H. T. M. van der Voort

Three-dimensional confocal fluorescence microscopy.

Publisher Summary Biological material is organized in four dimensions: three spatial ones and a temporal one. Light microscopy is able to visualize biological objects in their natural watery condition and during their temporal development. Improved imaging is the optical sectioning property by which the contributions from out-of-focus areas in the specimen are effectively suppressed. In normal microscopy, these contributions lead to a strong reduction in the available image contrast. A three-dimensional microscope is obtained where each data point as collected represents the quantity of the specific contrast parameter used at a certain point in space. Deconvolution techniques have been developed for eliminating the out-of-focus information from conventional fluorescence microscopy. Confocal microscopy can deliver directly clear optical sections without the use of time-consuming image reconstruction algorithms. Image processing can also be used to enhance the confocal images. Computer-generated stereoscopic images are also used for the visualization of the three-dimensional biological information. This chapter discusses the number of aspects of confocal imaging, especially the advantages and drawbacks of the various scanning approaches.

Three-dimensional structure of living chloroplasts as visualized by confocal scanning laser microscopy

SummaryThe newly developed confocal scanning laser microscope, together with image processing by computer, has been used to obtain three-dimensional information on the organization of grana in chloroplasts in living plant tissue. Chloroplasts are ideally suited for such studies because their pigments show bright autofluorescence. The high-resolution stereo images bridge a gap between classic light microscopy and electron microscopy. Our preliminary observations on several plant species resemble most the early observations of Strugger (1951: Die Strukturordnung im Chloroplasten. Ber Deutsch Bot Ges 64: 69–83) and suggest that the 3-D technique might well be suitable to solve discrepancies in the interpretation of classical light microscopic and electron microscopic observations.

Size and Shape of The Confocal Spot: Control and Relation to 3D Imaging and Image Processing

A confocal microscope can be considered as a 3D sampling instrument for collecting data from spatial structures, especially biological ones. Optimal data collection in confocal microscopes requires the adaptation of the dimensions of the sampling volume to the lateral and axial raster parameters employed during data collection. It is shown how, in principle, the collection volume can be partly manipulated by the use of variable pinholes both in the illumination and detection paths. The effective confocal spot will depend on the optics used, the degree of aberration, and the alignment of the instrument. Measurements of the axial response both in fluorescence and reflection for some high N.A. lens systems as a function of the above factors are presented. The use of variable pinholes in computer-controlled instruments is discussed, especially in relation to operation in fluorescence. It is indicated that proper interpretation and processing of 3D confocal data requires at least approximate knowledge of the applicable 3D response function.

Fast volume render techniques for interactive analysis

Without graphics hardware, interactive volume rendering is almost impossible with the current generation of computers and software. We describe the implementation of a volume renderer for interactive analysis of confocal images. We propose several techniques to accelerate the rendering of grey-value volumes. We propose to illuminate the volume selectively with ray templates to get a proper shadow cue in the shortest feasible time. In the viewing phase, rendering is distinctively accelerated for four user interactions: (1) a total change by successive adaptive refinement, (2) an unknown change in the view with this refinement strategy combined with suspended interpolation, (3) a known change in the view by recalculating only that part and (4) a view translation by recalculating the uncovered part.

High-resolution confocal scanning light microscopy in biology

Abstract Confocal scanning light microscopy provides an appreciable improvement in resolving power compared with classical light microscopy; the amount of information that can be extracted from a specimen is increased by a factor of 3. When a laser serves as light source and u.v. wavelengths are used, resolutions of 140 nm are possible. Applications are the study of biological structures in the sub-μm region, for instance, the replicating nucleoid (DNA) morphology of live Escherichia coli and eukaryotic chromosomes.

Illumination And Detection Strategies For Confocal Microscopy

A confocal microscope can be considered as a 3-D sampling instrument to collect data from spatial structures, especially biological ones. Optimal performance requires the adaptation of the dimensions of the sampling volume to the lateral and axial raster parameters employed during data collection. It will be shown that the shape of the sampling volume can be controlled through optical means by a combination of specific illumination and detection parameters. The use of these techniques in computer controlled instruments is discussed, especially in relation to operation in fluorescence.

Modelling Of 3-D Confocal Imaging At High Numerical Aperture In Fluorescence

The imaging properties of a model high aperture fluorescence confocal microscope are studied by way of numerical methods. The model consists of an aberration-free high aperture objective illuminated by a monochromatic linear or circular polarized light source, and a detection system equipped with a finite sized pinhole. The computation of the intensity distribution near the confocal plane is based on electromagnetic diffraction theory. We study the 3-D imaging properties of the model by examining the images of three different objects; points, lines and planes. We calculate the resolving power for these objects as a function of their orientation and the size of the detector pinhole. The results indicate the necessity for image analysis schemes to take these effects into account.

Confocal Scanning Laser Fluorescence And Reflection Microscopy: Measurements Of The 3-D Image Formation And Applications In Biology

A confocal scanning laser microscope of the on-axis type, directly coupled to an image processing system is described. Results of measurements of the actual response functions, both in fluorescence and reflection are presented. Applications of the confocal technique in the area of biology in combination with image processing are demonstrated.

A 3-D model for chromatin organisation of G1 and G2 populations from quantitative confocal image analysis

A study on the chromatin organisation of synchronised G1 and G2 populations of maize root cell nuclei is reported using 3-D images acquired with a confocal fluorescence microscope. The analysis is based on the concept of accessibility. Accessibility of a position x is the effort to arrive at x, when choosing the minimum effort path to arrive at x from the nuclear border. The effort is then taken to be proportional to the amount of all mass encountered on the path, and computed by a technique called the grey valued distance transform. The approach relies heavily on quantitative analysis of the intensity information. Hence, considerable attention was paid to the quantitative modification of the confocal intensity values by diffraction, absorption and scatter corrections. Three texture features are extracted from the accessibility maps: the global object inaccessibility, the relative object accessibility, and the object homogeneity. On the basis of individual texture features, no distinction between the G1 and G2 populations could be established. However, the three features combined did show a clear difference with a high significance.

Test pattern for fluorescence microscopy

A fluorescent test pattern with submicron dimensions has been produced. With this test pattern the optical characteristics of a confocal scanning laser microscope can be determined. The fabrication procedure of the test pattern and the raw data obtained in a measurement of the transfer function of the confocal scanning laser microscope are presented.