection and visualization of apoptosis in fetal limbs and embryos which were approximately 500 microns thick.Spectroscopic imaging capacity has been incorporated into all manufacturers' confocal microscopes. The LT spectra had a maximum peak around 610nm while the fixative, glutaraldehyde (Glut), had a maximum peak around 450nm. Glut was added primarily to preserve the tissue morphology, but also provided molecules emitting in the green fluorescence range that helped to visualize the morphology of the tissue. The understanding of the spectra derived from the tissue was extremely useful in optimizing the staining protocol. We have continued to incrementally improve the tissue staining and sample preparation techniques to achieve better quality 3D images.

Flow and image cytometers can provide useful fluorescence data that can be used to quantify fluorescence. 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 CVs, peak channels and histogram 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 of the system. A low CV indicates a good alignment while a high CV indicates either an alignment problem or a fluidic problem. Increasing the 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 2 nd test monitors the cleanliness of the system and the amount of background light scatter. This test is related to the alignment of the system. Failure to obtain good values in these tests will compromise the systems ability to detect dim fluorescence from background and will affect the precision of the system. 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 sensitivity test will be used to evaluate the machine contamination and ability to measure 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 when using the machine. When the machine is not aligned correctly the data will be effected. 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-dimensional 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 microscopy to image whole organs and embryos is the penetration of laser light into tissues. Good images begins by optimizing the sample preparation technique (1-2). In addition, the confocal microscope acquisition variables need to 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 (3-6).

The confocal laser-scanning microscope (CLSM) has enormous potential in many biological fields. The reliability of the CLSM to obtain specific measurements and quantify fluorescence data is dependent on using a correctly aligned machine that contains a stable laser power. For many applications it is useful to know the CLSM system's performance prior to acquiring images so the necessary resolution, sensitivity and precision can be obtained in the experiment. Applications of deconvolution, FRET and quantification necessitate that the confocal is correctly configured and operating at the highest performance levels to achieve the desired results. One of the most common methods that many laboratories use to measure system performance involves the use of a histological slide to create a "pretty picture". Although this test evaluates many parameters that can influence a CLSM image in a crude manner (i.e. laser power, field illumination and lateral resolution), the interpretation of this histological image is very subjective making the range of what constitutes an acceptable image and performance extremely variable. In fact, many confocal microscopes can obtain "pretty pictures" even when they are operating at sub-optimal levels. It is impossible to compare the performance of similar machines if only an image is used as the reference standard. If a scientist wants to determine whether the CLSM is indeed working at appropriate performance levels, it is essential that these types of CLSM QA performance tests be applied on their system. This tutorial will illustrate a number of different tests that can be applied to a CLSM to determine its functionality and performance. These tests methods have been devised on the Leica TCS-SP1 confocal microscope systems to ensure that it is operating correctly. The CLSM tests measure the following: field illumination, lens functionality and lens clarity, spectral registration, total laser power, laser stability, dichroic reflectance, spectral registration of the beams, axial resolution, scanning stability, overall machine stability, galvanometer function colocalization, and system noise (1-3). It is anticipated that by applying these QA tests, different performance standards for the confocal microscopes will be determined, thus eliminating some of the current subjectivity that exists in evaluating CLSM performance. Hopefully, these tests will serve as guidelines for other investigators to assess both the performance of their machines and the quality of their data. These confocal tests have also been applied successfully to Zeiss 510 confocal system. It is anticipated that with minor modifications they may be adaptable to the other CLSM manufactures' machines.