Coherent backscatter enhances reflection confocal microscopy

When thin optically transparent specimens are grown on reflective substrates, contrast in reflection confocal microscopy is markedly enhanced. This enhanced contrast allows for the visualization of the thin filopodia and organelles contained within the neuritic processes of PC12 cells in culture. The characteristics of this contrast enhancement suggest that it arises because of interference between light scattered from the specimen and coherently backscattered illumination reflected off the substrate. This technique provides a method for visualizing living cells or other similarly transparent objects on opaque substrates in a nondestructive manner.     

Intracellular fluorescent probe concentrations by confocal microscopy

A general method is described that takes advantage of the optical sectioning properties of a confocal microscope to enable measurement of both absolute and relative concentrations of fluorescent molecules inside cells. For compartments within cells that are substantially larger than the point spread function, the fluorescence intensity is simply proportional to the concentration of the fluorophore. For small compartments, the fluorescence intensity is diluted by contributions from regions outside the compartment. Corrections for this dilution can be estimated via calibrations that are based on the intensity distribution found in a computationally synthesized model for a cell or organelle that has been blurred by convolution with the microscope point spread function. The method is illustrated with four test cases: estimation of intracellular concentration of a fluorescent calcium indicator; estimation of the relative distribution between the neurite and soma of a neuronal cell of the InsP3 receptor on the endoplasmic reticulum; estimation of the distribution of the bradykinin receptor along the surface of a neuronal cell; and relative distribution of a potentiometric dye between the mitochondria and cytosol as a means of assaying mitochondrial membrane potential.     

In vivo confocal microscopy of Fuchs' endothelial dystrophy

PURPOSE: The purpose of this study is to analyze in vivo confocal microscopic findings of corneas with Fuchs' endothelial dystrophy. METHODS: Central corneas of 17 eyes of 11 patients aged 41-86 years were examined using in vivo scanning slit confocal microscopy after being diagnosed with Fuchs' endothelial dystrophy. The cellular structure of the corneas was analyzed morphologically and quantitatively and compared to control results from 22 healthy corneas. RESULTS: Bullae were detected in the basal epithelial layer of one eye. Eight of 17 eyes (47%) exhibited an abnormal Bowman's layer: diffuse bright reflection and absence of nerves. Eleven eyes (65%) exhibited abnormal anterior stroma: lacunae and diffuse increased light reflection due to edema. In 12 eyes (71%), lacunae or dark bands 5-20 microm wide against increased background reflection were noted in the posterior stroma. Descemet's membrane was thickened in all eyes. Dark bands were detected in six eyes (35%). Guttae (137-1,231/mm2) 20-40 microm in diameter were found in every endothelial cell layer. The mean endothelial cell count was 1,202 +/- 850 (cells/mm2 +/- SD; range, 0-2,735). There was a positive correlation between endothelial cell counts obtained by specular microscopy and those obtained by confocal microscopy (r = 0.95). CONCLUSION: In vivo confocal microscopic findings of Fuchs' endothelial dystrophy are described for the first time in a series of cases. Pathological changes in Fuchs' dystrophy were detected in all corneal layers, more frequently in the posterior layers. Endothelial cell counts obtained with confocal microscopy were statistically similar to those obtained with standard specular microscopy.     

Efficient Monte Carlo simulation of confocal microscopy in biological tissue

A variance-reduction technique is described that greatly improves the efficacy of Monte Carlo simulations of reflection-mode confocal microscopy in anisotropically scattering media. The efficiency gain is large enough that the performance of confocal microscopes probing as deep as 5 scattering lengths can be simulated with a desktop computer. We use the technique to simulate the response of a true confocal microscope probing biological tissue, a problem that has been impractical to undertake by using conventional Monte Carlo methods. Our most important finding is that operation of a confocal microscope in the true confocal mode enables much more effective rejection of undesired scattered light than operation in the partially coherent mode, but the maximum probing depths of microscopes operated in either mode are similar (2-3) scattering lengths) in practice because of sensitivity limitations.     

Applications of confocal laser scanning microscopy to dental bonding

Groups of photosensitive adult female blackheaded buntings were exposed to various ultrashort days photoperiodic regimes for 60 days in which a fixed 3 h photophase was coupled with dark phases in cycles of 22 to 32 hours duration. One group of buntings was kept in long days (15L:9D) as control. Significant increase in ovarian weight and circulating plasma estradiol concentration was marked in the cycles of 30 h (3L:27D), and 32 h (3L:29D) photoperiodic schedules as well as in control group (15L:9D), whereas there was no response in the cycles of 22 h (3L:19D) and 24 h (3L:21D). It seems that the response to ultrashort day cycles is due to a phase advance or delay in photosensitivity of the response system repeatedly shows coincidence of the external photophase (3 h) with the photoinducible phase of an endogenous circadian rhythm. Therefore, the present result appears to be consistent with Bünning hypothesis suggesting the involvement of an endogenous circadian component in the female blackheaded bunting.     

共焦激光扫描显微镜在冶金中的应用

共焦激光扫描显微镜（CLSM）是普通光学显微镜与激光、计算机相结合的产物,具有比普通光学显微镜更高的分辨率,并且可以实现对样品的分层扫描,进而进行动态原位观察。利用CLSM可以直接观察金属的晶体长大过程,高温相变过程以及凝固过程中第二相粒子的各种行为。本文综述了CLSM系统的组成和原理以及其在冶金领域的应用,如夹杂物的碰撞、聚集、长大,在钢渣界面的扩散过程及在凝固前沿的捕捉/推进行为,析出物的形核析出过程等。     

Quantitative pH imaging in cells using confocal fluorescence lifetime imaging microscopy

In guinea pigs, activity of glutathione peroxidase in most organs is markedly lower than in organs of other rodents despite comparable dietary intakes and tissue levels of selenium. To determine if metabolism of selenium with respect to other selenoproteins also differs in guinea pigs, we measured the effects of selenium intake on thyroid hormone metabolism. Weanling male Hartley Albino guinea pigs were fed a selenium-deficient Torula yeast-based diet, or the same diet supplemented with 0.5 mg selenium/kg diet as sodium selenate for 72 d. Growth was impaired in guinea pigs fed the unsupplemented diet. Activity of glutathione peroxidase was higher in tissues and plasma of supplemented guinea pigs than in selenium-deficient animals. However, it was still far lower than reported values for other rodent species. In selenium deficiency, activity of type 1 5'-iodothyronine deiodinase was 60% less in liver and 45% less in kidney. Concentration of thyroxine was 68% lower in kidney of selenium-deficient animals, and levels of 3,3',5-triiodothyronine in kidney and plasma were 44 and 31% lower, respectively. Thus, with the exception of thyroxine concentrations, thyroid hormone metabolism responds to selenium deficiency in guinea pigs as it does in rats, although the magnitude of that response is not as great.     

Quantitative three-dimensional confocal microscopy of synaptic structures in living brain tissue

In order to study changes in synaptic structure that accompany learning and memory, we have developed optical methods to visualize dendritic spines and presynaptic terminals in living, electrically monitored brain slices maintained in vitro. Focal microapplication of the fluorescent lipophilic dye DiI provides Golgi-like staining of small numbers of cells and processes that can be resolved clearly using confocal microscopy; viability of stained cells is established by exclusion of the fluorescent DNA-binding dye ethidium bromide. Serial optical sections are enhanced by deconvolution and other image processing methods. The resulting high-resolution images are combined in an automated procedure to generate three-dimensional reconstructions, in which submicron synaptic structures can be viewed and measured. These unbiased methods allow volume changes in individual, living synaptic structures to be assessed quantitatively over periods of hours or days in development or in response to stimulation, drug application, or other perturbations.     

Heterogeneous photobleaching in confocal microscopy caused by differences in refractive index and excitation mode

Changes in brain 5-HT turnover which have been associated with portal-systemic encephalopathy (PSE) in man were studied in rats with experimental PSE for intervals up to 15 weeks following the surgical construction of end-to-side portacaval shunts (PCS). These were compared to changes measured in portacaval transposed rats (PCT) which, show little hepatic dysfunction or cerebral abnormalities but, in common with the PCS rat, sustain total portal-systemic diversion. Thus any differences between these two groups were indicative of hepatic dysfunction and not the systemic diversion of portal blood. After 15 weeks, sustained increases were measured in brainstem and cerebral concentrations of the catabolite of 5-hydroxytryptamine (5-HT), 5-hydroxyindole acetic acid (5-HIAA), from 0.25+/-0.01 to 0.68+/-0.01*** microg g(-1) brain and from 0.18+/-0.01 to 0.31+/-0.03*** microg g(-1) brain respectively in PCS rats and were statistically greater to those measured in the brainstem and cerebrum of PCT and control rats. Sustained increases in cerebral concentrations alone of 5-hydroxytryptophan (5-HTP), the precursor of 5-HT, from 0.17+/-0.01 to 0.23+/-0.02 microg g(-1) brain were measured in PCS rats and were significantly*** greater than in PCT control rats after 15 weeks. Some early increases in 5-HTP were measured in PCS above control rats but these were not significant after 15 weeks. No sustained significant differences between the 3 groups were measured in 5-HT after 15 weeks. These data confirm previous evidence that the elevations in 5-HTP and 5-HIAA concentrations observed in experimental chronic liver failure and PSE are due to liver dysfunction and not portal-systemic diversion and may contribute additional information regarding the role of derangements in central 5-HT turnover as one of the causes of PSE. ***p<0.001, Newman-Keuls ANOVAR followed by Student's unpaired t-test for individual comparisons, (data shown are mean +/- SEM).     

Assessment of cornea viability by confocal laser scanning microscopy and MTT assay

PURPOSE: Determination of excised cornea viability is of interest for transplant-storage evaluation, but also for in vitro diffusion-study design and ocular-toxicity assessment. By using simultaneous vital staining by calcein AM (CAM) and ethidium homodimer-1 (EH-1), as "live" and "dead" probes, respectively, we developed a confocal laser scanning microscopy (CLSM) assay to determine epithelial and endothelial viability and estimate cornea thickness. METHODS: New Zealand White rabbit corneas were stored in phosphate-buffered saline (PBS) or Optisol at 4 degrees C or at room temperature. At various times, corneas were stained with an EH-1/CAM solution and observed, without further treatment, by CLSM. Storage effects on the cornea were also assessed by using an MTT assay. RESULTS: Stromal swelling, shedding of the upper epithelial layers, and severe endothelial damage were observed after 4 h in PBS at room temperature. After 8 h, lower epithelial cell death was observed, along with loss of endothelial structure. Corneas stored in similar conditions in Optisol were indistinguishable from controls. Storage in Optisol at 4 degrees C affected the superficial layers of the corneal epithelium similarly at both 7 and 14 days. Extensive epithelial shedding and wing-cell death were observed at 25 days, but the basal layer remained approximately 50% healthy. Significant endothelial cell loss was observed at 25 days. MTT results were consistent with CLSM data in the medium-term storage study only. CONCLUSIONS: This CAM/EH-1 CLSM fluorescence assay is a sensitive index of viability in cornea, and thus may prove useful in investigations in which maintenance of vital functions in different cell layers is critical.     

The application of confocal microscopy to the study of living systems

A unique tandem confocal microscope (TSCM) has been developed that permits noninvasive imaging in vivo of the eye and many other organ systems in real time in situ. The application to the study of microphysiological processes in vivo is described and illustrated for the cornea, kidney, liver, epididymis, muscle, and adipose tissue. Novel applications are shown for studying the healing of wounds in four dimensions (x, y, z, t) in single animals over time at the cellular level. Application to clinical diagnostic use in humans is also demonstrated. When combined with Laser Scanning Confocal fluorescence microscopy, the TSCM offers a unique new imaging paradigm for experimental biology and medicine with great potential for use in neuroscience and many other disciplines.     

Cytologic application of p53 overexpression using immunocytochemistry and confocal laser scanning microscopy

OBJECTIVE: p53 Protein plays an important role in cellular growth control. This study investigated p53 protein expression in cell smears of endometrial carcinomas supplemented by confocal laser scanning microscopy (CLSM). STUDY DESIGN: Imprints from surgical specimens of 20 endometrial carcinomas were used. p53 Protein expression was investigated immunocytochemically using the monoclonal antibody pAb1801. Using CLSM, three-dimensional morphology was studied. RESULTS: Of the 20 cases of endometrial carcinoma, 8 stained positively for p53 protein. p53 Showed heterogeneous intranuclear localization, which appeared to be associated with chromatin structure. CONCLUSION: Immunocytochemical detection of p53 overexpression in cell samples is practical, and CLSM has vast potentials in studying the intranuclear arrangement of chromatin.     

Cell kinetic analysis of intact rat colonic crypts by confocal microscopy and immunofluorescence

BACKGROUND & AIMS: Precise quantitative and spatial analysis of cell cycle-related biomarkers in colonic crypts is often vital for studies of colon carcinogenesis and cancer prevention. To overcome the limitations of histology, confocal laser microscopy of microdissected whole crypts was used to quantitate S phase and mitotic cells. METHODS: Microdissected distal colonic crypts were studied in a modified rat starvation refeeding model. S phase cells were labeled in vivo with 5-bromodeoxyuridine. Mitotic cells were labeled with MPM2 (antibody to mitosis-specific epitope) and also assessed for chromatin morphology with propidium iodide. Sequential optical crypt sections, produced by confocal microscopy, were digitally imaged. S phase labeling indices per whole crypt were also compared with those derived by conventional immunohistochemistry. RESULTS: S phase and mitotic cells were clearly discriminated without background staining. The labeled S phase cell number and fraction per whole crypt were significantly decreased with starvation and increased with refeeding. Variability in the labeling index between whole crypts analyzed by confocal microscopy was significantly smaller than between histological crypt sections. Consequently, the intervention contributed to 92.2% of the total variability of the labeling index in whole crypts but only to 59% of the variability in histological sections. CONCLUSIONS: Major limitations of histology are overcome by crypt microdissection and confocal microscopic analysis. The total crypt cell population as well as labeled M phase and S phase cells can be imaged, localized, and quantitated with improved precision.     

4D confocal microscopy of Dictyostelium discoideum morphogenesis and its presentation on the Internet

Methods to present three-dimensional (3D) and time series of 3D datasets (4D) are demonstrated using the recent advances in confocal microscopy and computer visualization. The process of cell sorting during tip formation in the slime mould Dictyostelium discoideum is examined as an example by in vivo confocal microscopy of spectrally different green fluorescent protein (GFP) variants as reporters of cell-type specific gene expression. Also, cell sorting of the co-aggregating slime mould species D. discoideum and D. mucoroides is observed using a GFP variant and a spectrally distinguishable fluorescent vital stain. The confocal data are handled as 3D and 4D datasets, their processing and the advantages of different methods of visualization are discussed step by step. Selected sequences of the experiments can be viewed on the Internet, giving a much better impression of the complex cellular movements during Dictyostelium morphogenesis than printed photographs.     

Recording of mitochondrial transmembrane potential and volume in cultured rat osteoclasts by confocal laser scanning microscopy

Osteoclasts are multinuclear bone-resorbing cells which contain abundant mitochondria. Morphological studies have suggested that a correlation may exist between mitochondrial concentration and bone resorption by osteoclasts. However, investigation of mitochondrial transmembrane potential (delta psi) and volume has been hampered by the difficulty in obtaining a sufficient number of osteoclasts for assessing these characteristics by flow cytometric analysis. In this study, we have used confocal laser scanning microscopy after loading the cells with Rhodamine 123 and 10-nonyl Acridine Orange to record mitochondrial delta psi and volume, respectively, in isolated rat osteoclasts cultured on bovine bone slices. Optimal staining conditions were found to be 10 micrograms ml-1 for 40 min for Rhodamine, and 1 microM for 10 min for the 10-nonyl Acridine Orange derivative. Two osteoclast populations, whose shape seemed to reflect bone resorption and migratory functions, were identified depending on their shape and on the distribution of the two dye probes. 'Round-shaped' osteoclasts had significantly higher mitochondrial delta psi and volume in the apical regions than in the basolateral portions (p < 0.00001). In contrast, mitochondrial delta psi and volume in 'irregular-shaped' osteoclasts were rather evenly distributed in both these regions (p > 0.05). Our results indicate that there is an apical polarization of mitochondria in osteoclasts corresponding to the energy demands associated with bone resorption.     

Applications of laser scanning confocal microscopy to the observation of human oocytes and embryos

Continuity of care beyond the walls of the acute hospital setting has always been a major emphasis in nursing. There is concern that the care needs of older adults at the time of discharge have been increased by shortened hospital stays. Yet little is known about the specific and changing health care needs of older adults during the early days at home following discharge from acute care, particularly those who are discharged without community referrals. To learn more about the experiences of this population, the College of Nursing at the University of Southern Maine, in collaboration with the Nursing Service Department at Maine Medical Center, conducted a demonstration project. This project involved follow-up home visits to older adults who were discharged to their homes from an acute care setting.     

Tight junctional permeability in living cells: dynamic changes directly visualized by confocal laser microscopy

Confocal microscopy was used to study the tight junctional permeability in living rat parotid and submandibular glands. The interstitial space of the tissue was perfused with medium containing fluorescent tracers Lucifer Yellow (anionic: MW 457), Propidium Iodide (cationic: MW 668) and dextrans labeled with FITC or RITC (anionic and neutral: MW 3K, 10K, 40K, 70 K and 500 K) to monitor whether or not these tracers permeate into the lumen across the junction. In the acini of normal glands, fluorescence was detected in the basolateral space but not in the luminal space up to 30 min. However, when secretion was induced by isoproterenol or carbachol, fluorescence appeared in the luminal space within 2 to 5 min. This did not involve the disruptive changes in tight junction ultrastructure, nor was it irreversible; the luminal fluorescence disappeared again when the secretagogues were removed. Tracers up to MW 40 K for isoproterenol and MW 10 K for carbachol revealed the luminal fluorescence in parotid acini, with little indications of the charge preference characteristics. The luminal fluorescence also appeared by anoxia, enzymatic cell dissociation and the cytochalasin D treatment. It was suggested that the tight junctions in salivary acini dynamically alter their permeability and modulate the passage of large molecules through the paracellular pathway. Oxygen supply, extracellular matrices and cytoskeletons were suggested to influence these regulations.     

Quantitation of microvascular plasma perfusion and neuronal microtubule-associated protein in ischemic mouse brain by laser-scanning confocal microscopy

In an exposition of the technique of calculating distribution volumes from laser-scanning confocal microscopic (LSCM) data, three-dimensional images of the distribution of one or two fluorescent markers in mouse brain specimens were generated by LSCM and processed by a system developed for morphometric analysis of fixed and stained serial brain histologic samples. To determine the volume of perfused cerebral capillaries, one of two fluorescent plasma markers, either fluorescein isothiocyanate (FITC)-dextran or Evans blue, was intravenously administered to mice subjected to 1 hour of embolic middle cerebral artery (MCA) occlusion (n = 9) and to mice that were not operated on (n = 3); after 1 minute of circulation, brains were removed, immersion-fixed, and processed for LSCM. In some of these animals (n = 5), the volume of endogenous microtubule-associated protein-2 (MAP2) fluorescence was also determined using immunohistochemical staining. For mice that were not operated on, this methodology yielded highly localized volumes of (1) microvascular plasma, which agree with those determined for rodents by other techniques, and (2) MAP2 expression, which appears physiologically and morphologically reasonable. After 1 hour of MCA occlusion, the MAP2 volumes of distribution were less than 10% of normal in the ipsilateral hemisphere in which plasma perfusion essentially ceased. In conclusion, precise colocalization and quantitation of early ischemic neuronal damage and cerebral plasma perfusion deficit can be done with this three-dimensional, microphysiologic and microanatomic methodology.