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 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.     

Biophysical methods for biochip analysis. Use of wide-field digital fluorescence microscopy

This paper discusses the development of biophysical methods for biochip analysis. A scheme and construction of a biochip analyzer based on wide-field digital fluorescence microscopy are described. The analyzer is designed to register images of biological microchips labeled with fluorescent dyes. The device developed is useful for high-sensitivity throughput recording of analyses with biochips after interaction of immobilized probes with fluorescently labeled sample molecules as well as it provides a higher rate of the analysis compared with laser scanning devices. With this analyzer, the scope where biological microchips can be applied becomes wider, development of new protocols of the analyses is possible and standard analyses run faster with the use of biochips, the expenses for performing routine analyses can be reduced.     

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.     

Multiple wavelength fluorescence enhancement on glass substrates for biochip and cell analyses

In microarrays experiments, a serious limitation is the unreliability of low signal intensities data and the lack of reproducibility for the resulting ratios between samples and controls. Most of the light emitted by a fluorophore at the air/glass interface of a glass slide is absorbed by the glass so just a part of the emitted fluorescence is detected. To improve the sensitivity of the fluorescence detection of both common fluorophores Cy3 and Cy5 in DNA microarrays and fluorescent cell analyses, we have designed a multi layer mirror with alternative thin layers of SiO2 and HfO2. This mirror (MOTL) prevents fluorescence absorption, allows the simultaneous enhancement of the fluorescence signals and increases the dynamic range of the slides. Using MOTL slides, Cy3 and Cy5 intensities are enhanced by 5-8-fold, consequently, the fluorescence analysis becomes easier and should allow the detection of low copy number genes or weakly fluorescent cells. With the same approach, other multiple optical thin layer slides could be designed for other series of fluorophores, extending the field of their applications.     

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 microscopy for modeling electron microbeam irradiation of skin

For radiation exposures employing targeted sources such as particle microbeams, the deposition of energy and dose will depend on the spatial heterogeneity of the sample. Although cell structural variations are relatively minor for two-dimensional cell cultures, they can vary significantly for fully differentiated tissues. Employing high-resolution confocal microscopy, we have determined the spatial distribution, size, and shape of epidermal keratinocyte nuclei for the full-thickness EpiDerm™ skin model (MatTek, Ashland, VA). Application of these data to calculate the microdosimetry and microdistribution of energy deposition by an electron microbeam is discussed.     

Bridging fluorescence microscopy and electron microscopy

Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy in biology is the ability to follow specific targets on or in living cells, revealing dynamic localization and/or function of target molecules. One of the inherent limitations of fluorescence microscopy is the resolution. Several efforts are undertaken to overcome this limit. The traditional and most well-known way to achieve higher resolution imaging is by electron microscopy. Moreover, electron microscopy reveals organelles, membranes, macromolecules, and thus aids in the understanding of cellular complexity and localization of molecules of interest in relation to other structures. With the new probe development, a solid bridge between fluorescence microscopy and electron microscopy is being built, even leading to correlative imaging. This connection provides several benefits, both scientifically as well as practically. Here, I summarize recent developments in bridging microscopy.     

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 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 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.     

Interaction between presenilin 1 and ubiquilin 1 as detected by fluorescence lifetime imaging microscopy and a high-throughput fluorescent plate reader

Presenilin 1 (PS1) in its active heterodimeric form is the catalytic center of the gamma-secretase complex, an enzymatic activity that cleaves amyloid precursor protein (APP) to produce amyloid beta (Abeta). Ubiquilin 1 is a recently described PS1 interacting protein, the overexpression of which increases PS1 holoprotein levels and leads to reduced levels of functionally active PS1 heterodimer. In addition, it has been suggested that splice variants of the UBQLN1 gene are associated with an increased risk of developing Alzheimer disease (AD). However, it is still unclear whether PS1 and ubiquilin 1 interact when expressed at endogenous levels under normal physiological conditions. Here, we employ three novel fluorescence resonance energy transfer-based techniques to investigate the interaction between PS1 and ubiquilin 1 in intact cells. We consistently find that the ubiquilin 1 N terminus is in close proximity to several epitopes on PS1. We show that ubiquilin 1 interacts both with PS1 holoprotein and heterodimer and that the interaction between PS1 and ubiquilin 1 takes place near the cell surface. Furthermore, we show that the PS1-ubiquilin 1 interaction can be detected between endogenous proteins in primary neurons in vitro as well as in brain tissue of healthy controls and Alzheimer disease patients, providing evidence of its physiological relevance.     

High-throughput fluorescence microscopy for systems biology

In this post-genomic era, we need to define gene function on a genome-wide scale for model organisms and humans. The fundamental unit of biological processes is the cell. Among the most powerful tools to assay such processes in the physiological context of intact living cells are fluorescence microscopy and related imaging techniques. To enable these techniques to be applied to functional genomics experiments, fluorescence microscopy is making the transition to a quantitative and high-throughput technology.