Artefacts in restored images due to intensity loss in three-dimensional fluorescence microscopy

Computational algorithms for three-dimensional deconvolution have proven successful in reducing blurring and improving the resolution of fluorescence microscopic images. However, discrepancies between the imaging conditions and the models on which such deconvolution algorithms are based may lead to artefacts and/or distortions in the images restored by application of the algorithms. In this paper, artefacts associated with a decrease of fluorescence intensity with time or slice in three-dimensional wide-field images are demonstrated using simulated images. Loss of intensity, whether due to photobleaching or other factors, leads to artefacts in the form of bands or stripes in the restored images. An empirical method for correcting the intensity losses in wide-field images has been implemented and used to correct biological images. This method is based on fitting a decreasing function to the slice intensity curve computed by summing all pixel values in each slice. The fitted curve is then used for the calculation of correction factors for each slice.     

Fully automated intensity compensation for confocal microscopic images

One well-recognized problem in three-dimensional (3D) confocal microscopic images is that the intensities in deeper slices are generally weaker than those in shallower slices. The loss of intensity with depth hampers both qualitative observation and quantitative measurement of specimens. Two major types of methods exist to compensate for this intensity loss: the first is based on the geometrical optics inside the specimen, and the second applies an empirical parametric intensity decay function (IDF) of depth. A common feature shared by both methods is that they are parameter-dependent. However, for the optics-based method there are as yet no fully automated parameter-setting approaches; and for the IDF method the traditional profile-fitting approach cannot provide proper parameters if the presumed IDF model does not match the experimental intensity–depth profile of the 3D image. In this paper, we propose a novel maximum-entropy (ME) approach to fully automated parameter-setting. In principle the ME approach is suitable for any compensation method as long as it is parameter-dependent. The basic assumption is that without intensity loss an ideal 3D image should be generally homogeneous with respect to depth and this axial homogeneity can be represented by the entropy of a normalized intensity–depth profile. Experiments on real confocal images showed that such a profile was consistent with visual evaluation of axial intensity homogeneity and that the ME approach could provide proper parameters for both compensation methods mentioned above. Moreover, for the IDF method, experiments on both real and simulated data showed that the ME approach could provide more precise parameters than with traditional profile-fitting. The Appendix provides a proof that under certain conditions the global maximization of the profile-entropy is guaranteed.     

Strategies for the compensation of specimen-induced spherical aberration in confocal microscopy of skin

We consider various strategies for confocal imaging of human skin which seek to reduce the effects of the specimen-induced aberrations. We calculate the spherical aberration introduced by the stratified structure of skin and show how the confocal signal is affected when attempting to image at various depths within the dermis. Using simple methods it is shown how images might be improved by compensating for the induced aberration. The methods include the use of an iris to reduce the pupil area, changing the refractive index of the immersion medium and using a lens with variable coverglass correction.     

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.     

Double-pulse fluorescence lifetime imaging in confocal microscopy

A theoretical analysis of a new technique for fluorescence lifetime measurement, relying on (near steady state) excitation with short optical pulses, is presented. Application of the technique to confocal microscopy enables local determination of the fluorescence lifetime, which is a parameter sensitive to the local environment of fluorescent probe molecules in biological samples. The novel technique provides high time resolution, since it relies on the laser pulse duration, rather than on electronic gating techniques, and permits, in combination with bilateral confocal microscopy and the use of a (cooled) CCD, sensitive signal detection over a large dynamic range. The principle of the technique is discussed within a theoretical framework. The sensitivity of the technique is analysed, taking into account: photodegradation, the effect of the laser repetition rate and the effect of non-steady-state excitation. The features of the technique are compared to more conventional methods for fluorescence lifetime determination.     

Advances in laser sources for confocal and multiphoton microscopy

The illumination source for all high-resolution, optical sectioning, scanning microscopes is crucially important to the overall performance of the system. We examine advances that have been made in laser sources for both confocal and multiphoton microscopy where the emphasis has been on the development of potentially low-cost, easy to use sources. Growing interest in temporally and spatially resolved techniques has directed laser research towards addressing these challenges. We present the most recent developments in sources for confocal and multiphoton microscopy along with the considerations that should be made when a new source is being considered.     

浅谈共聚焦显微技术

共聚焦显微镜以其高对比度、高分辨率及可重建三维图像的独特优势,在生物医学研究、微细加工、半导体和高分子材料的生产检测等领域获得广泛应用。常用的共聚焦技术方法有:传统的激光扫描共聚焦显微镜(LSCM),其特点是获得的图像对比度和分辨率高,但需要逐点扫描,帧成像时间长,系统复杂,体积大,价格昂贵;碟片共聚焦显微镜(SDCM)是采用多光束扫描的方法来获得共聚焦图像,速度可以大大提高,但牺牲了共聚焦图像的分辨率,系统更为复杂,且不能调整轴向分辨率;结构光显微镜(SIM)具有方法简单,可模块化设计,成本低,成像质量接近于激光扫描共聚焦显微镜,成像速度快,性价比较高。     

用于激光共聚焦显微镜的光谱扫描机构

随着生物医学技术的发展,组织样本经常被多种荧光标记物标记,需要通过光谱成像的方法区分出样本中不同的成分。本文在共聚焦显微镜基础上,介绍了一种由精密丝杠和步进电机控制的狭缝机构实现光谱成像的方法,讨论了狭缝缝片的具体设计和狭缝运动精度对光谱带宽和波长准确度的影响。     

Compensation of inhomogeneous fluorescence signal distribution in 2D images acquired by confocal microscopy

In images acquired by confocal laser scanning microscopy (CLSM), regions corresponding to the same concentration of fluorophores in the specimen should be mapped to the same grayscale levels. However, in practice, due to multiple distortion effects, CLSM images of even homogeneous specimen regions suffer from irregular brightness variations, e.g., darkening of image edges and lightening of the center. The effects are yet more pronounced in images of real biological specimens. A spatially varying grayscale map complicates image postprocessing, e.g., in alignment of overlapping regions of two images and in 3D reconstructions, since measures of similarity usually assume a spatially independent grayscale map. We present a fast correction method based on estimating a spatially variable illumination gain, and multiplying acquired CLSM images by the inverse of the estimated gain. The method does not require any special calibration of reference images since the gain estimate is extracted from the CLSM image being corrected itself. The proposed approach exploits two types of morphological filters: the median filter and the upper Lipschitz cover. The presented correction method, tested on images of both artificial (homogeneous fluorescent layer) and real biological specimens, namely sections of a rat embryo and a rat brain, proved to be very fast and yielded a significant visual improvement. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Parallel deconvolution of large 3D images obtained by confocal laser scanning microscopy

Various deconvolution algorithms are often used for restoration of digital images. Image deconvolution is especially needed for the correction of three‐dimensional images obtained by confocal laser scanning microscopy. Such images suffer from distortions, particularly in the Z dimension. As a result, reliable automatic segmentation of these images may be difficult or even impossible. Effective deconvolution algorithms are memory‐intensive and time‐consuming. In this work, we propose a parallel version of the well‐known Richardson–Lucy deconvolution algorithm developed for a system with distributed memory and implemented with the use of Message Passing Interface (MPI). It enables significantly more rapid deconvolution of two‐dimensional and three‐dimensional images by efficiently splitting the computation across multiple computers. The implementation of this algorithm can be used on professional clusters provided by computing centers as well as on simple networks of ordinary PC machines. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser

One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor‐photobleaching methods for FRET have mainly used wide‐field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser‐scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF‐Receptor‐Associated‐Factors or TRAFs) that are known to homo‐ and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non‐bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser‐scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.     

Limitations in tandem scanning confocal microscopy as a diagnostic tool for microbial keratitis

One potential application of tandem scanning confocal microscopy is the detection of in vivo pathogens. Our study of an experimental model of Acanthamoeba keratitis demonstrates that while this technology can successfully detect certain organisms, there are currently limitations. These limitations relate to instrument configuration, movement of either the tissue or the microscope, difficulty in reproducibly returning to the area of interest for serial examination, the lack of a distinctive morphology of some pathogens, and limited resolution of the microscope.     

Preliminary confocal scanning laser microscopy study of fluid inclusions in quartz

The initial results of the first dedicated confocal scanning laser microscopy (CSLM) study of fluid inclusions in quartz are presented. CSLM imaging of a large inclusion shows the quartz crystal to contain numerous small (< 1 μm), highly reflective inclusions arranged along planes in at least two directions that are not readily visible in transmitted light. The technique allows measurements to be made of the angular intersection and orientation of the planes in both two and three dimensions. Results suggest that larger inclusions (> 10 μm) occur where two planes of small inclusions intersect, and that the shape of the large inclusions is controlled by the angular relationship between intersecting planes.     

In vivo confocal microscopy of human skin: A new design for cosmetology and dermatology

In-depth exploration of cellular structures in living human skin in situ is possible with the tandem scanning microscope (TSM). However, the rigid design of the microscope limited observations to the arms, hands, and fingers. A mobile version allowing the investigation of any parts of the body has been designed. The head containing the Nipkow disk and the optical path were the only part saved from the original TSM. This prototype can be used to observe, in real time, the different skin structures down to a depth of 200 μm and to measure the thickness of the different layers with micron precision level. The hydration of the stratum corneum (SC) could be assessed. For example, lengthy immersion of the hand in water led to an increase in SC thickness without affecting that of the living epidermis. Occlusive patch tests also showed that water and, even more so, propylene glycol, led to transient swelling of the SC. In dermatology, the example of psoriasis illustrated the value of the TSM for describing, measuring, and assessing pathologic skin changes. The availability of this noninvasive method for observing changes with time in a given skin site should prove useful for monitoring treatment efficacy. This tool opens up new insight for the investigation of cutaneous pathophysiology.     

Cosmetic assessment of the human hair by confocal microscopy

The optical sectioning property of the confocal microscope offers a breakthrough from the classic observation of the hair in a scanning electron microscope (SEM). Confocal microscopy requires minimal sampling preparation, and the hair can be observed in its natural environment with less damage than by other microscopic methods such as SEM. While used in the reflection mode, the true morphology of the cuticle and the various exogenous deposits at the surface can be identified and quantified. This relatively noninvasive, nondestructive technique is routinely used by us to monitor the efficiency of cleansing shampoos, to assess the homogeneity of layering polymers, and to evaluate the changes they induce in the optical properties of the hair surface in terms of opacity, transparency, and brilliancy. A second important field of investigation uses the fluorescence channel which reveals the internal structure of the hair. Fluorescent probes (rhodamine and its derivatives) demonstrate the routes of penetration and outline the geometry of cortical cells and of the medulla according to their lipophilic or hydrophilic properties. A volume rendering of a hair cylinder provides a better understanding of the interrelationships between cuticle cells, cortical cells, and the medullar channel. This recent technology is becoming an invaluable tool for the cosmetic assessment of the hair.     

Quantitative determination of the reduction of phototoxicity and photobleaching by controlled light exposure microscopy

Phototoxicity and photobleaching are major limitations in live-cell fluorescence microscopy. They are caused by fluorophores in an excited singlet or triplet state that generate singlet oxygen and other reactive oxygen species. The principle of controlled light exposure microscopy (CLEM) is based on non-uniform illumination of the field of view to reduce the number of excited fluorophore molecules. This approach reduces phototoxicity and photobleaching 2- to 10-fold without deteriorating image quality. Reduction of phototoxicity and photobleaching depends on the fluorophore distribution in the studied object, the optical properties of the microscope and settings of CLEM electronics. Here, we introduce the CLEM factor as a quantitative measure of reduction in phototoxicity and photobleaching. Finally, we give a guideline to optimize the effect of CLEM without compromising image quality.