Confocal laser scanning microscopy

Many technological advancements of the past decade have contributed to improvements in the photon efficiency of the confocal laser scanning microscope (CLSM). The resolution of images from the new generation of CLSMs is approaching that achieved by the microscope itself because of continued development in digital imaging methods, laser technology and the availability of brighter and more photostable fluorescent probes. Such advances have made possible novel experimental approaches for multiple label fluorescence, live cell imaging and multidimensional microscopy.     

Confocal microscopy of hair

Confocal microscopy is an excellent method for studying the localization of fluorescent stains. Used in this way, superior 3D images can be obtained from multiple optical sections with very shallow depth of field. The main advantage of this technique is that the sample is not damaged. We have taken serial confocal sections of hair and via specific image enhancement routines have obtained high-quality 3D images enabling the visualization of cuticle scale and its pattern of distribution. This has been done on various types of hair: bleached, permed and in certain pathological conditions. This first step will allow us to characterize the hair surface in terms of its roughness, and the distribution and form of cuticular scale, parameters that have potential in the assessment of dermocosmetic efficacy.     

Confocal scanning microscopy: three-dimensional biological imaging

Confocal scanning optical microscopy, arguably the most significant in biological light microscopy in this decade, enables one to obtain quantitative non-invasive optical sections through labelled biological specimens, virtually free from out-of-focus blur. A set of these optical sections collected at a series of focal levels through an object constitutes a three-dimensional image which may then be processed digitally for display as a computer reconstruction, a stereo pair or an animation sequence.     

Confocal laser scanning microscopy as an analytical tool in chromatographic research

In recent years, confocal laser scanning microscopy has been developed into a non-invasive tool to probe intra-particle profiles of protein in chromatographic adsorbents. A necessary prerequisite when using this technique lies in the labeling of proteins with fluorescent probes. The quality of the obtained results is thus strongly dependent on the probes used, its sensitivity on experimental parameters and the change of protein characteristics upon binding. In this review, the fundamental issues when using fluorescent probes are described, before giving a critical evaluation on published literature in the field of confocal laser scanning microscopy for the analysis of chromatographic principles.     

Confocal fluorescence microscopy of plant cells

Summary The confocal laser scanning microscope (CLSM) has become a vital instrument for the examination of subcellular structure, especially in fluorescently stained cells. Because of its ability to markedly reduce out-of-focus flare, when compared to the conventional wide-field fluorescence microscope, the CLSM provides a substantial improvement in resolution along the z axis and permits optical sectioning of cells. These developments have been particularly helpful for the investigation of plant cells and tissues, which because of their shape, size, and optical properties have been difficult to analyze at high resolution by conventional means. We review the contribution that the CLSM has made to the study of plant cells. We first consider the principle of operation of the CLSM, including a discussion of image processing, and of lasers and appropriate fluorescent dyes. We then summarize several studies of both fixed and live plant cells in which the instrument has provided new or much clearer information about cellular substructure than has been possible heretofore. Attention is given to the visualization of different components, including especially the cytoskeleton, endomembranes, nuclear components, and relevant ions, and their changes in relationship to physiological and developmental processes. We conclude with an effort to anticipate advances in technology that will improve and extend the performance of the CLSM. In addition to the usual bibliography, we provide internet addresses for information about the CLSM.     

Confocal microscopy and plant cell biology: A perfect match

Abstract

The present short introduction to confocal microscopy in plant cell biology is meant to be a reference addressed to those who are approaching these studies. Optical and genetic techniques developed over the last years have revolutionised plant cell biology. They enable in vivo studies of cell organisation, mainly based on the use of confocal microscopy and derivatives of the Green Fluorescent Protein (GFP). Such fluorescent proteins are extremely useful tools for directly monitoring gene expression and protein localisation, and for selectively labelling sub-cellular structures. GFP-expressing plants can be directly examined by confocal microscopy to obtain high-resolution optical sections of intact tissues and to allow time-lapse observation of dynamic processes. The application of such approaches to the investigation of root cell responses during arbuscular mycorrhizal colonisation is discussed.     

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.     

Confocal microscopy of germinal vesicle-stage equine oocytes

The objectives were to compare cumulus type with nucleus form in equine cumulus oocyte complexes (COCs), to define the percentage of germinal vesicle (GV)-stage oocytes within a population of mares, and to further define GV nucleus shapes of equine oocytes. Cumulus types were as follows: 1) compact (56/208, 26.9%), 2) slightly expanded (37/208, 17.8%), 3) moderately expanded (27/208, 13.0%), 4) greatly expanded (15/208, 7.2%), or 5) denuded (73/208, 35.1%). One hundred thirty of 208 COCs (62.5%) were GV-stage, 21/208 (10.1%) were condensed chromatin-stage, 8/208 (3.8%) were polar body-stage, 40/208 (19.2%) were negative (nonstaining), and 9/208 (4.3%) were fragmented. Cumulus types were associated with nucleus forms because higher proportions (P < 0.05) of GV-stage oocytes occurred in compact (42/56, 75.0%), slightly expanded (30/37, 81.1%), moderately expanded (16/27, 59.3%), or denuded (40/73, 54.8%) COCs than in greatly expanded (2/15, 13.3%) COCs. In contrast, lower proportions (P<0.05) of condensed chromatin-stage oocytes occurred in compact (3/56, 5.4%), slightly expanded (0/37, 0.0%), moderately expanded (3/27, 11.1%) or denuded (9/73, 12.3%) COCs than in greatly expanded (6/15, 40.0%) COCs, and lower proportions (P < 0.05) of polar body-stage oocytes occurred in compact (0/56, 0.0%) or denuded (2/73, 2.7%) COCs than in greatly expanded (3/15, 20.0%) COCs. Germinal vesicle-stage equine oocytes had 4 distinct shapes, with higher proportions (P<0.05) having large-regular (54/130, 41.5%) than scattered (10/130, 7.7%), small-round (29/130, 22.3%), or large-irregular (37/130, 28.5%) shapes. Lower proportions (P<0.05) of large-regular GVs occurred in compact (11/42, 26.2%) COCs than in slightly expanded (15/30, 50.0%), or moderately expanded (12/16, 75.0%) COCs. Therefore oocytes with the large-regular GV shape are probably more advanced in development.     

Confocal scanning optical microscopy and three-dimensional imaging

Summary— Confocal scanning optical microscopy has significant advantages over conventional fluorescence microscopy: it rejects the out-of-locus light and provides a greater resolution than the wide-field microscope. In laser scanning optical microscopy, the specimen is scanned by a diffraction-limited spot of laser light and the fluorescence emission (or the reflected light) is focused onto a photodetector. The imaged point is then digitized, stored into the memory of a computer and displayed at the appropriate spatial position on a graphic device as a part of a two-dimensional image. Thus, confocal scanning optical microscopy allows accurate non-invasive optical sectioning and further three-dimensional reconstruction of biological specimens. Here we review the recent technological aspects of the principles and uses of the confocal microscope, and we introduce the different methods of three-dimensional imaging.     

Confocal fluorescence microscopy in modern cell biology

Confocal fluorescence microscopy has become a major tool in modern cell biology. The paper explains the basic principles and especially the depth discrimination properties of confocal microscopy. An important application is described briefly and outlined with some figures. The paper concludes with remarks on features to be expected in the near future.     

Confocal reader for biochip screening and fluorescence microscopy

We developed a fluorescence reader for the sensitive detection of surface-generated fluorescence. The system is applicable for high resolution imaging as well as for the readout of large biochips. The surface of a microscope coverslip is scanned with a laser beam focused to a waist diameter of 500 nm (FWHM) by means of a single aspheric lens. Scanning large areas with a focused beam usually evokes the need of automatic control elements to adjust the laser spot to the designated position at the surface. Due to the special design of the reader, the focus keeps at the plane of the surface even when scanning large areas, obviating the requirement of any real time control. Thus the instrument is straightforward and inexpensive. Nevertheless it features a high sensitivity and high optical resolution. The versatility of the instrument is demonstrated by imaging cells and reading out a DNA-chip. The excellent sensitivity is shown by detecting single fluorescently labeled antibodies.     

Confocal microscopy via reciprocal optical fibre image detection

We describe a compact form of confocal scanning microscope using a semiconductor laser. Confocal operation is ensured by the use of a single mode optical fibre for both launching the light into the microscope and collecting the signal from the object. The collected light is allowed to re-enter the laser and the image is detected as a modulation on the signal from the laser power monitor diode. Images are compared with those obtained from traditional point detectors. The alignment tolerances of the reciprocal scheme are found to be greatly reduced over conventional confocal systems.     

Confocal laser scanning microscopy of dextran-rice starch mixtures

The microstructure of rice starch and dextran-rice starch mixtures was studied using confocal laser scanning microscopy (CSLM) and scanning electron microscopy (SEM). Surface pores and channels of rice starch were looked for. Channels could not be found in rice starch granules after reaction with 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde (CBQCA). Fluorescein-labeled dextran was mixed with rice starch in order to locate hydrocolloid molecules in hydrocolloid-rice starch mixtures. The results showed that FITC-dextran (ave. Mw 4000; FD4) penetrated into raw rice starch granules, with the degree of penetration varying from granule to granule. FD4 could penetrate throughout cooked rice starch granules. FITC-dextran with an average Mw of more than 10,000 could not penetrate either raw or cooked rice starch granules.