A guided tour into subcellular colocalization analysis in light microscopy

It is generally accepted that the functional compartmentalization of eukaryotic cells is reflected by the differential occurrence of proteins in their compartments. The location and physiological function of a protein are closely related; local information of a protein is thus crucial to understanding its role in biological processes. The visualization of proteins residing on intracellular structures by fluorescence microscopy has become a routine approach in cell biology and is increasingly used to assess their colocalization with well‐characterized markers. However, image‐analysis methods for colocalization studies are a field of contention and enigma. We have therefore undertaken to review the most currently used colocalization analysis methods, introducing the basic optical concepts important for image acquisition and subsequent analysis. We provide a summary of practical tips for image acquisition and treatment that should precede proper colocalization analysis. Furthermore, we discuss the application and feasibility of colocalization tools for various biological colocalization situations and discuss their respective strengths and weaknesses. We have created a novel toolbox for subcellular colocalization analysis under ImageJ, named JACoP, that integrates current global statistic methods and a novel object‐based approach.     

Evaluation of epoxy resin sealer after three root canal filling techniques by confocal laser scanning microscopy

The aim of this study was to evaluate the penetration of endodontic sealer into the dentin tubules, the integrity of the sealer layer perimeter, and the sealer area at the apical third after different filling techniques by confocal laser scanning microscopy (CLSM). Forty‐five mandibular premolars were mechanically prepared with ProTaper files, until F5 file. Thereafter, they were filled with an epoxy‐resin sealer (AH Plus) mixed with Rhodamine B dye (0.1% proportion) and allocated in three groups: Group 1, single master cone; Group 2, cold lateral compaction; and Group 3, Thermafil. For confocal laser scanning microscopy analysis, the specimens were transversely sectioned at 4 mm from the apex. The images at ×10 and ×40 were analyzed by Imagetool 3.0 software. Significant differences were not found among the three experimental groups according the dentin‐impregnate area by the sealer (P = 0.68) and between the sealer and root canal perimeter (P = 0.18). However, root canal filling techniques were significantly different when apical sealer areas were compared (P = 0.001). Thermafil group showed smaller sealer areas (8.09%) while cold lateral compaction and gutta‐percha master cone showed similar areas (17.37 and 21.18%, respectively). The dentin‐impregnated area was not dependent on the root canal filling technique. Single master cone, cold lateral condensation and Thermafil techniques presented integrity of the sealer perimeter close to 100% and Thermafil resulted in a significantly thinner sealer layer. Microsc. Res. Tech. 75:1277–1280, 2012. © 2012 Wiley Periodicals, Inc.     

Confocal laser scanning microscopy of chondrocytes in vitro: Cytoskeletal changes after quinolone treatment

The use of quinolone antibiotics would be significant for chronically diseased children (e.g., cysticfibrosis) as a prophylactic long-term treatment. However, quinolones were shown to cause cartilage damage in experimental animals when administered during certain developmental stages. In the present study, the effect of quinolones on chondrocytes was studied in a cell culture model in order to avoid animal experiments, to investigate the influence of single factors, and to open up the possibility to test human tissue. Chondrocytes were obtained from hipjoint cartilage of 3 to 4-weeks-old rats and cultured in control medium or quinolone-supplemented medium. It was shown that quinolones heavily disturbed adhesion of chondrocytes to the culture dish, accompanied by changes in cell shape and cytoskeletal morphology. Reduction of filamentous actin (stress fibers) and disintegration of vimentin fibers was demonstrated by immunofluorescence and evaluated by con-focal laser scanning microscopy. In contrast, distribution and amount of the adhesion molecule integrin α did not change. Results of the present study indicate that quinolones disturb the adherence mechanism of chondrocytes and lead to cytoskeleton changes.     

Assessment of In vivo behavior of polymer tube nerve grafts simultaneously with the peripheral nerve regeneration process using scanning electron microscopy technique

In this study, scanning electron microscopy (SEM) has been applied for instantaneous assessment of processes occurring at the site of regenerating nerve. The technique proved to be especially useful when an artificial implant should have been observed but have not yet been extensively investigated before for assessment of nerve tissue. For in vivo studies, evaluation of implant's morphology and its neuroregenerative properties is of great importance when new prototype is developed. However, the usually applied histological techniques require separate and differently prepared samples, and therefore, the results are never a 100% comparable. In our research, we found SEM as a technique providing detailed data both on an implant behavior and the nerve regeneration process inside the implant. Observations were carried out during 12‐week period on rat sciatic nerve injury model reconstructed with nerve autografts and different tube nerve grafts. Samples were analyzed with haematoxylin‐eosin (HE), immunocytochemical staining for neurofillament and S‐100 protein, SEM, TEM, and the results were compared. SEM studies enabled to obtain characteristic pictures of the regeneration process similarly to TEM and histological studies. Schwann cell transformation and communication as well as axonal outgrowth were identified, newly created and matured axons could be recognized. Concurrent analysis of biomaterial changes in the implant (degradation, collapsing of the tube wall, migration of alginate gel) was possible. This study provides the groundwork for further use of the described technique in the nerve regeneration studies. SCANNING 35: 232‐245, 2013. © 2012 Wiley Periodicals, Inc.     

Three-dimensional analysis of cell nucleus structures visualized by confocal scanning laser microscopy

Studies of the three-dimensional (3-D) organization of cell nuclei are becoming increasingly important for the understanding of basic cellular events such as growth and differentiation. Modern methods of molecular biology, including in situ hybridization and immunofluorescence, allow the visualization of specific nuclear structures and the study of spatial arrangements of chromosome domains in interphase nuclei. Specific methods for labelling nuclear structures are used to develop computerized techniques for the automated analysis of the 3-D organization of cell nuclei. For this purpose, a coordinate system suitable for the analysis of tri-axial ellipsoidal nuclei is determined. High-resolution 3-D images are obtained using confocal scanning laser microscopy. The results demonstrate that with these methods it is possible to recognize the distribution of visualized structures and to obtain useful information regarding the 3-D organization of the nuclear structure of different cell systems.     

Preliminary confocal scanning laser microscopy study of fluid inclusions in quartz

The initial results of the first dedicated confocal scanning laser microscopy (CSLM) study of fluid inclusions in quartz are presented. CSLM imaging of a large inclusion shows the quartz crystal to contain numerous small (< 1 μm), highly reflective inclusions arranged along planes in at least two directions that are not readily visible in transmitted light. The technique allows measurements to be made of the angular intersection and orientation of the planes in both two and three dimensions. Results suggest that larger inclusions (> 10 μm) occur where two planes of small inclusions intersect, and that the shape of the large inclusions is controlled by the angular relationship between intersecting planes.     

Time-lapse imaging of In Vitro myogenesis using atomic force microscopy

Myoblast therapy relies on the integration of skeletal muscle stem cells into distinct muscular compartments for the prevention of clinical conditions such as heart failure, or bladder dysfunction. Understanding the fundamentals of myogenesis is hence crucial for the success of these potential medical therapies. In this report, we followed the rearrangement of the surface membrane structure and the actin cytoskeletal organization in C2C12 myoblasts at different stages of myogenesis using atomic force microscopy (AFM) and confocal laser scanning microscopy (CLSM). AFM imaging of living myoblasts undergoing fusion unveiled that within minutes of making cell–cell contact, membrane tubules appear that unite the myoblasts and increase in girth as fusion proceeds. CLSM identified these membrane tubules as built on scaffolds of actin filaments that nucleate at points of contact between fusing myoblasts. In contrast, similarly behaving membrane tubules are absent during cytokinesis. The results from our study in combination with recent findings in literature further expand the understanding of the biochemical and membrane structural rearrangements involved in the two fundamental cellular processes of division and fusion.     

Penetrability of AH plus and MTA fillapex after endodontic treatment and retreatment: A confocal laser scanning microscopy study

The aim of the study was to assess the penetrability of two endodontic sealers (AH Plus and MTA Fillapex) into dentinal tubules, submitted to endodontic treatment and subsequently to endodontic retreatment. Thirty ex vivo incisors were prepared using ProTaper rotary system up to F3 instrument and divided in three groups according to the endodontic sealer used for root canal filling: AH Plus (AHP), MTA Fillapex (MTAF), and control group (CG) without using EDTA previously to the root canal filling. Rhodamine B dye (red) was incorporated to the sealers in order to provide the fluorescence which will enable confocal laser scanning microscopy (CLSM) assessment. All specimens were filled with gutta‐percha cones using the lateral compaction technique. The specimens were submitted to endodontic retreatment using ProTaper Retreatment system, re‐prepared up to F5 instruments and filled with gutta‐percha cones and the same sealer used during endodontic retreatment. Fluorescein dye (green) was incorporated to the sealer in order to distinguish from the first filling. The roots were sectioned 2 mm from the apex and assessed by CLSM. No difference was found between the two experimental groups (P > 0.05). On the other hand, in the control group the sealers were not capable to penetrate into dentinal tubules after endodontic treatment (P > 0.05). In retreatment cases, none of the sealers were able to penetrate into dentin tubules. It can be concluded that sealer penetrability is high during endodontic treatment. However, MTA Fillapex and AH Plus do not penetrate into dentinal tubules after endodontic retreatment. Microsc. Res. Tech. 77:467–471, 2014. © 2014 Wiley Periodicals, Inc.     

Exploration of the optimisation algorithms used in the implementation of adaptive optics in confocal and multiphoton microscopy

We report on the introduction of active optical elements into confocal and multiphoton microscopes in order to reduce the sample-induced aberration. Using a flexible membrane mirror as the active element, the beam entering the rear of the microscope objective is altered to produce the smallest point spread function once it is brought to a focus inside the sample. The conventional approach to adaptive optics, commonly used in astronomy, is to utilise a wavefront sensor to determine the required mirror shape. We have developed a technique that uses optimisation algorithms to improve the returned signal without the use of a wavefront sensor. We have investigated a number of possible optimisation methods, covering hill climbing, genetic algorithms, and more random search methods. The system has demonstrated a significant enhancement in the axial resolution of a confocal microscope when imaging at depth within a sample. We discuss the trade-offs of the various approaches adopted, comparing speed with resolution enhancement.     

Confocal mosaicing microscopy in skin excisions: a demonstration of rapid surgical pathology

Precise micro-surgical removal of tumour with minimal damage to the surrounding normal tissue requires a series of excisions, each guided by an examination of frozen histology of the previous. An example is Mohs surgery for the removal of basal cell carcinomas (BCCs) in skin. The preparation of frozen histology is labour-intensive and slow. Confocal microscopy may enable rapid detection of tumours directly in surgical excisions with minimal need for frozen histology. Mosaicing of images enables observation of nuclear and cellular morphology in large areas of surgically excised tissue. In skin, the use of 10–1% acetic acid as a reflectance contrast agent brightens nuclei in 0.5–5 min and enhances nuclear-to-dermis contrast and detectability of BCCs. A tissue fixture was engineered for precisely mounting surgical excisions to enable mosaicing of 36 × 36 images to create a field of view of 12 × 12 mm. This large field of view displays the excision at 2× magnification, similar to that routinely used by Mohs surgeons when examining frozen histology. Comparison of mosaics to histology demonstrates detectability of BCCs. Confocal mosaicing presently requires 9 min, instead of 20–45 min per excision for preparing frozen histology, and thus may provide a means for rapid pathology-at-the-bedside to expedite and guide surgery.     

Confocal laser scanning microscopy of hamster cerebellum using FM4-64 as intracellular staining

The FM4-64, a member of the family of fluorescent dyes, has been applied to the cerebellar cortex to evaluate its properties as an intracellular stain and intracortical tracer. Slabs of hamster cerebellum, 1-2 mm thick, were incubated in 10, 30, and 100 microns solutions of FM4-64 in sodium phosphate buffer and observed in a slow scan confocal laser scanning microscope. Mossy and climbing fibers were traced in the cerebellar white and gray substances. They exhibited a high fluorescence signal at the level of the myelin sheath. Mossy fibers were identified in the granular layer by their typical rosette formation and dichotomous bifurcation pattern. Climbing fiber bundles were observed crossing the granular layer and giving collateral branches around Golgi cell bodies. They ascend to the Purkinje cell layer on their way to the molecular layer. Cerebellar macroneurons (Golgi and Purkinje cells) and microneurons (granule, basket, and stellate cells) showed optimal intracellular staining of cell soma, axonal, and dendritic processes. The z-series of stacks of optodigital sections allowed us to explore in depth the cytoarchitectonic arrangement, nerve and glial cell morphology, and the topographic relationship with the afferent fibers.     

Three-dimensional reconstruction of aqueous channels in human trabecular meshwork using light microscopy and confocal microscopy

Conventional two-dimensional imaging of the trabecular meshwork (TM) provides limited information about the size, shape, and interconnection of the aqueous channels within the meshwork. Understanding the three-dimensional (3-D) relationships of the channels within this tissue may give insight into its normal function and possible changes present in the eye disease glaucoma. The purpose of our study was to compare laser scanning confocal microscopy with standard 1 μm Araldite-embeddedhistologic sections for 3-D analysis of the trabecular meshwork. In addition, the study was done to determine whether computerized 3-D reconstruction could isolate the fluid spaces of the trabecular meshwork and determine the size of interconnections between the fluid spaces. Confocal microscopy appears comparable to 1 μm Araldite-embedded tissue sections and has the advantage of inherent registration of the serial tissue sections. Three-dimensional reconstruction allowed the isolation of the fluid spaces within the trabecular meshwork and revealed the presence of numerous interconnections between larger fluid spaces. The distribution of these interconnections was randomly arranged, with no predilection for specific regions within the trabecular meshwork. This distribution of constrictions and “expansion chambers” may provide a clue to the mechanism by which subtle histologic changes are associated with increased ocular pressure in glaucoma.     

Multicolour analysis and local image correlation in confocal microscopy

Multiparameter fluorescence microscopy is often used to identify cell types and subcellular organelles according to their differential labelling. For thick objects, the quantitative comparison of different multiply labelled specimens requires the three-dimensional (3-D) sampling capacity of confocal laser scanning microscopy, which can be used to generate pseudocolour images. To analyse such 3-D data sets, we have created pixel fluorogram representations, which are estimates of the joint probability densities linking multiple fluorescence distributions. Such pixel fluorograms also provide a powerful means of analysing image acquisition noise, fluorescence cross-talk, fluorescence photobleaching and cell movements. To identify true fluorescence co-localization, we have developed a novel approach based on local image correlation maps. These maps discriminate the coincident fluorescence distributions from the superimposition of noncorrelated fluorescence profiles on a local basis, by correcting for contrast and local variations in background intensity in each fluorescence channel. We believe that the pixel fluorograms are best suited to the quality control of multifluorescence image acquisition. The local image correlation methods are more appropriate for identifying co-localized structures at the cellular or subcellular level. The thresholding of these correlation maps can further be used to recognize and classify biological structures according to multifluorescence attributes.     

Quantitative microscopy reveals 3D organization and kinetics of endocytosis in rat hepatocytes

In order to demonstrate the power of quantitative microscopy, the endocytic apparatus of rat hepatocytes was reexamined using in situ liver and short term cultured hepatocyte couplets that were allowed to internalize endocytic markers for various time intervals. Correlative confocal light and electron microscopy demonstrate a tubulovesicular reticulum representing the endocytic apparatus. Volume and membrane area account for 2% of cell volume and 30% plasma membrane surface. Colocalization analysis demonstrated that pathway-specific ligands and fluid-phase markers enter EEA1-positive vesicles, the early endosomal compartment, immediately after internalization. These vesicles are translocated rapidly from basolateral to perinuclear and apical locations. Ligands are sorted within 5 min to their respective pathways. Sequential colocalization of an asialoglycoprotein-pulse with rab7 and lamp3 demonstrates that early endosomes change into or fuse with late endosomes and lysosomes. Alternatively, markers are sequestered into the common endosome consisting of rab11-positive, long tubules that originate from early endosomes and show an affinity for the transcytotic marker pIgA and its receptor. This compartment mediates transcytosis by delivering the receptor-ligand complex to the subapical compartment, a set of apical, rab11-positive vesicles, which are connected to the tubular reticulum. We conclude that vesicular traffic between preexisting compartments, maturation or fusion of endocytic organelles, and transport in tubules act in concert and together mediate transport between compartments of a tubulovesicular endocytic apparatus. In addition, we show that quantitative microscopy using high resolution data sets can detect and characterize kinetics of various parameters thus adding a dynamic component to 3D information.     

Remote focusing in confocal microscopy by means of a modified Alvarez lens

Alvarez lenses are actuated lens‐pairs which allow one to tune the optical power by mechanical displacement of subelements. Here, we show that a recently realized modified Alvarez lens design which does not require mechanical actuation can be integrated into a confocal microscope. Instead of mechanically moving them, the sublenses are imaged onto each other in a 4f‐configuration, where the lateral image shift leading to a change in optical power is created by a galvo‐mirror. The avoidance of mechanical lens shifts leads to a large speed gain for axial (and hence also 3D) image scans compared to classical Alvarez lenses. We demonstrate that the suggested operation principle is compatible with confocal microscopy. In order to optimize the system, we have drawn advantage of the flexibility a liquid‐crystal spatial light modulator offers for the implementation. For given specifications, dedicated diffractive optical elements or freeform elements can be used in combination with resonant galvo‐scanners or acousto‐optic beam deflectors, to achieve even faster z‐scans than reported here, reaching video rate.     

Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin

We consider various strategies for confocal imaging of human skin which seek to reduce the effects of the specimen-induced aberrations. We calculate the spherical aberration introduced by the stratified structure of skin and show how the confocal signal is affected when attempting to image at various depths within the dermis. Using simple methods it is shown how images might be improved by compensating for the induced aberration. The methods include the use of an iris to reduce the pupil area, changing the refractive index of the immersion medium and using a lens with variable coverglass correction.     

Automated assessment of keratocyte density in clinical confocal microscopy of the corneal stroma

Cell density in the corneal stroma is typically determined by counting the number of bright objects, presumably keratocyte nuclei, in images from clinical confocal microscopy. We present a program that identifies bright objects and counts those that most likely represent cells. Selection variables were determined from 125 normal corneas with cell densities that had been assessed manually. The program was tested on 17 corneas of patients before and at several intervals to 5 years after laser in situ keratomileusis (LASIK) surgery. In these corneas, which showed a decrease in cell density after surgery, the program identified cells as well as human observers did.