Application Of Confocal Laser Scanning Microscopy Research Articles

Tools of the Trade: Image Analysis Programs for Confocal Laser-Scanning Microscopy Studies of Biofilms and Considerations for Their Use by Experimental Researchers.

Confocal laser-scanning microscopy (CLSM) is the bedrock of the microscopic visualization of biofilms. Previous applications of CLSM in biofilm studies have largely focused on observations of bacterial or fungal elements of biofilms, often seen as aggregates or mats of cells. However, the field of biofilm research is moving beyond qualitative observations alone, toward the quantitative analysis of the structural and functional features of biofilms, across clinical, environmental, and laboratory conditions. In recent times, several image analysis programs have been developed to extract and quantify biofilm properties from confocal micrographs. These tools not only vary in their scope and relevance to the specific biofilm features under study but also with respect to the user interface, compatibility with operating systems, and raw image requirements. Understanding these considerations is important when selecting tools for quantitative biofilm analysis, including at the initial experimental stages of image acquisition. In this review, we provide an overview of image analysis programs for confocal micrographs of biofilms, with a focus on tool selection and image acquisition parameters that are relevant for experimental researchers to ensure reliability and compatibility with downstream image processing.

Confocal laser scanning microscopy (CLSM) as a new tool for morphological characterisation of both newly collected and museum voucher specimens of the Trypanorhyncha Diesing, 1863 (Platyhelminthes: Cestoda).

Taxonomic issues within Trypanorhyncha, e.g., the inaccurate light microscopic visualisation of the hook patterns, are solvable by confocal laser scanning microscopy (CLSM). We applied CLSM imaging to study Trygonicola macropora (Shipley et Hornell, 1906) and Dollfusiella michiae (Southwell, 1929) from Neotrygon caeruliopunctata Last, White et Séret from Bali, Indonesia. To illustrate the strength and limitations of CLSM, images of Otobothrium cysticum (Mayer, 1842) and Symbothriorhynchus tigaminacantha Palm, 2004, both permanent mounts from a collection, were also processed. The CLSM created image stacks of many layers, and edited with IMARIS Software, these layers resulted in three-dimensional images of the armature patterns and internal organs of both species. BABB (benzylalcohol and benzylbenzuolate) clearing was applied to T. macropora. We conclude that trypanorhynch cestodes stained with Mayer-Schuberg's acetic carmine permanently mounted in Canada balsam are suitable for CLSM, allowing detailed analyses of museum type-material as well as freshly collected and processed worms. BABB resulted in imaging the testes in detail, suggesting other stains to be used for CLSM in trypanorhynch cestode research. Application of CLSM for studies of other cestode groups is highly recommended.

Fluorescence spectroscopy of ancient sedimentary organic matter via confocal laser scanning microscopy (CLSM)

Fluorescence spectroscopy via confocal laser scanning microscopy (CLSM) was used to analyze ancient sedimentary organic matter, including Tasmanites microfossils in Devonian shale and Gloecapsomorpha prisca (G. prisca) in Ordovician kukersite from North American basins. We examined fluorescence emission as a function of excitation laser wavelength, sample orientation, and with respect to location within individual organic entities and in transects across bedded organic matter. Results from spectral scans of the same field of view in Tasmanites with different laser lines showed progressive red-shift in emission maxima with longer excitation wavelengths. This result indicates steady-state Tasmanites fluorescence emission is an overlapping combination of emission from multiple fluorophore functions. Stokes shift decreased with increasing excitation wavelength, further suggesting the presence of multiple fluorophore functions with different S1 → S0 transition energies. This observation also indicates that at longer excitation wavelengths, less absorbed light energy is dissipated via collisional transfer than at shorter excitation wavelengths and may suggest fewer polar functions are preferentially absorbing. Confirming earlier results, emission spectra observed from high fluorescence intensity regions (fold apices) in individual Tasmanites are blue-shifted relative to emission from other locations in the same microfossil. We suggest high intensity emission is from photoselective alignment of polarized excitation with the fluorophore absorption and emission transition moment. The blue shift observed in regions of high intensity emission may be due to relative absence or realignment of polar species, e.g., bridging ether or ester functions, although variations in O abundance could not be confirmed with preliminary time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis. Tasmanites occurring in consolidated sediments are flattened from original spherical morphology and, in optical microscopy, this burial deformation results in generally parallel extinction (strain-influenced) and positive elongation. The deformation also induces fluorescence anisotropy observed as variations in emission wavelength when individual Tasmanites are measured from their long axis parallel to bedding, whereas this effect is absent in bedding-normal view. Transects from G. prisca-rich source layers into adjacent reservoir layers show decrease in fluorescence intensity and spectral red-shift (increase in full-width half-maximum with increasing red portion of the half-width). These results may suggest an increase in fluorescence quenching across the source-to-reservoir transition zone, consistent with an increase in aromaticity following petroleum expulsion and migration. These observations are supported by increasing reflectance values measured across similar micro-scale transects. Our results highlight the applicability of CLSM as a broad and under-utilized approach for the characterization of sedimentary organic matter and are discussed with perspective toward petroleum processes and thermal indices research.

Visualization of materials using the confocal laser scanning microscopy technique.

The development of materials science always benefits from advanced characterizations. Currently, imaging techniques are of great technological importance in both fundamental and applied research on materials. In comparison to conventional visualization methods, confocal laser scanning microscopy (CLSM) is non-invasive, with macroscale and high-contrast scanning, a simple and fast sample preparation procedure as well as easy operation. In addition, CLSM allows rapid acquisition of longitudinal and cross-sectional images at any position in a material. Therefore, the CLSM-based visualization technique could provide direct and model-independent insight into material characterizations. This review summarizes the recent applications of CLSM in materials science. The current CLSM approaches for the visualization of surface structures, internal structures, spatial structures and reaction processes are discussed in detail. Finally, we provide our thoughts and predictions on the future development of CLSM in materials science. The purpose of this review is to guide researchers to build a suitable CLSM approach for material characterizations, and to open viable opportunities and inspirations for the development of new strategies aiming at the preparation of advanced materials. We hope that this review will be useful for a wide range of research communities of materials science, chemistry, and engineering.

Application of Confocal Laser Scanning Microscopy in Bioseparation Studies: Visual Characterization of Protein Adsorption Behaviors

Confocal laser scanning microscopy (CLSM) as an innovative technique has been widely used in biological research and it offers unique opportunities to visualize distribution and transportation of proteins in various media. For protein chromatographic purification studies, CLSM can further provide direct and detailed information about protein adsorption behaviors. This review highlighted the application of CLSM in investigating protein adsorption processes including single-component and multi-component studies in finite bath adsorption and real-time observation of protein adsorption in the micro-column with various adsorbents. Factors such as liquid phase conditions and the limitation of CLSM application in protein adsorption were discussed. The adsorption profiles in different adsorbents, such as the widely observed shrinking core profile, the overshoot adsorption profile as well as some special profiles were compared and discussed. Keywords: Chromatography, confocal laser scanning microscopy, protein chromatography, protein adsorption behaviors.

Application of confocal laser-scanning microscopy (CLSM) to autofluorescent organic and mineral matter in peat, coals and siliciclastic sedimentary rocks — A qualitative approach

Organic matter occurring in the form of macerals and natural solid bitumens in peats, coals, and siliciclastic sediments of various stratigraphic ages and locations was studied by means of conventional incident light microscopy and CLSM. Classical optical microscopic investigations are restricted by conventional 2-D imaging and are often hindered by the associated out-of-focus-blur effects. The qualitative application of CLSM overcomes these limitations, permitting to reveal precisely 3-D morphology and detailed microstructure of autofluorescent organic matter. The in depth examinations along Z-axis in XY, XZ, or YZ scanning modes at increments of 250nm are capable to reconstruct 3-D internal configuration and arrangement occurring in autofluorescent macerals. The non-invasive and non-destructive laser-raster technique is applied to characterize in situ autofluorescent organic particles at micron to sub-micron scale resolution in polished whole rock pellets. In this study, we demonstrate that CLSM is suitable to (i) detect fine-scale microstructure of autofluorescent organic matter, (iii) reconstruct 3-D morphology at cellular and tissue level, (iii) analyze maceral composition in terms of its relicts and fragments beyond the classical optical assignment limits, (iv) detect and examine inorganic components enclosed in the embedding mineral-bituminous groundmass, and (v) differentiate mineral constituents from the remaining organic matter.CLSM as an optical microscopic technique was applied to generate high-resolution 2-D and 3-D images, projections as well as 3-D reconstructions of autofluorescent objects excited with aid of laser-induced light operating at variable visible wavelengths. CLSM is applicable to support microscopic studies of thermally immature and mature organic matter within the oil and early gas windows enclosed either within siliciclastic and carbonate sediments or in peat and low to medium rank coals.The application of this technique will be of great interest to a wide range of geology related disciplines such as petroleum geology, biostratigraphy, marine geology, peat petrography, and archeology.

Non-invasive in vivo imaging by confocal laser scanning microscopy of gingival tissues following natural plaque deposition

Imaging with Confocal Laser Scanning Microscopy (CLSM) generates high-resolution images and may be well suited for basic research in Periodontology and Implant Dentistry. The present study was aimed to explore the in vivo application of CLSM in experimentally induced gingivitis. Ten subjects were recruited and were advised to stop any oral hygiene of the upper front teeth for 7 days. The gingival tissues were observed using a Heidelberg Retina Tomograph combined with a Rostock Cornea Module at baseline and day 7. The system used a laser of 670 nm and the contrast was given by backscattering from different tissues. Each examination created 800-1200 images that were descriptively analysed. After 7 days of abandoned oral hygiene, plaque scores and bleeding frequencies increased. By using CLSM images tooth hard substances, cells and plaque deposits were distinguishable. Increased epithelial cell irregularities, the apical migration of the sulcular epithelium, cellular infiltrates within the sulcus and plaque deposits were observed at day 7. The present study showed for the first time that CLSM is suitable for in vivo imaging of the gingival sulcus and adjacent tissues.

In vivoconfocal microscopy in dermatology: from research to clinical application

Confocal laser scanning microscopy (CLSM) represents an emerging technique for the noninvasive histomorphological analysis of skin in vivo and has shown its applicability for dermatological research as well as its value as an adjunct tool in the clinical management of skin cancer patients. Herein, we aim to give an overview on the current clinical indications for CLSM in dermatology and also highlight the diverse applications of CLSM in dermatological research.

Use of confocal microscopy for nanoparticle drug delivery through skin

Confocal laser scanning microscopy (CLSM) is a well-used microscopic tool that provides valuable morphological and functional information within cells and tissues. The application of CLSM to skin and the topical penetration of nanoparticles (NP) will be addressed. First, we describe the advantages of confocal microscopy compared to other techniques and its use relative to skin research. Second, we discuss the ability of CLSM to detect single NP. Regarding their interaction with skin, the appropriate method to retain nanoparticle localization in the tissue with minimal fixation is critically important. Also, the interaction of several different types of NP (quantum dots, fullerene and dendrimers) and their interaction with skin detected by CLSM under various conditions (flexed, tape stripped and abraded skin) is reviewed. Finally, human epidermal keratinocytes and dendritic cells that serve as appropriate in vitro models for skin cell interactions and cellular uptake of NP are also discussed. In conclusion, the unique functions of CLSM such as the ability to detect fluorescence, optical sectioning, three dimensional remodeling, as well as its use in the reflection mode in tandem with other methods, provides great promise with broad applications regarding the interactions of nanomaterials with skin.

Use of Confocal Microscopy as a Nondestructive Method in the Study of Ink Jet Dot Formation

The formation of the printing ink dot on the substrate is the final and the most critical phase in the printing process. The ink distribution on the printing surface and the drying mechanism depend on ink characteristics as well as on many other factors, for example, surface energy, roughness, sizing, and porosity. Suitable dot gain and high circularity (near unity) of printed dots predict the final print quality. High deviation from ideal circularity could cause undesired phenomena like wicking and bleeding. The aim of the present study is to determine ink dot formation by three different microscopic methods, optical microscopy (OM), scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). The main goal of the authors research was to evaluate the applicability of CLSM as a nondestructive method for three-dimensional visualization in the analysis of ink dot formation on UV ink jet prints. To validate the feasibility of such means in a three-dimensional context, the images obtained are compared to those obtained by traditional two-dimensional imaging systems such as OM and SEM. The authors show that the CLSM produces a replica of the cross-sectioned dot profile as seen in SEM. This means that the CLSM technique can be used to rapidly assess the dot profile without physical sectioning.

In vivo imaging of intradermal tattoos by confocal scanning laser microscopy

In vivo confocal laser scanning microscopy (CLSM) is a new method for high-resolution imaging of intact skin in situ. Horizontal mapping of the outer skin is provided (magnification x 1000). Tattooing is popular all over the world; however, tattooed skin has not been studied in using CLSM. Tattoos in two volunteers were studied using the Vivascope1500 of Lucid Inc. Subepidermal massive deposits of dense, clustered pigment granules up to about 3 mum in size corresponding to black tattoos, and more scarce and diffuse deposits, corresponding to red, blue and green tattoos, were observed. Diffuse pigment granules tended to accumulate in the outer dermis underneath the level of the basement membrane zone. Dermal pigments from tattoos can be imaged in vivo using CLSM. This application of CLSM has an important future potential for pre-evaluation of tattoos before laser removal, predicting good or poor outcome of laser removal.

Confocal laser scanning microscopy in orthopaedic research

Confocal laser scanning microscopy (CLSM) is a type of high-resolution fluorescence microscopy that overcomes the limitations of conventional widefield microscopy and facilitates the generation of high-resolution 3D images from relatively thick sections of tissue. As a comparatively non-destructive imaging technique, CLSM facilitates the in situ characterization of tissue microstructure. Images generated by CLSM have been utilized for the study of articular cartilage, bone, muscle, tendon, ligament and menisci by the foremost research groups in the field of orthopaedics including those teams headed by Bush, Errington, Guilak, Hall, Hunziker, Knight, Mow, Poole, Ratcliffe and White. Recent evolutions in techniques and technologies have facilitated a relatively widespread adoption of this imaging modality, with increased “user friendliness” and flexibility. Applications of CLSM also exist in the rapidly advancing field of orthopaedic implants and in the investigation of joint lubrication.

New insights in the spatially resolved dynamic pH measurement in macroscopic large absorbent particles by confocal laser scanning microscopy

Both, experimental investigation of protein adsorption processes and mathematical models describing such processes indicate, that the pH in the absorbent particle might be the key factor for an improved understanding of these chromatographic processes. Thus, a technique aiming at the spatially resolved pH measurement in macroscopic large absorbent particles is presented. The first application of this method, being based on confocal laser scanning microscopy (CLSM), revealed an apparent dependence of the pH calibration curve on the scanning depth. By a model-based approach, factors distorting the measurement signal are identified: The wavelength-dependent light scattering and the re-absorption of emitted light. The resulting consequences for further development and application of CLSM based techniques to measure pH in macroscopic large absorbent particles are illustrated and discussed.

Three-dimensional imaging of proton gradients at microelectrode surfaces using confocal laser scanning microscopy

The first use of fluorescence confocal laser scanning microscopy (CLSM) to image three-dimensional pH gradients at electrode surfaces is described, using the reduction of benzoquinone (BQ) to hydroquinone in aqueous solution as an example. The associated local pH changes accompanying the process have been imaged using a trace amount of fluorescein, which has a pH-dependent fluorescent signal. Images recorded in x–y–z space, allow pH profiles to be obtained as a function of applied electrode potential. Experimentally determined profiles measured when BQ is reduced at a diffusion-limited rate are shown to be in good agreement with predictions from numerical simulation. Future applications of CLSM for pH imaging at electrode surfaces and its use in scanning electrochemical microscopy (SECM) are highlighted briefly.

The contribution of quantitative confocal laser scanning microscopy in cartilage research: chondrocyte insulin-like growth factor-1 receptors in health and pathology.

The use of immunohistochemical detection techniques and fluorescent molecular probes in light and fluorescence microscopy allows accurate and specific analysis of a great variety of cell and tissue components. However, when staining yields only low intensity levels, serious problems may arise with discrimination of specific signals against background staining. This problem is often inherent with articular cartilage research. Application of confocal laser scanning microscopy (CLSM) can circumvent these problems. The CLSM collects images that are almost free of out-of-focus signals, which results in improved spatial resolution and discrimination as compared with conventional microscopy. Moreover, CLSM allows optical sectioning of specimens and three-dimensional reconstruction of the microscopical object. Quantitative evaluation of microscopical images is hampered by out-of-focus signals because they interfere with specific signals in the image. Interference of these nonspecific signals can be diminished by application of CLSM; in CLSM only one single point in microscopical objects is illuminated at any time and this point is then imaged into the pinhole at the entrance of the photo-detector and subsequently digitized. The present review is a discussion of the present state of the art in digital imaging with the use of CLSM in cartilage research. This discussion includes aspects such as sensitivity, specificity, spatial resolution and accuracy of quantitative analysis in microscopical immunofluorescent objects.

Conversion of biocytin labelled cells and structures for the confocal laser-scanning method

The method for converting biocytin preparations of brain sections fills a gap in the application of confocal laser-scanning microscopy. Both neuronal and non-neuronal structures are converted. The background remains free of staining. The protocol can be applied to old and already existing biocytin-(diaminobenzidine)-nickel preparations which are then made accessible to evaluation with the laser-scanning microscope by the substitution of nickel with silver-gold. Sodium thiosulphate is used to remove the unbound silver. The reflection image of the laser-scanning microscopy provides more information than the transmission image.