A method to compensate for light attenuation with depth in three-dimensional DNA image cytometry using a confocal scanning laser microscope

A method to compensate for attenuation of detected light with increased depth of the collected optical section, and its application in three-dimensional (3-D) DNA image cytometry is described. The method is based on studying the stack of 2-D histograms that can be formed from each consecutive pair of sections in a stack of optical serial sections. An attenuation factor is calculated interactively and a new compensated section series is computed. Formalin-fixed paraffin-embedded rat tissue was stained with propidium iodide. Each cell nucleus is extracted by thresholding and its total intensity is calculated. The coefficient of variation (CV) of the total intensity of all cells in each stack is computed. For comparison the CV of the same cells is computed in the uncompensated stacks. This study shows a significantly lower CV for the compensated data, thus contributing to the accuracy of DNA quantification in 3-D DNA image cytometry.     

Adaptive-weighted cubic B-spline using lookup tables for fast and efficient axial resampling of 3D confocal microscopy images

Confocal laser scanning microscopy has become a most powerful tool to visualize and analyze the dynamic behavior of cellular molecules. Photobleaching of fluorochromes is a major problem with confocal image acquisition that will lead to intensity attenuation. Photobleaching effect can be reduced by optimizing the collection efficiency of the confocal image by fast z-scanning. However, such images suffer from distortions, particularly in the z dimension, which causes disparities in the x, y, and z directions of the voxels with the original image stacks. As a result, reliable segmentation and feature extraction of these images may be difficult or even impossible. Image interpolation is especially needed for the correction of undersampling artifact in the axial plane of three-dimensional images generated by a confocal microscope to obtain cubic voxels. In this work, we present an adaptive cubic B-spline-based interpolation with the aid of lookup tables by deriving adaptive weights based on local gradients for the sampling nodes in the interpolation formulae. Thus, the proposed method enhances the axial resolution of confocal images by improving the accuracy of the interpolated value simultaneously with great reduction in computational cost. Numerical experimental results confirm the effectiveness of the proposed interpolation approach and demonstrate its superiority both in terms of accuracy and speed compared to other interpolation algorithms.     

Out‐of‐focus background subtraction for fast structured illumination super‐resolution microscopy of optically thick samples

We propose a structured illumination microscopy method to combine super resolution and optical sectioning in three‐dimensional (3D) samples that allows the use of two‐dimensional (2D) data processing. Indeed, obtaining super‐resolution images of thick samples is a difficult task if low spatial frequencies are present in the in‐focus section of the sample, as these frequencies have to be distinguished from the out‐of‐focus background. A rigorous treatment would require a 3D reconstruction of the whole sample using a 3D point spread function and a 3D stack of structured illumination data. The number of raw images required, 15 per optical section in this case, limits the rate at which high‐resolution images can be obtained. We show that by a succession of two different treatments of structured illumination data we can estimate the contrast of the illumination pattern and remove the out‐of‐focus content from the raw images. After this cleaning step, we can obtain super‐resolution images of optical sections in thick samples using a two‐beam harmonic illumination pattern and a limited number of raw images. This two‐step processing makes it possible to obtain super resolved optical sections in thick samples as fast as if the sample was two‐dimensional.     

Absorption and scattering correction in fluorescence confocal microscopy

In three-dimensional (3-D) fluorescence images produced by a confocal scanning laser microscope (CSLM), the contribution of the deeper layers is attenuated due to absorption and scattering of both the excitation and the fluorescence light. Because of these effects a quantitative analysis of the images is not always possible without restoration. Both scattering and absorption are governed by an exponential decay law. Using only one (space-dependent) extinction coefficient, the total attenuation process can be described. Given the extinction coefficient we calculate within a non-uniform object the relative intensity of the excitation light at its deeper layers. We also give a method to estimate the extinction coefficients which are required to restore 3-D images. An implementation of such a restoration filter is discussed and an example of a successful restoration is given.     

A confocal laser microscope scanner for digital recording of optical serial sections

A confocal laser microscope scanner developed at our institute is described. Since an ordinary microscope is used, it is easy to view the specimen prior to scanning. Confocal imaging is obtained by laser spot illumination, and by focusing the reflected or fluorescent light from the specimen onto a pinhole aperture in front of the detector (a photomultiplier tube). Two rotating mirrors are used to scan the laser beam in a raster pattern. The scanner is controlled by a microprocessor which coordinates scanning, data display, and data transfer to a host computer equipped with an array processor. Digital images with up to 1024 × 1024 pixels and 256 grey levels can be recorded. The optical sectioning property of confocal scanning is used to record thin (～ 1 μm) sections of a specimen without the need for mechanical sectioning. By using computer-control to adjust the focus of the microscope, a stack of consecutive sections can be automatically recorded. A computer is then used to display the 3-D structure of the specimen. It is also possible to obtain quantitative information, both geometric and photometric. In addition to confocal laser scanning, it is easy to perform non-confocal laser scanning, or to use conventional microscopic illumination techniques for (non-confocal) scanning. The design has proved reliable and stable, requiring very few adjustments and realignments. Results obtained with this scanner are reported, and some limitations of the technique are discussed.     

Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy

Fluorescent signal intensities from confocal laser scanning microscopes (CLSM) suffer from several distortions inherent to the method. Namely, layers which lie deeper within the specimen are relatively dark due to absorption and scattering of both excitation and fluorescent light, photobleaching and/or other factors. Because of these effects, a quantitative analysis of images is not always possible without correction. Under certain assumptions, the decay of intensities can be estimated and used for a partial depth intensity correction. In this paper we propose an original robust incremental method for compensating the attenuation of intensity signals. Most previous correction methods are more or less empirical and based on fitting a decreasing parametric function to the section mean intensity curve computed by summing all pixel values in each section. The fitted curve is then used for the calculation of correction factors for each section and a new compensated sections series is computed. However, these methods do not perfectly correct the images. Hence, the algorithm we propose for the automatic correction of intensities relies on robust estimation, which automatically ignores pixels where measurements deviate from the decay model. It is based on techniques adopted from the computer vision literature for image motion estimation. The resulting algorithm is used to correct volumes acquired in CLSM. An implementation of such a restoration filter is discussed and examples of successful restorations are given.     

Measuring the surface area of a cell by the method of the spatial grid with a CSLM—a demonstration

The spatial grid is a method for estimating the surface area of particles. A stack of perfectly registered sections is the essential prerequisite for its use. The confocal scanning light microscope provides such a stack by optical sectioning. The spatial grid method is briefly described and applied to an osteocyte lacuna in dry mineralized human mandible. This type of cell was chosen because of its very complex shape. The variance of the area estimate is studied and compared with the results of a simulation.     

Reconstruction and display of curvilinear objects from optical section data using 3-D curve fitting algorithms

Biological objects resembling filaments are often highly elongated while presenting a small cross-sectional area. Examination of such objects requires acquisition of images from regions large enough to contain entire objects, but at sufficiently high resolution to resolve individual filaments. These requirements complicate the application of conventional optical sectioning and volume reconstruction techniques. For example, objective lenses used to acquire images of entire filaments or filament networks may lack sufficient depth ( Z ) resolution to localize filament cross-sections along the optical axis. Because volume reconstruction techniques consider only the information represented by a single volume element (voxel), views of filament networks reconstructed from images obtained at low Z -resolution will not accurately represent filament morphology. A possible solution to these problems is to simultaneously utilize all available information on the path of an object by fitting 3-D curves through data points localized in 2-D images. Here, we present an application of this approach to the reconstruction of microtubule networks from 2-D optical sections obtained using confocal microscopy, and to synthesized curves which have been distorted using a simple mathematical model of optical sectioning artefacts. Our results demonstrate that this strategy can produce high resolution 3-D views of filamentous objects from a small number of optical sections.     

Automated compensation of light attenuation in confocal microscopy by exact histogram specification

Confocal laser scanning microscopy (CLSM) enables us to capture images representing optical sections on the volume of a specimen. The images acquired from different layers have a different contrast: the images obtained from the deeper layers of the specimen will have a lower contrast with respect to the images obtained from the topmost layers. The main reasons responsible for the effects described above are light absorption and scattering by the atoms and molecules contained in the volume through which the light passes. Also light attenuation can be caused by the inclination of the observed surface. In the case of the surfaces that have a steep inclination, the reflected light will have a different direction than the one of the detector. We propose a technique of digital image processing that can be used to compensate the effects of light attenuation based on histogram operations. We process the image series obtained by CLSM by exact histogram specification and equalization. In this case, a strict ordering among pixels must be induced in order to achieve the exact histogram modeling. The processed images will end up having exactly the specified histogram and not a histogram with a shape that just resembles to the specified one, as in the case of classical histogram specification algorithms. Experimental results and theoretical aspects of the induced ordering are discussed, as well as a comparison between several histogram modeling techniques with respect to the processing of image series obtained by confocal microscopy. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

A new method to correlate acoustic spectroscopic microscopy (30 MHz) and light microscopy

A powerful new method is used to investigate the correlation between light microscopic and acoustic properties of biological tissues. Specimens of liver were sectioned into successive slices, 250 μm and 10 μm thick. The thick sections were investigated acoustically, the thin sections by means of light microscopy. Markers that could be detected and located, both optically and acoustically, were used to find and reconstruct corresponding regions in the acoustic and optical sections (2·5 × 2·5 mm). Parameter images were reconstructed from the sections investigated acoustically. The acoustic parameters were attenuation at 30 MHz, the slope of the attenuation spectrum (between 10 and 50 MHz), backscattering at 30 MHz, the slope of the backscattering spectrum (between 10 and 50 MHz) and the local ultrasound velocity. Acoustic images were obtained in the frequency range from 10 to 50 MHz, yielding a lateral resolution of about 50 μm. The sections for light microscopy were stained according to the Goldner trichrome staining technique. The histological composition was determined quantitatively, using digital image segmentation techniques. The percentage of collagen-rich fibrous tissue, luminal structure and interstitial spaces, and the number of nuclei were calculated for regions of 250 × 250 μm. These histological features were correlated with the acoustic parameters obtained from the corresponding regions in adjacent sections. It was thus possible to find the histological components responsible for acoustic parameters.     

Central and peripheral nervous structures as seen at the confocal scanning laser microscope

Central neurons and peripheral nervous structures, e.g. cutaneous free endings, perifollicular nets, Meissners corpuscles and intramuscular fibres, were studied using various impregnation methods. The confocal scanning laser microscopes (CSLMs) used were equipped with different laser sources, in order to evaluate their limitations and advantages with these techniques and to contribute to a better understanding of the general morphology of the nervous system. When staining with silver sections with clouds of tiny silver granules which are beyond the resolution power of the conventional light microscope but which show a high reflectivity with the CSLM are obtained. Golgi-Cox mercuric impregnation, however, provides specimens which are precipitate-free, thus ensuring the reliability of information obtained. It does, however, have the disadvantage of being applicable only to the central nervous system. In all cases it is an advantage for the instrument to be fitted with different lasers (e.g. Ar and He–Ne), so as to optimize the images of samples impregnated with different methods. Notwithstanding the possibility that artefacts may distort the geometry of the sample and reduce the resolution, the images presented in this paper show that with careful selection of optical sectioning distances, the use of a suitable stack of sections and, if necessary, the aid of false electronic colours and of partial or complete rotation, it is possible to achieve a more precise interpretation of the morphology and organization of complex structures, such as those of the nervous system.     

En bloc optical sectioning of resin-embedded specimens using a confocal laser scanning microscope

Reconstruction of 3D structures of specimens embedded for light or electron microscopy is usually achieved by cutting serial sections through the tissues, then assembling the images from each section to reconstruct the original structure or feature. This is both time-consuming and destructive, and may lead to areas of particular interest being missed. This paper describes a method of examining specimens which have been fixed in glutaraldehyde and embedded in epoxy resin, by utilising the autofluorescence preserved or enhanced by aldehyde fixation, and by using a confocal laser scanning microscope to section optically such specimens in the block down to a depth of about 200 μm. In this way, the accurate estimation of the depth of particular features could be used to facilitate subsequent sectioning at the light microscope or electron microscope level for more detailed studies, and 3D images of tissues/structures within the block could be easily prepared if required.     

Wavelength considerations in confocal microscopy of botanical specimens

We have used a multiple-laser confocal microscope with lines at 325, 442, 488, 514 and 633 nm to investigate optical sectioning of botanical specimens over a wide range of wavelengths. The 442-nm line allowed efficient excitation of Chromomycin A3, with minimal background autofluorescence, to visualize GC-rich heterochromatin as an aid to chromosome identification. Sequential excitation with 442- and 488-nm light enabled ratio imaging of cytosolic pH using BCECF. The red HeNe laser penetrated deep into intact plant tissues, being less prone to scattering than shorter blue lines, and was also used to image fluorescent samples in reflection, prior to fluorescence measurements, to reduce photobleaching. Chromatic corrections are more important in confocal microscope optics than in conventional microscopy. Measured focus differences between blue, green and red wavelengths, for commonly used objectives, were up to half the optical section thickness for both our multi-laser system and a multi-line single-laser instrument. This limited high-resolution sectioning at visible wavelengths caused a loss in signal. For ultraviolet excitation the focus shift was much larger and had to be corrected by pre-focusing the illumination. With this system we have imaged DAPI-stained nuclei, callose in pollen tubes using Aniline Blue and the calcium probe Indo-1.     

Shack-Hartmann wave front measurements in cortical tissue for deconvolution of large three-dimensional mosaic transmitted light brightfield micrographs

We present a novel approach for deconvolution of 3D image stacks of cortical tissue taken by mosaic/optical‐sectioning technology, using a transmitted light brightfield microscope. Mosaic/optical‐sectioning offers the possibility of imaging large volumes (e.g. from cortical sections) on a millimetre scale at sub‐micrometre resolution. However, a blurred contribution from out‐of‐focus light results in an image quality that usually prohibits 3D quantitative analysis. Such quantitative analysis is only possible after deblurring by deconvolution. The resulting image quality is strongly dependent on how accurate the point spread function used for deconvolution resembles the properties of the imaging system. Since direct measurement of the true point spread function is laborious and modelled point spread functions usually deviate from measured ones, we present a method of optimizing the microscope until it meets almost ideal imaging conditions. These conditions are validated by measuring the aberration function of the microscope and tissue using a Shack‐Hartmann sensor. The analysis shows that cortical tissue from rat brains embedded in Mowiol and imaged by an oil‐immersion objective can be regarded as having a homogeneous index of refraction. In addition, the amount of spherical aberration that is caused by the optics or the specimen is relatively low. Consequently the image formation is simplified to refraction between the embedding and immersion medium and to 3D diffraction at the finite entrance pupil of the objective. The resulting model point spread function is applied to the image stacks by linear or iterative deconvolution algorithms. For the presented dataset of large 3D images the linear approach proves to be superior. The linear deconvolution yields a significant improvement in signal‐to‐noise ratio and resolution. This novel approach allows a quantitative analysis of the cortical image stacks such as the reconstruction of biocytin‐stained neuronal dendrites and axons.     

Scanning interference and confocal microscopy

The form of the interference term image in scanning confocal and scanning conventional interference microscopes is identical in all respects including optical sectioning. This observation is used to obtain confocal images and surface profiles from conventional scanning interference microscope images.     

Correcting the axial shrinkage of skeletal muscle thick sections visualized by confocal microscopy

Confocal microscopy is a suitable method for measurements and visualization of skeletal muscle fibres and the neighbouring capillaries. When using 3D images of thick sections the tissue deformation effects should be avoided. We studied the deformation in thick sections of the rat skeletal muscle from complete stacks of images captured with confocal microscope. We measured the apparent thickness of the stacks and compared it to the slice thickness deduced from calibrated microtome settings. The ratio of both values yielded the axial scaling factor for every image stack. Careful sample preparation and treatment of the tissue cryosections with cold Ringer solution minimize the tissue deformation. We conclude that rescaling by the inverse of the axial scaling factor of the stack of optical slices in the direction of the microscope optical axis satisfactorily corrects the axial deformation of skeletal muscle samples.     

Automated correction of linear deformation due to sectioning in serial micrographs

This paper describes an objective and automatic method for detection and correction of sectioning deformations in digitized micrographs, as well as an evaluation of the method applied to light and electron microscopic images of semi-thin and ultra-thin serial sections from brain cortex. The detection is based on matching of image subregions and the deformation model is bi-linear, i.e. two first-order polynomials are used for modelling compression/expansion in perpendicular directions. The procedure is applicable to prealigned serial two-dimensional sections and is primarily aimed at three-dimensional reconstruction of tissue samples consisting of a large number of cells with random distribution and morphology.     

Can you trust your cryostat? Reproducibility of cryostat section thickness

Reproducibility of cryostat section thickness is required for valid quantitative microscopy. This is generally pursued by motorized sectioning using a low but constant speed. The purpose of our study was to compare variation in section thickness between motorized and manual cryostat sectioning. Serial sections were cut from a frozen block of homogenized tissue on different days. Lactate dehydrogenase activity was histochemically detected and calibrated absorbance measurements were taken. The coefficients of variation of measurements was 9.7% for motorized sectioning and 3.3% for manual sectioning. In conclusion, section thickness is similarly reproducible after manual sectioning compared with motorized sectioning, if not better.     

An FFT-based method for attenuation correction in fluorescence confocal microscopy

A problem in three-dimensional imaging using a confocal scanning laser microscope (CSLM) in the (epi)fluorescence mode is the darkening of the deeper layers due to absorption and scattering of both the excitation and the fluorescence light. A new method is proposed to correct for these effects. The approach, valid for weak attenuation, consists of multiplying the measured fluorescence intensity by a correction factor involving a convolution integral of the measured signal, which can be computed efficiently by the fast Fourier transform. Analytical and numerical estimates are given for the degree of attenuation under which the method is valid, and the method is applied to various test images. A real CSLM image is restored. Finally, the method is compared with a recent iterative method with regard to numerical accuracy and computational efficiency.