Use Of Confocal Laser Scanning Microscopy Research Articles

Integrated FRET Polymers Spatially Reveal Micro- to Nanostructure and Irregularities in Electrospun Microfibers.

A spatial view of macroscopic polymer material properties, in terms of nanostructure and irregularities, can help to better understand engineering processes such as when materials may fail. However, bridging the gap between the molecular-scale arrangement of polymer chains and the spatially resolved macroscopic properties of a material poses numerous difficulties. Herein, an integrated messenger material that can report on the material micro- to nanostructure and its processes is introduced. It is based on polymer chains labeled with fluorescent dyes that feature Förster resonance energy transfer (FRET) dependent on chain conformation and concentration within a host polymer material. These FRET materials are integrated within electrospun polystyrene microfibers, and the FRET is analyzed by confocal laser scanning microscopy (CLSM). Importantly, the use of CLSM allows a spatial view of material nanostructure and irregularities within the microfibers, where changes in FRET are significant when differences in fiber geometries and regularities exist. Furthermore, changes in FRET observed in damaged regions of the fibers indicate changes in polymer conformation and/or concentration as the material changes during compression. The system promises high utility for applications where nano-to-macro communication is needed for a better understanding of material processes.

A Feasibility Study for Immediate Histological Assessment of Various Skin Biopsies Using Ex Vivo Confocal Laser Scanning Microscopy.

Digitally stained ex vivo confocal laser scanning microscopy (CLSM) scans are a possible alternative to formalin-fixed and paraffin-embedded (FFPE) and hematoxylin-eosin (H&E) stained slides. This study explores the diagnostic accuracy of digitally-stained CLSM scans in comparison to H&E-stained slides in various dermatologic diseases in a real-life setting. Samples of patients out of one selected dermatologic office were primarily scanned via CLSM; a diagnosis was made afterwards using FFPE- and H&E-stained slides by two experienced dermatopathologists. Primary outcomes were sensitivity and specificity of diagnosis in digitally stained CLSM scans in three separate diagnostic groups. CLSM evaluation of epithelial tumors (n = 132) demonstrated a sensitivity of 64.3%/83.9% and a specificity of 84.2%/71.1%. Diagnosis of melanocytic tumors (n = 86) showed a sensitivity of 19.1%/85.1% and a specificity of 96.3%/66.7%. In the diagnosis of other tumors/cysts and inflammatory dermatoses (n = 42), a sensitivity of 96.4%/96.8% and a specificity of 57.1%/45.5% was reached. This study shows the possibilities and limitations of a broad use of CLSM. Because of a partly low diagnostic accuracy, such an application does not seem to be recommendable at present for every indication.

Confocal laser scanning microscopy as a novel tool of hyperspectral imaging for the localization and quantification of fluorescent active principles in pharmaceutical solid dosage forms

Confocal laser scanning microscopy (CLSM) supported with multivariate analysis is proposed as hyperspectral imaging technique to identify, locate, and quantify, in a direct way, active pharmaceutical ingredients (APIs) in synthetic tablets, with the aim of establishing a new analysis methodology that collects qualitative and quantitative information from the surface and inner layers of solid dosage forms. This method is proposed as a novel, non-destructive, rapid, and highly sensitive hyperspectral imaging technique, with an excellent spatial resolution at the microscopic level. Two chemical systems comprising pharmaceutical mixtures of acetaminophen–caffeine–excipients and digoxin–excipients were analyzed by multivariate curve resolution-alternating least squares (MCR-ALS) using emission spectra obtained from microscopic images with the pixel sizes of 512 μm × 512 μm to localize the APIs in the tablets. In addition, partial least squares (PLS) regression models were developed and used for calibration to obtain the concentration of the fluorescent active ingredients. The analysis by MCR-ALS delivered excellent results in localizing the fluorescent compounds; meanwhile, PLS achieved good error parameters for prediction of the external validation set for the quantification of caffeine and less successful quantification of digoxin. The performance of CLSM was evaluated by estimation of the analytical figures of merit of the technique to assess the quantification of APIs, including calculations of the uncertainty in the signal, sensitivity, analytical sensitivity, and ranges of detection and quantification limits. Use of CLSM as hyperspectral imaging attempts to increase the application of this technique in direct analysis of solid samples, showing that autofluorescent compounds can be analyzed for qualitative and quantitative purposes in presence of interferents by application of chemometrics.

Pericapillary Edema Assessment by Means of the Nailfold Capillaroscopy and Laser Scanning Microscopy.

Edema, i.e., fluid accumulation in the interstitial space, accompanies numerous pathological states of the human organism, including heart failure (HF), inflammatory response, and lymphedema. Nevertheless, techniques for quantitative assessment of the edema’s severity and dynamics are absent in clinical practice, and the analysis is mainly limited to physical examination. This fact stimulates the development of novel methods for fast and reliable diagnostics of fluid retention in tissues. In this work, we focused on the possibilities of two microscopic techniques, nailfold video capillaroscopy (NVC) and confocal laser scanning microscopy (CLSM), in the assessment of the short-term and long-term cutaneous edema. We showed that for the patients with HF, morphological parameters obtained by NVC—namely, the apical diameter of capillaries and the size of the perivascular zone—indicate long-term edema. On the other hand, for healthy volunteers, the application of two models of short-term edema, venous occlusion, and histamine treatment of the skin, did not reveal notable changes in the capillary parameters. However, a significant reduction of the NVC image sharpness was observed in this case, which was suggested to be due to water accumulation in the epidermis. To verify these findings, we made use of CLSM, which provides the skin structure with cellular resolution. It was observed that for the histamine-treated skin, the areas of the dermal papillae become hyporefractive, leading to the loss of contrast and the lower visibility of capillaries. Similar effect was observed for patients undergoing infusion therapy. Collectively, our results reveal the parameters can be used for pericapillary edema assessment using the NVC and CLSM, and paves the way for their application in a clinical set-up.

Potential Penetration of CTAB- and MUDA-coated Gold Nanorods into Tooth Enamel

Gold nanorods (GNRs) have gained interest as a promising carrier for antibiotics. Gold nanorods may reduce the development of antimicrobial resistance in certain microbial species. Although applications of GNRs to mitigate oral biofilms are under development, their use in the oral cavity may have adverse effects. The aim of this study was to evaluate the potential penetration of GNRs into the tooth enamel structure using confocal laser scanning microscopy (CLSM) and scanning transmission electron microscopy (STEM). Our approach was to synthesize GNRs with cationic [cetyltrimethylammoniumbromide (CTAB)] and anionic [11-mercaptoundecanoic acid (MUDA)] surface coatings. We hypothesized that penetration would be surface coating dependent. Regardless of the chemical modification of the GNRs of size ∼20 nm × 8 nm, exposure of these materials did not result in superficial penetration into the enamel. Within the limitations of this study, it is concluded that the use of CLSM and STEM is a feasible approach to investigate the penetration of nanomaterials into the tooth structure. Exposure of the enamel with chemically modified GNRs of size ∼20 nm × 8 nm will not result in superficial penetration into the enamel.

Global microfossil changes through the Precambrian-Cambrian phosphogenic event: The Shabakta Formation of the phosphorite-bearing Maly Karatau Range, South Kazakhstan

Subtidal cherts from phosphorite-bearing basal strata of the Early Cambrian Shabakta (Dzhylandy) Formation, Tamda Group of the Maly Karatau Range of South Kazakhstan, contain a diverse assemblage of acanthomorph acritarchs and other well-preserved organic-walled microfossils. Acanthomorphs of the Shabakta lowermost layers are taxonomically essentially identical to those of many similarly aged Early Cambrian organic-walled compression-preserved and permineralized microbiotas preserved in rocks deposited in relatively open marine settings and are biostratigraphically correlative with acanthomorph-containing microfloral assemblages of the Vergale and Rausve Horizons (Regional Stages) of the East European Platform as well as with other assemblages known worldwide from the upper Atdabanian (Series 2 of the International Union of Geological Sciences [IUGS]). Although lower Atdabanian deposits of the Talsy (Lükati) horizon are missing from the Maly Karatau succession studied here, Nemakit-Daldynian through Tommotian (the IUGS Terranovian) microbiotas both of the Shabakta-underlying Berkuta Member of the Kyrshabakta Formation and phosphoritic Chulaktau Formation are comparable to those of the pre-trilobite Rovno and Lontova Horizons (Regional Stages) of the East European Platform. Thus, the phosphatized and chert-permineralized Shabakta assemblages date from of the Early Cambrian part of the Precambrian-Cambrian phosphogenic event, an interpretation supported also by associated small shelly fossils.The studies reported here of the Shabakta microfossils – based on the use of optical microscopy, confocal laser scanning microscopy (CLSM) and Raman spectroscopy – provide information in three dimensions at high spatial resolution about their organismal morphology, cellular anatomy, kerogenous composition, taphonomy, and mode and fidelity of preservation. The combined use of CLSM and Raman, techniques introduced to Precambrian paleobiology only rather recently, is shown to reveal morphological characters of taxonomic, biostratigraphic and palaeoenvironmental significance.

An experimental investigation of the effects of post-depositional damage on current quantitative use-wear methods

This paper describes a new method of estimating stone tool function using a laser-scanning confocal microscope. The primary benefit of the quantitative approach used here, and others like it, is that it increases reproducibility while reducing subjectivity and inter/intra-observer error. Although the use of laser-scanning confocal microscopy in functional analyses is not novel, the method presented here was “stress tested” using an experimental assemblage to explore the impact of post-depositional damage on its results. The findings reveal that this method is still viable with lightly and moderately damaged specimens. It is therefore likely that methods like this one are no more vulnerable to post-depositional damage than conventional use-wear methods and can confidently be applied to archaeological specimens.

Concealed Androconial Scales in Metalmark Butterflies (Lepidoptera: Riodinidae): New Insights from Confocal Laser Scanning Microscopy

Abstract Confocal scanning microscopy is widely used in the biological sciences—from tissue protein expression to the study of fossil amber inclusions. However, it is rarely used in insect taxonomy despite its unique feature for the examination of structural detail in small samples. In this study, we use confocal laser scanning microscopy (CLSM) to image certain poorly studied male secondary sexual organs, namely androconial scales, in a group of riodinid butterflies (Lepidoptera: Riodinidae). This group shows cryptic and mimetic morphology, so pheromones are thought to play an important role in mate choice. We found significant interspecific structural differences in androconial patches. The use of CLSM provides a simple, nondestructive and highly effective method to examine complex morphological characters, which could be applied to other structures and taxa across Lepidoptera and other insects.

Morphological, ultrastructural and functional imaging of frozen/thawed and vitrified/warmed human ovarian tissue retrieved from oncological patients.

Which is the best method for human ovarian tissue cryopreservation: slow freezing/rapid thawing (SF/RT) or vitrification/warming (V/W)? The conventional SF/RT protocol used in this study seems to better preserve the morpho-functional status of human cryopreserved ovarian tissue than the used open carrier V/W protocol. Cryopreservation of human ovarian tissue is generally performed using the SF/RT method. However, reduction in the follicular pool and stroma damage are often observed. An emerging alternative procedure is represented by V/W which seems to allow the maintenance of the morphological integrity of the stroma. This is a retrospective cohort study including six patients affected by oncological diseases and enrolled from January to December 2014. Ovarian tissue was laparoscopically harvested from the right and left ovaries and was cryopreserved using a routinary SF/RT protocol or a V/W method, involving tissue incubation in two solutions (containing propylene glycol, ethylene glycol and sucrose at different concentrations) and vitrification in an open system. For each patient, three pieces from each ovary were collected at the time of laparoscopy (fresh tissue) and after storage (SF/RT or V/W) and processed for light microscopy (LM) and transmission electron microscopy (TEM), to assess the morphological and ultrastructural features of follicles and stroma, and for laser scanning confocal microscopy (LSCM), to determine the functional energetic/redox stroma status. The preservation status of SF/RT and V/W ovarian tissues was compared with that of fresh ones, as well as between them. By LM and TEM, SF/RT and V/W samples showed cryodamage of small entity. Interstitial oedema and increased stromal cell vacuolization and chromatin clumping were observed in SF/RT samples; in contrast, V/W samples showed oocyte nuclei with slightly thickened chromatin and irregular shapes. The functional imaging analysis by LSCM revealed that the mitochondrial activity and intracellular reactive oxygen species levels were reduced both in SF/RT and in V/W samples compared with fresh samples. The study also showed progressive dysfunction of the mitochondrial activity going from the outer to the inner serial section of the ovarian cortex. The reduction of mitochondrial activity of V/W samples compared with fresh samples was significantly higher in the inner section than in the outer section. The results report the bioenergetic and oxidative status assessment of fresh and cryopreserved human ovarian tissue by LSCM, a technique recently applied to tissue samples. The use of LSCM on human ovarian tissues after SF/RT or V/W is a new application that requires validation. The procedures for mitochondrial staining with functional probes and fixing are not yet standardized. Xenografting of the cryopreserved ovarian tissue in severe combined immunodeficient mice and in vitro culture have not yet been performed. The identification of a cryopreservation method able to maintain the morpho-functional integrity of the ovarian tissue and a number of follicles comparable with those observed in fresh tissue might optimize results in clinical practice, in terms of recovery, duration of ovarian function and increased delivery outcomes after replanting. The SF/RT protocol allowed better morpho-functional tissue integrity than the V/W procedure. Funding was provided by Fondazione del Monte di Bologna e Ravenna, Italy. Dr N.A.M. was granted by the project ONEV MIUR PONa3 00134-n.254/R&C 18 5 2011 and the project GR-2011-02351396 (Ministry of Health, Young Researchers Grant 2011/2012). There are no competing interests. Clinical trial 74/2001/0 (approved:13 2 2002): 'Pilot study on cryopreservation of human ovarian tissue: morphological and immunohistochemical analysis before and after cryopreservation'.

Goat milk CSN1S2 is able to decrease the severity scoring, TNF-a, and RAGE expression in complete Freund's adjuvant-induced rheumatoid arthritis model of rats

This study aimed to elucidate whether CSN1S2 protein of goat's milk was able to inhibit pro-inflammatory cytokine [interleukin (IL)-17 and tumor necrosis factor (TNF)-α], RAGE, and caspase-3 expression in rheumatoid arthritis (RA) rats. A total of 24 Wistar female rats, were randomly assigned into four groups: control group (C), CSN1S2 protein of goat's milk group (CM), CFA-induced RA-rats group (RA), and the RA group treated by CSN1S2 protein of goat's milk (RAM). The severity of erythema and swelling in lower extremities were counted by scoring. IL-17, TNF-α, RAGE, and caspase-3 expression in synovial membranes were analyzed by confocal laser scanning microscopy (CLSM) and western blotting. Erythema and swelling in the RA group was significantly attenuated by goat's milk CSN1S2 (p < 0.05), but did not reach the level in the control group (p < 0.05). The use of CLSM, CSN1S2 protein of goat's milk could decrease the TNF-α, caspase-3, and the number of hyperplasia cells in comparison with the RA group (p < 0.05), to reach the level in the control group (p > 0.05). Western blotting analysis showed that the expression of IL-17, RAGE, TNF-α, and caspase-3 were higher in the RA group compared with the control group. CSN1S2 protein of goat's milk decreased RAGE and TNF-α expression, but increased the IL-17 and caspase-3 expression. In conclusion, CSN1S2 protein of goat's milk decreased erythema, swelling, and inflammation in lower extremities. The CSN1S2 protein of goat's milk also decreased TNF-α and RAGE expression in the synovial membrane of ankle joints. Unfortunately, CSN1S2 protein of goat's milk may induce the production of IL-17. Therefore CSN1S2 protein of goat's milk may provide a nutritional therapy for attenuating the inflammation found in rheumatoid arthritis.

In Vivo Laser Scanning Confocal Microscopy of the Ocular Surface in Glaucoma

Over the past decade, knowledge about the ocular surface in glaucoma has significantly increased through the use of in vivo laser scanning confocal microscopy (LSCM). This in vivo imaging method can show modifications at the cellular level induced by anti-glaucoma drugs on ocular surface structures and adnexa in the eye. High-quality images of the conjunctiva, cornea, limbus, meibomian glands, and lymphoid structures during therapy can be obtained. In addition, LSCM opened new fields of research on the patho-physiology of aqueous humor (AH) hydrodynamics in untreated, and in medically or surgically treated glaucomatous patients. In these conditions, an enhancement of the trans-scleral AH outflow contributed to clarification of the mechanism of action of different anti-glaucoma medications and surgical approaches. Finally, the use of LSCM represented a huge advance in evaluation of bleb functionality after filtration surgery, defining the hallmarks of AH filtration through the bleb-wall and distinguishing functional from nonfunctional blebs. Thus, signs seen with LSCM may anticipate clinical failure, guiding the clinician in planning the appropriate timing of the various steps in bleb management. In this review we summarize the current knowledge about in vivo LSCM of the ocular surface in glaucoma.

Confocal laser scanning microscopy as a tool to validate the efficiency of membrane cleaning procedures to remove biofilms

The efficiency of six different cleaning protocols was studied and quantified by measuring ATP and carbohydrate (CH) concentrations. The detailed characterization of changes in the biofilm structure due to the application of various cleaning agents was performed by means of confocal laser scanning microscopy (CLSM). Biofilm samples were cultivated over a period of 28days under well-defined conditions in space and time to mimic seriously biofouled membrane surfaces which were tested under reproducible conditions. Quantitative structural parameters were calculated from CLSM image stacks. Imaging data revealed visually and quantitatively differences at the microscale in the behavior of cleaning procedures applied. Moreover, these data showed correlations with quantitative biomass parameters ATP and CH at the macroscale. Hence, this study supports the use of CLSM as a research tool to understand in detail the effects of selected cleaning agents on biofilm structure and presents supportive data for the efficacy of certain cleaning protocols.

Characterization of the stem anatomy of the Eocene fern Dennstaedtiopsis aerenchymata (Dennstaedtiaceae) by use of confocal laser scanning microscopy

Permineralization provides the most faithful known mode of three-dimensional preservation of the morphology and cellular anatomy of fossil plants. Standard optical microscopic documentation of such structures can provide only an approximation of their true three-dimensional form and is incapable of revealing fine-structural (<300 nm) details, deficiencies that can be addressed by the use of confocal laser scanning microscopy (CLSM). To demonstrate the usefulness of CLSM in such studies, we compare confocal laser scanning micrographs and optical photomicrographs of the permineralized tissues of rhizomes and petioles of the Eocene fern Dennstaedtiopsis aerenchymata preserved in cherts of the Clarno Formation of Oregon, USA, and the Allenby Formation (Princeton chert) of British Columbia, Canada. The laser-induced fluorescence detected by CLSM produces crisp high-resolution images of the three-dimensionally permineralized tissues of Dennstaedtiopsis aerenchymata. Tissues analyzed include the epidermis and epidermal hairs, cortex, aerenchyma, endodermis, vascular tissue, and pith-for each of which, CLSM yields results superior to those of standard optical microscopy. CLSM and previous Raman spectroscopic analyses of the same specimens provide evidence consistent with original biochemistry. Use of CLSM to characterize the morphology and cellular anatomy of permineralized fossil plants can provide accurate data in two and three dimensions at high spatial resolution, information that can be critically important to taxonomic, taphonomic, and developmental interpretations. Results presented here from this first detailed CLSM-based study of permineralized plant axes indicate that this nonintrusive, nondestructive technique should be widely applicable in paleobotany.

Confocal laser scanning microscopy technique for the study of internal genitalia and external morphology of eriophyoid mites (Acari: Eriophyoidea)

Confocal laser scanning microscopy (CLSM) is a modern powerful technique that can be used for studying the externaland internal anatomy of arthropods. CLSM has seldom been used in acarology and very rarely for studying eriophyoidmites. It allows the capture of precise digital images of the fine details of external and internal chitinous structures, whichcan be further analysed using various computer programs. CLSM can serve as an effective tool for comparing closelyrelated and/or cryptic species, correcting diagnoses of poorly described taxa, studying immature instars, and particularly,for studying the structures and the functioning of the internal genitalia of adult females and males. In this paper, thepotential use of CLSM for the study of eriophyoids is demonstrated using specimens of 13 mite species and eight generafrom the families Phytoptidae Murray 1877 and Eriophyidae Nalepa 1898. This study showed that freshly mountedspecimens on microscope slides appeared to be the most appropriate for CLSM as older specimens tended to have reducedautofluorescence. The best choice for studying the external morphology and internal genital apparatus of eriophyoid mitesappeared to be the blue laser. Green and light blue wavelengths (488 nm and 532 nm) were found to be less useful. Thequality of CLSM images depended on the slide-mounting medium used. Among those compared, Hoyer’s medium wasfound to be the most appropriate whereas Heinze medium and media including Iodium gave poorer results. The empodiaand proximal parts of setae were shown to have very weak autofluorescence signals, but they reflected red (635 nm) and blue (405 nm) laser light, which could be detected with CLSM.

Use of laser scanning confocal microscopy for morphological taxonomy and the potential for digital type specimens (e-types)

The use of laser scanning confocal microscopy (LSCM) for creating taxonomic descriptions of copepods is investigated. A new technique is described, which employs a contour filter to process digital LSCM images, allowing taxonomic information to be quickly and accu- rately distilled into a simple illustration. LSCM allows the imaging of whole specimens, which can be rotated and viewed from any angle—a major benefit over light microscopy. Using this tech- nique, it is suggested that taxonomic descriptions can be rapidly produced in a fraction of the time required to produce similar descriptions using traditional light microscopy and hand drawing techniques. Good staining of specimens is, however, essential to produce accurate descriptions and more research is required in this area. The use of LSCM for morphological taxonomy shows great potential, not only for producing taxonomic descriptions, but also providing a comple - mentary adjunct to traditional type specimens in the form of 3D digital 'e-types' deposited in recognised international databases.

QUANTITATIVE CONFOCAL LASER SCANNING MICROSCOPY

This paper discusses recent advances in confocal laser scanning microscopy (CLSM) for imaging of 3D structure as well as quantitative characterization of biomolecular interactions and diffusion behaviour by means of one- and two-photon excitation. The use of CLSM for improved stereological length estimation in thick (up to 0.5 mm) tissue is proposed. The techniques of FRET (Fluorescence Resonance Energy Transfer), FLIM (Fluorescence Lifetime Imaging Microscopy), FCS (Fluorescence Correlation Spectroscopy) and FRAP (Fluorescence Recovery After Photobleaching) are introduced and their applicability for quantitative imaging of biomolecular (co-)localization and trafficking in live cells described. The advantage of two-photon versus one-photon excitation in relation to these techniques is discussed.

The Effect of Milk Processing on the Microstructure of the Milk Fat Globule and Rennet Induced Gel Observed Using Confocal Laser Scanning Microscopy

Confocal laser scanning microscopy (CLSM) was successfully used to observe the effect of milk processing on the size and the morphology of the milk fat globule in raw milk, raw ultrafiltered milk, and standardized and pasteurized milk prepared for cheese manufacture (cheese-milk) and commercial pasteurized and homogenized milk. Fat globule size distributions for the milk preparations were analyzed using both image analysis and light scattering and both measurements produced similar data trends. Changes to the native milk fat globule membrane (MFGM) were tracked using a MFGM specific fluorescent stain that allowed MFGM proteins and adsorbed proteins to be differentiated on the fat globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the identity of native MFGM proteins isolated from the surface of fat globules within raw, UF retentate, and cheese-milk preparations, whereas only casein was detected on the surface of fat globules in homogenized milk. The microstructure, porosity, and gel strength of the rennet induced gel made from raw milk and cheese-milk was also found to be comparable and significantly different to that made from homogenized milk. Our results highlight the potential use of CLSM as a tool to observe the structural details of the fat globule and associated membrane close to its native environment.

THE USE OF CONFOCAL LASER SCANNING MICROSCOPY TO ASSESS THE POTENTIAL SUITABILITY OF 3D SCAFFOLDS FOR TISSUE REGENERATION, BY MONITORING EXTRA‐CELLULAR MATRIX DEPOSITION AND BY QUANTIFYING CELLULAR INFILTRATION AND PROLIFERATION

The great many varieties of dermal regeneration materials currently being developed requires that sound in vitro assessments of their potential to be infiltrated with host cells are needed prior to any in vivo studies. The biocompatibility of scaffolds is conventionally determined by indirect biochemical assays as measures of cell number/viability within these materials. A disadvantage of these methods is the failure to provide information pertaining to the spatial distribution of cells in situ. In the case of dermal regeneration, if cells are only present in appreciable numbers on the outer portion of a scaffold, then critical aspects such as extra‐cellular matrix (ECM) production and vascularization will be limited, and the likelihood of successful tissue regeneration low. Thus, direct enumeration of cells within scaffolds, with respect to spatial distribution and time, is required to adequately assess the potential biocompatibility. In this article we demonstrate a systematic approach to the quantification of infiltration and proliferation of primary human dermal fibroblasts into the dermal regeneration scaffold, Integra® (the most frequently used dermal regeneration template in burns patients), using confocal laser scanning microscopy (CLSM). Scaffold samples were placed on top of confluent cell layers and infiltrated by cells migrating from the culture dish against gravity. Cells were then enumerated (by nuclear staining) with respect to their location within the scaffold and over a time period of up to 28 days. Large increases in cell numbers were observed on the surface of the scaffolds, together with measurable increases in cell numbers within their interiors. We also describe the immunofluorescent staining of fibroblasts and ECM components as well as the subsequent use of CLSM to qualitatively assess the potential for scaffold re‐modeling. Of particular note was the cellular deposition of a “front” of fibronectin matching the maximum extent of cell infiltration at each time point. We suggest that this key ECM component is a useful indicator of the extent of cellular re‐modeling of a biomaterial.

Confocal Microscopy of Unfixed Breast Needle Core Biopsies: A Comparison to Fixed and Stained Sections

BackgroundNeedle core biopsy, often in conjunction with ultrasonic or stereotactic guided techniques, is frequently used to diagnose breast carcinoma in women. Confocal scanning laser microscopy (CSLM) is a technology that provides real-time digital images of tissues with cellular resolution. This paper reports the progress in developing techniques to rapidly screen needle core breast biopsy and surgical specimens at the point of care. CSLM requires minimal tissue processing and has the potential to reduce the time from excision to diagnosis. Following imaging, specimens can still be submitted for standard histopathological preparation.MethodsNeedle core breast specimens from 49 patients were imaged at the time of biopsy. These lesions had been characterized under the Breast Imaging Reporting And Data System (BI-RADS) as category 3, 4 or 5. The core biopsies were imaged with the CSLM before fixation. Samples were treated with 5% citric acid and glycerin USP to enhance nuclear visibility in the reflectance confocal images. Immediately following imaging, the specimens were fixed in buffered formalin and submitted for histological processing and pathological diagnosis. CSLM images were then compared to the standard histology.ResultsThe pathologic diagnoses by standard histology were 7 invasive ductal carcinomas, 2 invasive lobular carcinomas, 3 ductal carcinomas in-situ (CIS), 21 fibrocystic changes/proliferative conditions, 9 fibroadenomas, and 5 other/benign; two were excluded due to imaging difficulties. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images and were comparable to standard histologic sections of the same tissue.ConclusionCSLM is a technique with the potential to screen needle core biopsy specimens in real-time. The confocal images contained sufficient information to identify stromal reactions such as fibrosis and cellular proliferations such as intra-ductal and infiltrating carcinoma, and were comparable to standard histologic sections of the same tissue. Morphologic and cellular features of benign and cancerous lesions were identified in the confocal images. Additional studies are needed to 1.) establish correlation of the confocal and traditional histologic images for the various diseases of the breast; 2.) validate diagnostic use of CSLM and; 3.) further define features of borderline lesions such as well-differentiated ductal CIS vs. atypical hyperplasia.

Assessment of Sarcoplasmic Reticulum Ca 2+ Depletion During Spontaneous Ca 2+ Waves in Isolated Permeabilized Rabbit Ventricular Cardiomyocytes

In this study, Ca 2+ release due to spontaneous Ca 2+ waves was measured both from inside the sarcoplasmic reticulum (SR) and from the cytosol of rabbit cardiomyocytes. These measurements utilized Fluo5N-AM for intra-SR Ca 2+ from intact cells and Fluo5F in the cytosol of permeabilized cells. Restricted subcellular volumes were resolved with the use of laser-scanning confocal microscopy. Local Ca 2+ signals during spontaneous Ca 2+ release were compared with those induced by rapid caffeine application. The free cytoplasmic [Ca 2+] increase during a Ca 2+ wave was 98.1% ± 0.3% of that observed during caffeine application. Conversion to total Ca 2+ release suggested that Ca 2+ release from a Ca 2+ wave was not significantly different from that released during caffeine application (104% ± 6%). In contrast, the maximum decrease in intra-SR Fluo-5N fluorescence during a Ca 2+ wave was 82.5% ± 2.6% of that observed during caffeine application. Assuming a maximum free [Ca 2+] of 1.1 mM, this translates to a 96.2% ± 0.8% change in intra-SR free [Ca 2+] and a 91.7% ± 1.6% depletion of the total Ca 2 +. This equates to a minimum intra-SR free Ca 2+ of 46 ± 7 μM during a Ca 2+ wave. Reduction of RyR2 Ca 2+ sensitivity by tetracaine (50 μM) reduced the spontaneous Ca 2+ release frequency while increasing the Ca 2+ wave amplitude. This did not significantly change the total depletion of the SR (94.5% ± 1.1%). The calculated minimum [Ca 2+] during these Ca 2+ waves (87 ± 19 μM) was significantly higher than control ( p < 0.05). A computational model incorporating this level of Ca 2+ depletion during a Ca 2+ wave mimicked the transient and sustained effects of tetracaine on spontaneous Ca 2+ release. In conclusion, spontaneous Ca 2+ release results in substantial but not complete local Ca 2+ depletion of the SR. Furthermore, measurements suggest that Ca 2+ release terminates when luminal [Ca 2+] reaches ∼50 μM.