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PubMed2005

Confocal microscopy used as the definitive, early diagnostic method in Chandler syndrome.

PURPOSE: To report the early, rapid diagnosis of the Chandler variant of the iridocorneal endothelial (ICE) syndrome using confocal light microscopy. METHODS: A 62-year-old man with a long history of unilateral glaucoma reported progressively blurred vision in his right eye. Examination of both eyes included visual acuity, slit-lamp examination, pneumotonometry, visual field, gonioscopy, and confocal microscopy. RESULTS: On examination, visual acuity was 20/80 and 20/20 and the IOPs were 26 and 12. The anterior segment OD revealed 1+ inferior and axial corneal edema, while the OS was normal to biomicroscopy and posterior segment. Chandler syndrome or Fuchs endothelial dystrophy was suspected. In the affected eye, confocal light microscopy clearly showed an "epithelium-like" transformation of the corneal endothelium with irregularly shaped cells and hyperreflective nuclei, establishing the diagnosis of Chandler syndrome. CONCLUSIONS: In the presence of corneal edema or haze, corneal endothelium can be clearly visualized by confocal microscopy. "Epithelium-like" endothelial cells with highly reflective nuclei characteristic of Chandler syndrome were easily identified by confocal light microscopy to establish the diagnosis, despite the presence of corneal edema. Thus, confocal microscopy is a sensitive tool for the rapid, early diagnosis of ICE syndrome and may help distinguish among its variants.

Corneal Diseases↗
PubMed2004

In vivo confocal microscopy: increased conjunctival or episcleral leukocyte adhesion in patients who wear contact lenses with lower oxygen permeability (Dk) values.

PURPOSE: Contact lens wear is known to threaten the health of the ocular surface. In vivo confocal microscopy (IVCM) to visualize leukocyte rolling and extravasation in inflammation was recently described. We tested the hypothesis that contact lens wear is associated with measurable inflammation in superficial vessels. METHODS: Leukocyte rolling and sticking (hallmarks of the inflammatory process) were recorded by IVCM. IVCM was performed on conjunctival or episcleral blood vessels bilaterally on 55 contact lens wearers (15 male, 40 female) and 22 non-contact lens wearers (8 male, 14 female). Data were analyzed in 2 ways. Considering each vessel as an independent variable resulted in 132 analyzable vessel segments (13 daily disposable contact lenses, 67 traditional contact lenses, 14 rigid gas-permeable lenses, and 38 controls). Considering each subject as an independent variable resulted in analyzable data for 47 subjects (5 daily disposable contact lens wearers, 22 traditional contact lens wearers, 5 rigid contact lens wearers, and 15 control patients). Free-flowing, sticking, and rolling cells were counted in the vessels. Multiple parameters including mean flow velocity, shear rate, rolling cells/mm/min, and sticking cells/mm were calculated. RESULTS: We found no significant difference in leukocyte adhesion between control patients and patients wearing daily disposable, traditional disposable, or rigid gas-permeable lenses in both types of statistical analyses. However, the data regarding vessel segments as an independent variable show that there were more rolling cells in patients who wore contact lenses with oxygen permeability values (Dk) less than 10 as compared to those who wore contact lenses with oxygen permeability values greater than 16 (P < 0.01) or compared to controls (P < 0.01). CONCLUSION: IVCM is a novel, powerful technique to recognize a critical but subclinical component of inflammation. Although our data indicate that contact lens wear does not markedly increase rolling and sticking of leukocytes in conjunctival or episcleral vessels, there may be subclinical inflammation in association with lenses with the lowest oxygen permeability.

Adult↗
PubMed1996

Subcellular properties of triggered Ca2+ waves in isolated citrate-loaded guinea-pig atrial myocytes characterized by ratiometric confocal microscopy.

1. Spatiotemporal aspects of subcellular Ca2+ signalling were studied in cultured adult guinea-pig atrial myocytes. A mixture of the Ca2+ indicators fluo-3 and Fura Red in combination with laser-scanning confocal microscopy was used for [Ca2+]i measurements while membrane currents were recorded simultaneously. 2. In citrate-loaded atrial myocytes not every Ca2+ current (ICa) could trigger Ca2+ release from the sarcoplasmic reticulum (SR). Two types of Ca2+ signals could be observed: Ca2+ transients resulting from (i) Ca2+ influx alone and (ii) additional Ca2+ release. 3. Ca2+ release elicited by voltage steps of 100-150 ms duration was either apparently homogeneous or propagated as Ca2+ waves through the entire cell. With brief ICa (50-75 ms), Ca2+ waves with limited subcellular propagation were observed frequently. These waves always originated from either end of the myocyte. 4. The time course of changes in Na(+)-Ca2+ exchange current (INaCa) depended on the subcellular properties of the underlying Ca2+ transient and on the particular cell geometry. Apparently homogeneous Ca2+ release was accompanied by an inward change of INaCa the onset phase of which was fused with ICa. Changes in INaCa caused by a Ca2+ wave propagating through the entire cell showed a W shape, which could be attributed to differences of the fractional surface-to-volume ratio in different cell segments during propagation of the Ca2+ wavefront. Those waves with limited spreading only activated a small component of INaCa. 5. The different subcellular patterns of Ca2+ release signals can be explained by spatial inhomogeneities in the positive feedback of the SR. This depends on the local SR Ca2+ loading state under the control of the local Ca2+ influx during activation of ICa. Due to the higher surface-to-volume ratio at the two ends of the myocyte, SR loading and therefore the positive feedback in Ca(2+)-induced Ca2+ release may be higher at the ends, locations where Ca2+ waves are preferentially triggered. 6. We conclude that the individual cell geometry may be an important determinant of subcellular Ca2+ signalling not only in cardiac muscle cells but presumably also in other types of cells that depend on Ca2+ signalling. In addition, the cell geometry in combination with varying subcellular Ca2+ release patterns can greatly affect the time course of Ca(2+)-activated membrane currents.

Animals↗
PubMed2006

[Detection of cyclin E in lymph node metastases of laryngeal squamous cell carcinomas by laser scanning confocal microscopy].

OBJECTIVE: To study the expression of Cyclin E proteins in carcinogenesis and development of the metastasis of laryngeal squamous cell carcinomas (LSCC). METHOD: The expression of Cyclin E protein were detected by laser scanning confocal microscopy (LSCM) and multiparameter flow cytometry (FCM) after immunofluorescence staining method in twenty-one surgically resected primary LSCC (7 LSCC with cervical lymph node metastasis, 14 LSCC without cervical lymph node metastasis). RESULT: The fluorescence intensity was 78.12 +/- 6.54 in LSCC with cervical lymph node metastasis and 74.93 +/- 8. 82 in LSCC without cervical lymph node metastasis (P > 0. 05); The fluorescence intensity in cervical lymph node metastasis was 93.65 +/- 7.48, it's more higher than that in cervical lymph node negative metastasis (P < 0.01) and both LSCC with cervical lymph node metastasis and without cervical lymph node metastasis (P < 0.05). CONCLUSION: It is suggested that Cyclin E proteins may play an important role in the carcinogenesis and development of the lymph node metastases of LSCC. Detection of Cyclin E maybe becomes an indication to evaluating the lymph node metastases of LSCC.

Carcinoma, Squamous Cell↗
PubMed1997

[Platelet calcium oscillation by laser scanning confocal microscopy].

OBJECTIVE: To observe platelet calcium oscillation and the influence of extracellular Ca2+ on it. METHODS: We observed a single platelet intracellular Ca++ change and the influence of extracellular Ca2+ on it by laser sanning confocal microscopy when various agonists of different concentrations stimulated platelets. RESULTS: The other agonists except thrombin induced platelet calcium oscillation. With increasing agonis concentrations, the frequence of calcium oscillation enhanced and calcium oscillation sustained longer. In the absence of Ca++, the amplitude of ADP-induced calcium oscillation was smaller without frequence variations, and 5-TH induced calcium oscillation was inhibited. CONCLUSIONS: Platelet calcium oscillation increased from fluctuation either in the entry of external calcium or the release from internal stores.

Adenosine Diphosphate↗
PubMed1992

Confocal microscopy of fertilization-induced calcium dynamics in sea urchin eggs.

Although confocal microscopy has typically been utilized in studies of fixed specimens, its potential for exploring dynamic processes in living cells is rapidly being realized. In this report, confocal laser scanning microscopy is used to analyze the calcium wave that occurs following fertilization in living sea urchin eggs microinjected with the calcium-sensitive fluorescent probes fluo-3 or calcium green. Time-lapse recordings of optical sections depicting calcium dynamics within the eggs are also subjected to volumetric reconstructions. Such analyses indicate that (1) cytoplasmic free calcium levels become elevated throughout the fertilized egg, (2) fertilization also causes the egg nucleus to undergo a transient increase in free calcium, and (3) normal cleavage can be obtained following time-lapse imaging of the calcium waves.

Aniline Compounds↗
PubMed1998

Confocal microscopy of the mitochondrial permeability transition in necrotic cell killing, apoptosis and autophagy.

Onset of the cyclosporin-A-sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca2+ ionophore toxicity and tumor necrosis factor alpha (TNF alpha) induced apoptosis, and may contribute to Reye's-related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca2+ and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF alpha-induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death.

Animals↗
PubMed2004

Evaluation of atlas selection strategies for atlas-based image segmentation with application to confocal microscopy images of bee brains.

This paper evaluates strategies for atlas selection in atlas-based segmentation of three-dimensional biomedical images. Segmentation by intensity-based nonrigid registration to atlas images is applied to confocal microscopy images acquired from the brains of 20 bees. This paper evaluates and compares four different approaches for atlas image selection: registration to an individual atlas image (IND), registration to an average-shape atlas image (AVG), registration to the most similar image from a database of individual atlas images (SIM), and registration to all images from a database of individual atlas images with subsequent multi-classifier decision fusion (MUL). The MUL strategy is a novel application of multi-classifier techniques, which are common in pattern recognition, to atlas-based segmentation. For each atlas selection strategy, the segmentation performance of the algorithm was quantified by the similarity index (SI) between the automatic segmentation result and a manually generated gold standard. The best segmentation accuracy was achieved using the MUL paradigm, which resulted in a mean similarity index value between manual and automatic segmentation of 0.86 (AVG, 0.84; SIM, 0.82; IND, 0.81). The superiority of the MUL strategy over the other three methods is statistically significant (two-sided paired t test, P < 0.001). Both the MUL and AVG strategies performed better than the best possible SIM and IND strategies with optimal a posteriori atlas selection (mean similarity index for optimal SIM, 0.83; for optimal IND, 0.81). Our findings show that atlas selection is an important issue in atlas-based segmentation and that, in particular, multi-classifier techniques can substantially increase the segmentation accuracy.

Algorithms↗
PubMed1996

Ca2+ signaling in endothelial cells stimulated by bradykinin: Ca2+ measurement in the mitochondria and the cytosol by confocal microscopy.

In this study we have monitored the change of intracellular Ca2+ concentrations in the cytosol ([Ca2+]c) and the mitochondria ([Ca2+]m) of single bovine endothelial cells following treatment with bradykinin (BK). Using laser scanning confocal microscopy, we have found that the Ca2+ indicator, Fluo-3, is compartmentalized in the mitochondria of endothelial cells loaded with Fluo-3/AM. After BK stimulation, the pattern of Ca2+ increase in the cytosol is different from that in the mitochondria. The amplitude of the Ca2+ rise in the mitochondria is higher than that in the cytosol. Further analysis using rapid scanning measurements indicates that the [Ca2+]c increase is very fast after BK addition and reaches a maxima level within 400 ms. In contrast, the [Ca2+]m increase appears to be biphasic with an initial rapid increase (concomitant with the [Ca2+]c increase) followed by a slower [Ca2+]m increase before reaching a maximal level (within 5 s of BK treatment). The differential Ca2+ signaling pattern between the cytosol and the mitochondria suggests that the intracellular Ca2+ concentrations needed to regulate various Ca(2+)-dependent enzymes located in these two compartments are different during BK-induced endothelial cell activation.

Aniline Compounds↗
PubMed1998

Confocal microscopy reveals persisting stromal changes after myopic photorefractive keratectomy in zero haze corneas.

AIMS: Micromorphological examination of the central cornea in myopic patients 8-43 months after excimer laser photorefractive keratectomy (PRK), using the slit scanning confocal microscope. METHODS: Patients were selected from a larger cohort of individuals on the basis of full corneal clarity (haze grading 0 to +1; mean 0.3) and their willingness to participate in the study. 15 eyes of 10 patients with myopic PRK (-4 to -11 D; mean 6.7) and an uneventful postoperative interval of 8-43 months (mean 26) were examined. Contact lenses had been worn by eight of the 10 patients for 4-11 years (mean 6.7) before surgery. Controls included the five untreated fellow eyes of PRK patients, 10 healthy, age matched volunteers without a history of ocular inflammation or contact lens wear, and 20 patients who had worn rigid gas permeable (n = 10) or soft contact lenses (n = 10) for 2-11 years. Subjects were examined with a real time flying slit, scanning confocal microscope using x25 and x50 objectives. RESULTS: In PRK treated patients and contact lens wearers, basal layer epithelial cells sporadically displayed enhanced reflectivity. The subepithelial nerve plexus was observed in all individuals, but was usually less well contrasted in the PRK group, owing to the presence of a very discrete layer of subepithelial scar tissue, which patchily enhanced background reflectivity. Within all layers of the stroma, two distinct types of abnormal reflective bodies were observed in all PRK treated eyes, but in none of the controls. One had the appearance of long (> = 50 microns), slender (2-8 microns in diameter) dimly reflective rods, which sometimes contained bright, punctate, crystal-like inclusions, arranged linearly and at irregular intervals. The other was shorter (< 25 microns), more slender in form (< 1 micron in diameter), and highly reflective; these so called needles were composed of crystal-like granules in linear array, with an individual appearance similar to the bright punctate inclusions seen in rods, but densely packed. Both of these unusual structures were confined, laterally, to the ablated area, but were otherwise distributed throughout all stromal layers, with a clear predominance in the anterior ones. These rods and needles were observed in all PRK treated corneas, irrespective of previous contact lens wear. On the basis of qualitative inspection, the incidence of rods and needles did not appear to correlate with either the volume of tissue ablated or the length of the postoperative interval. In contact lens wearing controls, highly reflective granules, reminiscent of those from which the needles were composed, were found scattered as isolated entities throughout the entire depth and lateral extent of the corneal stroma, but rods and needles were never encountered. The corneal endothelium exhibited no obvious abnormalities. CONCLUSION: Confocal microscopy 8-43 months after PRK revealed belated changes in the corneal stroma. These were manifested as two distinct types of abnormal reflective bodies, which had persisted beyond the stage when acute wound healing would have been expected to be complete. The clinical significance of these findings in the context of contrast visual acuity and long term status of the cornea is, as yet, unknown.

Adult↗
PubMed2006

Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 2. Altered vacuolar volume regulation in drug resistant malaria.

In the previous paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12400-12410], we reported on a customized Nipkow spinning disk confocal microscopy (SDCM) system and its initial application to DIC imaging of hemozoin within live, synchronized, intraerythrocytic Plasmodium falciparum malarial parasites. In this paper, we probe the biogenesis as well as the volume and pH regulation of the parasite digestive vacuole (DV), using the fluorescence imaging capabilities of the system. Several previous reports have suggested that mutant PfCRT protein, which causes chloroquine resistance (CQR) in P. falciparum, also causes increased acidification of the DV. Since pH and volume regulation are often linked, we wondered whether DV volume differences might be associated with CQR. Using fast acquisition of SDCM z stacks for synchronized parasites with OGd internalized into the DV, followed by iterative deconvolution using experimental point spread functions, we quantify the volume of the DV for live, intraerythrocytic HB3 (CQS), Dd2 (CQR via drug selection), GCO3 (CQS), and GCO3/C3(Dd2) (CQR via transfection with mutant pfcrt) malarial parasites as they develop within the human red blood cell. We find that relative to both CQS strains, both CQR strains show significantly increased DV volume as the organelle forms upon entry into the trophozoite stage of development and that this persists until the trophozoite-schizont boundary. A more acidic DV pH is found for CQR parasites as soon as the organelle forms and persists throughout the trophozoite stage. We probe DV volume and pH changes upon ATP depletion, hypo- and hypertonic shock, and rapid withdrawal of perfusate chloride. Taken together, these data suggest that the PfCRT mutations that cause CQR also lead to altered DV volume regulation.

Animals↗
PubMed1996

Improved resolution of fibronectin mRNA expression in the inner ear using laser scanning confocal microscopy.

We describe a modified in situ hybridization protocol for localizing and quantifying fibronectin gene expression at the cellular level in paraffin sections of rat temporal bone. When combined with a novel analytical approach using laser scanning confocal microscopy (LSCM), this protocol significantly improved the resolution, sensitivity, and specificity of existing procedures for evaluating fibronectin synthesis in developing inner ear. For simultaneous viewing of cochlear anatomy and the autoradiographic signal, transmitted light images of the cochlea were collected separately from LSCM reflected light images of the autoradiographic silver grains and then the two images were electronically merged. Within the first 2 microns below the surface of the emulsion, silver grains were clustered specifically over hybridized cells. In contrast, nonspecific silver grain development (i.e., background noise) was confined primarily to the lower 5 microns of the emulsion adjacent to the tissue section. Limiting the volume of the emulsion examined in the LSCM analysis, i.e., restricting the range of optical sectioning to the first 2 micron below the surface of the emulsion, effectively minimized nonspecific background noise and maximized the specificity of the hybridization signal. The improvements offered by the described methodological approaches are equally appropriate for non-calcified tissues.

Animals↗
PubMed1999

Measurements of vascular remodeling by confocal microscopy.

The introduction of myographs was crucial for the study of structure and function of resistance arteries. The ability to support and maintain small blood vessels paved the way for true microscopic studies of the vascular cells. However, even after decades of study, we still do not know very much about the "normal" arrangement of smooth muscle cells in the vascular wall and how their distribution affects function. It was clearly time for the next technological step forward. We have shown here how the combination of myography and confocal microscopy creates a platform for the study of vascular structure at the cellular level and in 3D. In addition, the possibility of using live myograph-mounted vessels in combination with LSCM opens a new field of research to assess vascular remodeling from a physiological point of view and to study vascular function at a level not achieved by any other method at present. Now that the hardware is in place it is time to concentrate on the software and improve the methods of analysis. We have used 2D analysis of 3D data sets to describe differences in vascular structure and, at the same time, developed methods to semiautomate the process. The success of the 3D methods will ultimately depend on the reliability and accuracy of the analysis routines. There are still problems to overcome en route to finding a complete solution. However, we believe that the search for a robust fully (or semi-) automated method of 3D analysis will be more than worthwhile. We have defined vascular remodeling to include any changes in cellular arrangement or morphology. However, on a more subtle level, changes in receptors, enzymes, and proteins leading to altered functionality could also be regarded as remodeling. In that respect it may be interesting to map the distribution of the many receptors, channels, and proteins that regulate vascular growth, death, and function. Currently, there is a growing list of fluorescent ligands and antibodies that can be used in conjunction with confocal microscopy. It is possible that multiple stains could be used and imaged at different wavelengths with a view to constructing full 3D models of various structures and their colocalization. It is our belief that the confocal approach will prove to be a major tool in unraveling the complexities of cell-cell interactions and arrangements and will allow a better understanding of the process of vascular remodeling and function.

Animals↗
PubMed1996

Three-dimensional confocal microscopy of the lens.

A confocal microscopic technique is described to obtain optical sections from the in situ ocular lens of the rabbit and mouse. Optical sections were obtained with blue light through the full thickness of the lens epithelium, and of the anterior lenticular fibers. An image showing the structure of an anterior suture was obtained from the murine lens. A three-dimensional volume visualization of lenticular fibers from the anterior region of the rabbit lens was reconstructed from a stack of optical sections. These in situ microscopic observations of unstained, unfixed specimens correspond to previous studies using electron microscopy.

Animals↗
PubMed2001

Guard cell volume and pressure measured concurrently by confocal microscopy and the cell pressure probe.

Guard cell turgor pressures in epidermal peels of broad bean (Vicia faba) were measured and controlled with a pressure probe. At the same time, images of the guard cell were acquired using confocal microscopy. To obtain a clear image of guard cell volume, a fluorescent dye that labels the plasma membrane was added to the solution bathing the epidermal peel. At each pressure, 17 to 20 optical sections (each 2 microm thick) were acquired. Out-of-focus light in these images was removed using blind deconvolution, and volume was estimated using direct linear integration. As pressure was increased from as low as 0.3 MPa to as high as 5.0 MPa, guard cell volume increased in a saturating fashion. The elastic modulus was calculated from these data and was found to range from approximately 2 to 40 MPa. The data allow inference of guard cell osmotic content from stomatal aperture and facilitate accurate mechanistic modeling of epidermal water relations and stomatal functioning.

Fabaceae↗
PubMed1999

Localization of beta-endorphin in tetrahymena by confocal microscopy. Induction of the prolonged production of the hormone by hormonal imprinting.

The unicellular Tetrahymena has hormone receptors and hormones which are characteristic of higher vertebrates, as well as similar signal transduction pathways. In the present experiments, immunocytochemistry and confocal microscopy were used to study the presence and localization of beta-endorphin in Tetrahymena pyriformis GL. Endorphin (or endorphin-like material) was localized in the cortical structures, oral field, cilia and nuclear envelope. One-hour treatment with beta-endorphin ('hormonal imprinting') increased the presence of immunocytochemically demonstrable endorphin immediately and after 24 h, and was especially strong after 96 h of treatment. Simultaneous treatment with naloxone, an opioid antagonist, did not inhibit endorphin effect, but had an additive effect on endorphin production. Naloxone alone induced a very intensive accumulation of endorphin 96 h after treatment. The results support the possibility of a hormone production being induced by the imprinting procedure, but the imprinter-like effect of naloxone also points to the importance in this case of non-discriminatory receptors also being involved in the process.

Animals↗
PubMed1996

Multiple pathways of organic anion secretion in renal proximal tubule revealed by confocal microscopy.

Previous studies with p-aminohippurate (PAH) and fluorescein (FL) have shown that cellular uptake and tubular secretion of organic anions is driven by indirect coupling to sodium. Here we used killifish proximal tubules and laser-scanning confocal microscopy to study the transport of a larger organic anion, fluorescein-methotrexate (FL-MTX, mol mass 923 Da). When tubules were incubated in medium containing 2 microM FL-MTX, dye accumulated in both cells and tubular lumens. At steady state, luminal fluorescence was 4-5 times higher than cellular fluorescence. Ouabain (0.1 mM) did not affect cellular or luminal fluorescence, and replacement of medium sodium by N-methylglucamine had only a modest effect; preincubation with glutarate had no effect. KCN did not affect cellular uptake but abolished secretion into the lumen. Uptake and secretion of FL-MTX were inhibited by micromolar concentrations of other organic anions (MTX, folate, probenecid, bromocresol green, bromosulfophthalein), but 1 mM PAH had a relatively small effect. FL-MTX secretion into the lumen was inhibited by leukotriene C4, cyclosporine A, and verapamil, none of which affected FL transport. Thus a substantial component of FL-MTX secretion is Na independent and ouabain insensitive. Both the basolateral and luminal steps in the Na-independent pathway differ from those usually associated with FL and PAH secretion.

Animals↗
PubMed2001

Evaluation of confocal microscopy system performance.

BACKGROUND: The confocal laser scanning microscope (CLSM) has been used by scientists to visualize three-dimensional (3D) biological samples. Although this system involves lasers, electronics, optics, and microscopes, there are few published tests that can be used to assess the performance of this equipment. Usually the CLSM is assessed by subjectively evaluating a biological/histological test slide for image quality. Although there is a use for the test slide, there are many other components in the CLSM that need to be assessed. It would be useful if tests existed that produced reference values for machine performance. The aim of this research was to develop quality assurance tests to ensure that the CLSM was stable while delivering reproducible intensity measurements with excellent image quality. METHODS: Our ultimate research objective was to quantify fluorescence using a CLSM. To achieve this goal, it is essential that the CLSM be stable while delivering known parameters of performance. Using Leica TCS-SP1 and TCS-4D systems, a number of tests have been devised to evaluate equipment performance. Tests measuring dichroic reflectivity, field illumination, lens performance, laser power output, spectral registration, axial resolution, laser stability, photomultiplier tube (PMT) reliability, and system noise were either incorporated from the literature or derived in our laboratory to measure performance. These tests are also applicable to other manufacturer's systems with minor modifications. RESULTS: A preliminary report from our laboratory has addressed a number of the QA issues necessary to achieve CLSM performance. This report extends our initial work on the evaluation of CLSM system performance. Tests that were described previously have been modified and new tests involved in laser stability and sensitivity are described. The QA tests on the CLSM measured laser power, PMT function, dichroic reflection, spectral registration, axial registration, system noise and sensitivity, lens performance, and laser stability. Laser power stability varied between 3% and 30% due to various factors, which may include incompatibility of the fiber-optic polarization with laser polarization, thermal instability of the acoustical optical transmission filter (AOTF), and laser noise. The sensitivity of the system was measured using a 10-microm Spherotech bead and the PMTs were assessed with the CV concept (image noise). The maximum sensitivity obtainable on our TCS-SP1 system measured on the 10-microm Spherotech beads was approximately 4% for 488 nm, 2.5% for 568 nm, 20% for 647 nm, and 19% for 365 nm laser light. The values serve as a comparison to test machine sensitivity from the same or different manufacturers. CONCLUSIONS: QA tests are described on the CLSM to assess performance and ensure that reproducing data are obtained. It is suggested strongly that these tests be used in place of a biological/histological sample to evaluate system performance. The tests are more specific and can recognize instrument functionality and problems better than a biological/histological sample. Utilization of this testing approach will eliminate the subjective assessment of the CLSM and may allow the data from different machines to be compared. These tests are essential if one is interested in making intensity measurements on experimental samples as well as obtaining the best signal detection and image resolution from a CLSM. Published 2001 Wiley-Liss, Inc.

Electric Power Supplies↗