Two-photon excitation laser scanning microscopy of human, porcine, and rabbit nasal septal cartilage.

Two-photon excitation laser scanning microscopy (TPM) was used to image human, porcine, and rabbit nasal septal cartilage. TPM provides optical sections of thick tissue specimens in situ without the use of exogenous dyes or need for tissue fixation. The cartilage tissue was imaged using near-infrared light generated by a mode-locked titanium/sapphire laser that was raster-scanned and coupled to an inverted microscope. Absorption of two photons by endogenous molecules and subsequent fluorescence was filtered to specific spectral bandwidths and detected with photomultiplier tubes. Two-photon stimulated fluorescence was detected with photomultiplier tubes optimized to specific spectral bandwidths. Signal intensity corresponds to the concentration of fluorophores, principally NADH, NADPH, and flavoproteins hence providing a means of redox imaging the cellular metabolic state. Specimens were scanned from the surface to a depth of about 150 microm. Image size was 50 x 50 microm with a diffraction limited pixel size of 0.4 microm. Cell membranes, nuclei, and matrix structures were identified in human, pig, and rabbit tissues. TPM provides a means to study three dimensional chondrocyte structure and matrix organization in situ at substantial depths, and permits longitudinal examination of cultured tissue explants without the need for exogenous dyes, tissue preparation, or fixation.     

Morphologic features of melanocytes, pigmented keratinocytes, and melanophages by in vivo confocal scanning laser microscopy.

Confocal scanning laser microscopy (CSLM) represents a novel imaging technique for in vivo microscopic analysis of skin lesions at a level of resolution that allows morphologic analysis of microanatomic structures. We investigated the feasibility of recognizing the cellular constituents of pigmented skin lesions, such as pigmented keratinocytes, melanocytes, and melanophages, by CSLM. Fifteen pigmented lesions (five pigmented seborrheic keratoses, and 10 compound melanocytic nevi) from 15 patients were studied, as well as normal skin. After the clinical lesions were imaged by CSLM, they were biopsied or excised for examination by conventional histology for comparison of the morphologic features. In images obtained by CSLM, pigmented keratinocytes were seen as polygonal cohesive cells with variably bright granular cytoplasm. Melanocytes appeared as bright round, oval, fusiform, or dendritic cells. The architectural growth pattern of melanocytes could be analyzed. Melanocytes were identified by their nested growth pattern as aggregates of bright round to oval structures at the dermoepidermal junction or in the superficial dermis. Melanocytes were also recognizable as single cells along the dermoepidermal junction, usually separated from each other by a variable number of keratinocytes. Melanophages appeared as large bright plump cells with ill-defined cytoplasmic borders, usually located around or near vessels of the superficial dermis. Our results demonstrate that the cellular constituents of pigmented lesions can be recognized by CSLM. This technique sets a new paradigm for noninvasive quasihistologic examination of pigmented lesions in vivo and merits further evaluation for diagnostic use.     

Improving the penetration depth in multiphoton excitation laser scanning microscopy

More than a threefold increase in multiphoton laser scanning microscopy depth penetration using a passive predispersion compensation system is reported. Using dispersion-controlled pulses to counteract the effects of positive group delay dispersion in the imaging platform, optical sectioning of fluorescent samples to depths in excess of 800 microm was observed, compared with only 240 microm using a noncompensated setup. Experimental results obtained from both the predispersion compensated and noncompensated systems are compared with theoretical values of pulse broadening in a laser scanning microscope. The observed improvement in depth profiling potentially widens the applications and user base of nonlinear microscopy techniques.     

Mitochondria in tumor cells studied by laser scanning confocal microscopy

We present here a confocal fluorescence microscopy study of mitochondria in sensitive and resistant carcinoma cells by using two potentiometric probes of mitochondria, rhodamine 123 (R123) and dimethylaminostyryl-methylpyridiniumiodine. We have found that active mitochondria in sensitive MCF-7 and multidrug resistant MCF-7/DX carcinoma cells are very different in localization and morphology. In sensitive cells active mitochondria are found in the perinuclear region, whereas in the multidrug resistance (MDR) subline they are confined to the cell periphery. Interestingly, the MDR revertant verapamil has been found to restore in MCF-7/DX cells the same pattern of active mitochondria seen in sensitive cells. We have also studied R123 in human lung carcinoma A549 cells, which display a low responsivity to doxorubicin, and overexpress the lung resistance-related protein. In addition to perinuclear mitochondria, peripheral mitochondria with weaker fluorescence can be seen in this cell line. Interestingly, in the two examined carcinoma lines we have been able to recognize by image analysis a common new star-lobed morphology. Our results indicate that in resistant carcinoma cells two populations of mitochondria coexist with different localization, morphology, and activity.     

Two-photon laser scanning fluorescence microscopy using photonic crystal fiber

We report the application of a simple yet powerful modular pulse compression system based on photonic crystal fibers that improves on incumbent two-photon laser scanning fluorescence microscopy techniques. This system provides more than a sevenfold increase in fluorescence yield when compared with a commercial two-photon microscopy system. From this, we infer pulses of IR radiation of less than 35 fs duration reaching the sample.     

Bound fibrinogen distribution on stimulated platelets. Examination by confocal scanning laser microscopy.

Previous studies have suggested that qualitative changes in platelet bound fibrinogen modulate platelet aggregation. The present study used confocal scanning laser microscopy to further evaluate post-ligand binding events over a 60-minute time course. When fluorescein isothiocyanate (FITC)-streptavidin was added to ADP-stimulated platelets 1 minute after biotinylated fibrinogen binding at 22 degrees C, bound fibrinogen was found in variously sized patches on the cell surface. When streptavidin was added 60 minutes later, bound fibrinogen had been cleared from the platelet surface and was observed in clusters penetrating into platelets to various extents. ADP-activated platelets did not stain with a monoclonal antibody against CD62 suggesting that platelets were not permeabilized during the experiment and had not released alpha-granules. Additional studies using either biotinylated fibrinogen that had been prelabeled with FITC-streptavidin or FITC-labeled fibrinogen revealed similar patterns of platelet-associated fibrinogen clearance and redistribution. Pretreatment of platelets with cytochalasin D prevented this redistribution. Dual labeling experiments using biotinylated fibrinogen and FITC-streptavidin as well as a monoclonal anti-GPIIIa antibody labeled with rhodamine-conjugated anti-mouse IgG demonstrated the co-localization of fibrinogen and GPIIIa. Similar observations were made with fibrinogen bound to thrombin-stimulated platelets. In contrast, fibronectin bound to thrombin-activated platelets retained a predominantly surface membrane distribution under identical experimental conditions. Since surface-cleared fibrinogen was accessible to exogenous FITC-streptavidin under conditions that did not lead to platelet permeabilization, the data suggest fibrinogen deposition in compartments that are accessible to the extracellular milieu. This is consistent with the ability of exogenous plasmin to completely remove cleared fibrinogen pools without detectable fibrinogen reexpression on the platelet surface or alpha-granule secretion. The data provide morphological evidence for the selective, GPIIb-IIIa mediated, actin-dependent clearance of bound fibrinogen from the activated platelet surface, suggesting a mechanism for preventing and limiting thrombus development.     

Analysis of three-dimensional architecture in Feulgen-stained fine-needle liver aspirates, using confocal scanning laser microscopy

Early and accurate diagnosis of hepatocellular carcinoma in liver nodules using fine-needle aspiration biopsy can still pose difficulties, particularly in cirrhotic nodules containing dysplastic hepatocytes. Loss of architectural clues, which may aid diagnosis, can be overcome by using confocal scanning laser microscopic examination of tissue fragments in smears without the need for further processing of slides. The basic fuchsin of the Feulgen method resulted in excellent confocal images without the need for further processing of the smears. Clear morphological differences in three-dimensional reconstructions of optically sectioned tissue fragments were demonstrated in normal, hyperplastic, and malignant smears as an aid to diagnosis.     

Confocal laser scanning microscopy and three-dimensional reconstruction of cell clusters in serous fluids

Thick cell clusters are a common finding in reactive and malignant effusions. In order to arrive at a diagnosis, clusters are evaluated for certain cytomorphologic features including size, shape, smooth vs. scalloped borders, and three-dimensional (3-D) configuration. By conventional microscopy, the image of these clusters is often blurred due to limitations in resolution. Consequently, the exact internal structure and cellular arrangement within these clusters cannot be adequately determined. Utilizing confocal laser scanning microscopy (CLSM), we examined serous fluids from a variety of conditions. Cases included mesothelioma, adenocarcinoma, and papillary adenocarcinoma. Smears were stained with 0.01% ethidium bromide and 1% eosin Y, followed by analysis with an ACAS 570™ image analyzer (Meridian Instruments, Inc. Okemos, MI). Serial confocal fluorescence images were acquired, which allowed 3-D reconstruction of the clusters. Mesothelioma clusters (excluding those with obvious central collagen cores by light microscopy) appeared to be formed of the following configurations: 1) randomly coiled cords of cells, 2) small papillae encompassing central cores, and 3) tissue fragments with pseudoacinar formation. In contrast, adenocarcinomas had a more orderly pattern, with tightly cohesive cells and true acinar formation. Diagn. Cytopathol. 1997;17: 272–279. © 1997 Wiley-Liss, Inc.     

Detection of macrophage activity in atherosclerosis in vivo using multichannel, high-resolution laser scanning fluorescence microscopy

Molecular and cellular mechanisms of atherogenesis and its treatment are largely being unraveled by in vitro techniques. We describe methodology to directly image macrophage cell activity in vivo in a murine model of atherosclerosis using laser scanning fluorescence microscopy (LSFM) and a macrophage-targeted, near-infrared fluorescent (NIRF) magnetofluorescent nanoparticle (MFNP). Atherosclerotic apolipoprotein E deficient (apoE -/-) mice (n=10) are injected with MFNP or 0.9% saline, and wild-type mice (n=4) are injected with MFNP as additional controls. After 24 h, common carotid arteries are surgically exposed and prepared for LSFM. Multichannel LSFM of MFNP-enhanced carotid atheroma (5x5-microm in-plane resolution) shows a strong focal NIRF signal, with a plaque target-to-background ratio of 3.9+/-1.8. Minimal NIRF signal is observed in control mice. Spectrally resolved indocyanine green (ICG) fluorescence angiograms confirm the intravascular location of atheroma. On ex vivo fluorescence reflectance imaging, greater NIRF plaque signal is seen in apoE -/- MFNP mice compared to controls (p<0.01). The NIRF signal correlates well with immunostained macrophages, both by stained surface area (r=0.77) and macrophage number (r=0.86). The validated experimental methodology thus establishes a platform for investigating macrophage activity in atherosclerosis in vivo, and has implications for the detection of clinical vulnerable plaques.     

Observation of dendritic cell morphology under light, phase-contrast or confocal laser scanning microscopy

Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs.     

The scanning laser ophthalmoscope--a review of its role in bioscience and medicine

The scanning laser ophthalmoscope (SLO) offers the potential for retinal imaging that is complementary both to that of the fundus camera and also the newly developing technique of optical coherence tomography (OCT). It has the ability to produce rapid images at low light levels using light of specific wavelengths. This permits temporal studies of fluorescent-labelled cells which offer a unique insight into inflammatory processes in the eye. The facility to image with several different wavelengths simultaneously offers the potential for spectral imaging of retinal tissue with the aim of revealing those early changes in tissue perfusion that indicate the onset of retinal disease, so increasing the probability of successful therapy.     

Application of confocal laser scanning microscopy to the deep pineal gland and other neural tissues.

The study of the deep pineal gland of the Mongolian gerbil and other neuronal tissue from the rat by means of confocal laser scanning microscopy (CLSM) is described. Opical serial sectioning was performed on thick (100-200 microns) sections of the deep pineal gland of the Mongolian gerbil stained immunohistochemically using antisera to S-antigen and tyrosine hydroxylase (TH). Both dual-stained and single-stained material was examined using the fluorochromes fluorescein isothiocyanate (FITC) and Texas Red. High resolution images were obtained showing that pinealocytes have 1-3 processes that extend primarily to other pinealocytes or presumptive pinealocytes. Pinealocytes are located within the deep pineal gland as well as adjacent to the posterior aspect of the medial habenular nuclei. Pinealocyte processes were not seen extending into the habenular nuclei, but rather ended within the deep pineal gland a significant distance from their perikarya. The TH-immunopositive fibers were distributed throughout the deep pineal gland, often forming "baskets" of fibers around pinealocytes rather than being associated primarily with blood vessels. Other uses of the confocal microscope are demonstrated on rat neural tissue reacted with peroxidase/diaminobenzidine (DAB) immunohistochemistry and FITC fluorescence immunohistochemistry (paraventricular nucleus) as well as Golgi-stained neuronal tissue (cerebral cortex). The HRP/DAB and Golgi-stained images were visualized using the reflected image mode of the confocal system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunofluorescence imaging diagnosis of Fabry heterozygotes using confocal laser scanning microscopy

Itoh K, Kotani M, Tai T, Suzuki H, Utsunomiya T, Inoue H, Yamada H, Sakuraba H, Suzuki Y. Immunofluorescence imaging diagnosis of Fabry heterozygotes using confocal laser scanning microscopy.
Clin Genet 1993: 44: 302–306. © Munksgaard, 1993
An immunofluorometric method was developed for the semiquantitative determination of trihexosylceramide in cultured fibroblasts from Fabry disease patients, using a laser scanning confocal imaging system. The accumulated glycolipid was detected as granular inclusions in the cells. Heterozygote identification was achieved both by counting of immunoreactive cells and by measuring the relative fluorescence intensity with a digital imaging system.     

Imaging of cardiovascular structures using near-infrared femtosecond multiphoton laser scanning microscopy

Multiphoton imaging represents a novel and very promising medical diagnostic technology for the high-resolution analysis of living biological tissues. We performed multiphoton imaging to analyzed structural features of extracellular matrix (ECM) components, e.g., collagen and elastin, of vital pulmonary and aortic heart valves. High-resolution autofluorescence images of collagenous and elastic fibers were demonstrated using multifluorophore, multiphoton excitation at two different wavelengths and optical sectioning, without the requirement of embedding, fixation, or staining. Collagenous structures were selectively imaged by detection of second harmonic generation (SHG). Additionally, routine histology and electron microscopy were integrated to verify the observed results. In comparison with pulmonary tissues, aortic heart valve specimens show very similar matrix formations. The quality of the resulting three-dimensional (3-D) images enabled the differentiation between collagenous and elastic fibers. These experimental results indicate that multiphoton imaging with near-infrared (NIR) femtosecond laser pulses may prove to be a useful tool for the nondestructive monitoring and characterization of cardiovascular structures.     

Confocal laser scanning microscopy as a tool for imaging cancellous bone

Understanding the bimodal structure of cancellous bone is important for tissue engineering in order to more accurately fabricate scaffolds to promote bone ingrowth and vascularization in newly forming bone. In this study, confocal laser scanning microscopy (CLSM) was used to create detailed images of the bimodally porous intertrabecular space of defatted and deproteinized cancellous canine bone taken from the epiphysis of the humerus. The bimodal pore structure was imaged using both reflective and fluorescent modes in CLSM, resulting in four different, but complementary image types: (1) a Z-stack overlay, (2) a phi-Z scan, (3) a topographical map, and (4) a contour map. Submerging the bone in rhodamine B dye prior to fluorescent imaging enhanced the pore surface details, giving a more accurate pore size measurement. The average macropore diameter was found to be 260 +/- 97 microm while the average micropore diameter was 13 +/- 10 microm. When compared with common techniques, including microcomputed tomography, magnetic resonance imaging, scanning electron microscopy, and environmental scanning electron microscopy, for imaging cancellous bone, CLSM was found to be an effective tool, given its ability to nondestructively image the surface and near-surface pore structure.