G. J. Brakenhoff

Confocal scanning light microscopy with high aperture immersion lenses

The imaging characteristics of a confocal scanning light microscope (CSLM) with high aperture, immersion type, lenses (N.A. = 1·3) are investigated. In the confocal arrangement the images of the illumination and detector pinholes are made to coincide in a common point, through which the object is scanned mechanically. Results show that for point objects the theoretically expected improved response by a factor of 1·4 in comparison with standard microscopy can indeed be realized. Low side lobe intensity and absence of glare permits the imaging at high resolution of weak details close to strong features. A further improvement by a factor of 1·25 in point resolution in CSLM is found after apodization with an annular aperture. Due to the scanning approach all possibilities of electronic image processing become available in light microscopy.

3‐D image formation in high‐aperture fluorescence confocal microscopy: a numerical analysis

The imaging properties of a confocal fluorescence microscope are considered on the basis of a theoretical model. The model takes into account high‐aperture objectives, the polarization state of the excitation light and a finite detector pinhole. Electromagnetic diffraction theory of the field near focus as developed by Richards and Wolf is used to compute the optical properties of the model. These are shown to be dependent on the polarization of the light. With the resulting three‐dimensional point spread function we have studied the imaging of point, line and plane objects as a function of their orientation with respect to the confocal plane. In addition, the effect of the pinhole size on the image formation of these objects is discussed.

Topography of peptidoglycan synthesis during elongation and polar cap formation in a cell division mutant of Escherichia coli MC4100

SUMMARY: A cell division mutant of Escherichia coli K12 lysA, the temperature sensitive ftsZ strain, was pulse-labelled with [3H]diaminopimelic acid (DAP) during growth in minimal salts medium both at the permissive (28°C) and restrictive (42°C) temperature. In contrast to other known cell division mutants, ftsZ filaments obtained during growth at 42°C show no sign of persisting or newly initiated constrictions. The location of the incorporated DAP in dividing cells and in filaments was analysed with an improved autoradiographic method in which preparations of well-spread sacculi are covered with a dry emulsion. From the populations of sacculi complete distributions were obtained, which compared well with those of the intact cells. The grain-density distributions of cells dividing at 28°C showed that the rate of surface synthesis was strongly increased at the site of constriction at the expense of the activity in the lateral wall, suggesting a redistribution of surface synthesis activity. In individual filaments elongating at 42°C no indication for the existence of narrow or broad growth zones was found, suggesting a dispersed mode of lateral wall synthesis. These observations are in accordance with theoretical predictions on the rate of surface synthesis during the constriction period in cells which elongate at a constant diameter.

Three-dimensional visualization methods for confocal microscopy

Three‐dimensional images of microscopic objects can be obtained by confocal scanning laser microscopy (CSLM). The imaging process in a CSLM consists of sampling a specific volume in the object and storing the result in a three‐dimensional memory array of a digital computer. Methods are needed to visualize these images. In this paper three methods are discussed, each suitable in a specific area of application. For purposes where realistic rendering of solid or semi‐transparent objects is required, an algorithm based on simulation of a fluorescence process is most suitable. When speed is essential, as for interactive purposes, a simple procedure to generate anaglyphs can be used. Both methods have in common that they require no previous interpretation or analysis of the image. When the study of an object imaged by CSLM involves analysis in terms of a geometrical model, sophisticated graphics techniques can be used to display the results of the analysis.

Imaging modes in confocal scanning light microscopy (CSLM)

The optical arrangement for confocal scanning light microscopy can be incorporated in various imaging modes. Light microscopical specimens can be imaged with contrast enhanced, under γ‐control, inverted, etc. In interference, conditions can be set such that either pure phase or pure amplitude images result. Stereoscopic images at arbitrary aspect ratios can be realized in CSLM by electronic processing of the data obtained when the specimen is sampled with more than one confocal point concurrently. Also forms of differential imaging either amplitude or phase are possible. The coupling of these imaging modes with the improved resolving powers of CSLM results in some unique imaging opportunities, especially of value for high resolution light microscopy of living specimens.

Image calibration in fluorescence microscopy.

A fluorescence image calibration method is presented based on the use of standardized uniformly fluorescing reference layers. It is demonstrated to be effective for the correction of non‐uniform imaging characteristics across the image (shading correction) as well as for relating fluorescence intensities between images taken with different microscopes or imaging conditions. The variation of the illumination intensity over the image can be determined on the basis of the uniform bleaching characteristics of the layers. This permits correction for the latter and makes bleach‐rate‐related imaging practical. The significant potential of these layers for calibration in quantitative fluorescence microscopy is illustrated with a series of applications. As the illumination and imaging properties of a microscope can be evaluated separately, the methods presented are also valuable for general microscope testing and characterization.

Three‐dimensional imaging in fluorescence by confocal scanning microscopy

The improved resolution and sectioning capability of a confocal microscope make it an ideal instrument for extracting three‐dimensional information especially from extended biological specimens. The imaging properties, also with finite detection pinholes are considered and a number of biological applications demonstrated.

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.

Image cytometric method for quantifying the relative amount of DNA in bacterial nucleoids using Escherichia coli.

An image cytometric method for quantifying integrated fluorescence was developed to measure the relative DNA contents of bacterial nucleoids. Image analysis was performed with newly developed macros in combination with the program Object‐Image, all downloadable from . Four aspects of the method were investigated. (i) Good linearity was found over a ten‐fold range of fluorescence intensity in a test with a calibration kit of fluorescent latex spheres. (ii) The accuracy of the method was tested with a narrowly distributed Escherichia coli population, which was obtained by growing cells into stationary phase. The width of the image cytometric distribution was approximately 6%, in good agreement with results obtained by flow cytometry. (iii) The error contribution of manual focusing could be kept below 2%, although a strong dependency between integrated fluorescence and focus position was observed. (iv) The results were verified with a flow cytometer, which gave similar distributions for the DNA contents per cell expressed in chromosome equivalents (4.8u2003fg of DNA). We used the presented method to evaluate whether the DNA conformation had any effect on the total fluorescence of bacterial nucleoids. Experiments using nucleoids with the same amount of DNA in either a dispersed or a compact conformation showed no significant difference in integrated fluorescence, indicating that it is possible to determine the DNA content per nucleoid independently of the actual organization of the DNA.

Optical far-field microscopy of single molecules with 3.4 nm lateral resolution.

Optical far‐field imaging of single molecules in a frozen solution at 1.2 K with a lateral resolution of 3.4 nm is reported. The mechanical stability of the fluorescence microscope, especially of the low‐temperature insert, allows for the localization of fluorescing molecules with a reproducibility of better than 5 nm within observation times up to 10 min. For observation times of 9 h the reproducibility of the lateral position is limited to about 20 nm due to mechanical drift. Lateral position and orientation of 314 single molecules, present within the confocal detection volume of ~10 µm3, are obtained. The possibility to correct for mechanical drift by monitoring the position of a spatial reference in the sample is demonstrated.