Fluorescence photobleaching-based shading correction for fluorescence microscopy

A thin fluorescent test layer, which is used in a practically mono-exponential bleaching regime, is employed to determine separately the excitation intensity and the fluorescence detection efficiency distributions in the field of view of a confocal fluorescence microscope. We demonstrate that once these distributions are known, it is possible to correct an image of a specimen for intensity variations which are caused by spatial nonuniformities of the illumination and the detection efficiency of the microscope. It is indicated that, provided a photophysically well-characterized fluorescent test layer is available, the method is potentially capable of quantifying the fluorescence intensities in an image of a specimen in terms of the fluorescence quantum yield, the absorption cross-section and the concentration of the fluorophore in the specimen.     

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.     

Quantitative image correction and calibration for confocal fluorescence microscopy using thin reference layers and SIPchart-based calibration procedures

The fluorescence intensity image of an axially integrated through-focus series of a thin standardized uniform fluorescent layer can be used for image intensity correction and calibration in sectioning microscopy. This intensity image is in fact available from the earlier introduced Sectioned Imaging Property (SIP) charts ( Brakenhoff et al. , 2005 ). It is shown that the integrated intensity of a z -stack from a biological sample, imaged under identical conditions as the layer, can be calibrated in terms of fluorescence layer units of the calibration layer. The imaging after such calibration becomes, as a first approximation, independent of the microscope system and imaging conditions. This is demonstrated on axially integrated images of standard fluorescent beads and standard BPAE Fluorocells. Corrections on the microscope imaging conditions include shading effects, imaging with different magnifications and objectives, and using different microscope systems. It is also shown that with the present approach the actual underlying three-dimensional (3D) fluorescence data set itself can be corrected for variations in point spread function (PSF) imaging efficiency over the imaging data cube. Realizing such calibration between imaging conditions or systems requires basically only the 2D fluorescer molecule density of the reference layers and the section distances with which the layer data are collected.     

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.     

Assessing the efficacy of low-level image content descriptors for computer-based fluorescence microscopy image analysis

The increasing prevalence of automated image acquisition systems and state-of-the-art information technology has enabled new types of microscopy experiments based on automatic processing of massive image data sets, and numerous methods of high-content screening using machine vision and pattern recognition methods have been proposed. However, as a relatively young discipline, it is important to validate these methods and ensure that the machine vision and pattern recognition techniques reliably reflect the actual morphology, and can be effectively used for finding and validating scientific discoveries. In this report we show that some of the previously reported experimental results using automatic microscopy image analysis might be biased, and discuss practices and methods that can be used to obtain objective and reliable automatic analysis of microscopy images.     

Characterization of sectioning fluorescence microscopy with thin uniform fluorescent layers: Sectioned Imaging Property or SIPcharts

Thin, uniformly fluorescing reference layers can be used to characterize the imaging conditions in confocal, or more general, sectioning microscopy. Through-focus datasets of such layers obtained by standard microscope routines provide the basis for the approach. A set of parameters derived from these datasets is developed for defining a number of relevant sectioned imaging properties. The main characteristics of a particular imaging situation can then be summarized in a Sectioned Imaging Property-chart or SIPchart. We propose the use of such charts for the characterization of imaging properties in confocal and multiphoton microscopy. As such, they can be the basis for comparison of sectioned imaging condition characteristics, quality control, maintenance or reproduction of sectioned imaging conditions and other applications. Such charts could prove useful in documenting the more relevant properties of the instrumentation used in microscopy studies. The method carries the potential to provide the basis for a general characterization of sectioned imaging conditions as the layers employed can be characterized and fabricated to standard specifications. A limited number of such thin, uniformly fluorescing layers is available from our group for this purpose. Extension of the method to multiphoton microscopy is discussed.     

Generalized approach for accelerated maximum likelihood based image restoration applied to three-dimensional fluorescence microscopy

For deconvolution applications in three-dimensional microscopy we derived and implemented a generic, accelerated maximum likelihood image restoration algorithm. A conjugate gradient iteration scheme was used considering either Gaussian or Poisson noise models. Poisson models are better suited to low intensity fluorescent image data; typically, they show smaller restoration errors and smoother results. For the regularization, we modified the standard Tikhonov method. However, the generic design of the algorithm allows for more regularization approaches. The Hessian matrix of the restoration functional was used to determine the step size. We compared restoration error and convergence behaviour between the classical line-search and the Hessian matrix method. Under typical working conditions, the restoration error did not increase over that of the line-search and the speed of convergence did not significantly decrease allowing for a twofold increase in processing speed. To determine the regularization parameter, we modified the generalized cross-validation method. Tests that were done on both simulated and experimental fluorescence wide-field data show reliable results.     

Evaluating performance in three-dimensional fluorescence microscopy

In biological fluorescence microscopy, image contrast is often degraded by a high background arising from out of focus regions of the specimen. This background can be greatly reduced or eliminated by several modes of thick specimen microscopy, including techniques such as 3-D deconvolution and confocal. There has been a great deal of interest and some confusion about which of these methods is 'better', in principle or in practice. The motivation for the experiments reported here is to establish some rough guidelines for choosing the most appropriate method of microscopy for a given biological specimen. The approach is to compare the efficiency of photon collection, the image contrast and the signal-to-noise ratio achieved by the different methods at equivalent illumination, using a specimen in which the amount of out of focus background is adjustable over the range encountered with biological samples. We compared spot scanning confocal, spinning disk confocal and wide-field/deconvolution (WFD) microscopes and find that the ratio of out of focus background to in-focus signal can be used to predict which method of microscopy will provide the most useful image. We also find that the precision of measurements of net fluorescence yield is very much lower than expected for all modes of microscopy. Our analysis enabled a clear, quantitative delineation of the appropriate use of different imaging modes relative to the ratio of out-of-focus background to in-focus signal, and defines an upper limit to the useful range of the three most common modes of imaging.     

Comparative evaluation of performance measures for shading correction in time‐lapse fluorescence microscopy

Time‐lapse fluorescence microscopy is a valuable technology in cell biology, but it suffers from the inherent problem of intensity inhomogeneity due to uneven illumination or camera nonlinearity, known as shading artefacts. This will lead to inaccurate estimates of single‐cell features such as average and total intensity. Numerous shading correction methods have been proposed to remove this effect. In order to compare the performance of different methods, many quantitative performance measures have been developed. However, there is little discussion about which performance measure should be generally applied for evaluation on real data, where the ground truth is absent. In this paper, the state‐of‐the‐art shading correction methods and performance evaluation methods are reviewed. We implement 10 popular shading correction methods on two artificial datasets and four real ones. In order to make an objective comparison between those methods, we employ a number of quantitative performance measures. Extensive validation demonstrates that the coefficient of joint variation (CJV) is the most applicable measure in time‐lapse fluorescence images. Based on this measure, we have proposed a novel shading correction method that performs better compared to well‐established methods for a range of real data tested.     

Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy

The imaging performance in single-photon (1-p) and two-photon (2-p) fluorescence microscopy is described. Both confocal and conventional systems are compared in terms of the three-dimensional (3-D) point spread function and the 3-D optical transfer function. Images of fluorescent sharp edges and layers are modelled, giving resolution in transverse and axial directions. A comparison of the imaging properties is also given for a 4Pi confocal system. Confocal 2-p 4Pi fluorescence microscopy gives the best axial resolution in the sense that its 3-D optical transfer function has the strongest response along the axial direction.     

Image scanning fluorescence emission difference microscopy based on a detector array

We propose a novel imaging method that enables the enhancement of three‐dimensional resolution of confocal microscopy significantly and achieve experimentally a new fluorescence emission difference method for the first time, based on the parallel detection with a detector array. Following the principles of photon reassignment in image scanning microscopy, images captured by the detector array were arranged. And by selecting appropriate reassign patterns, the imaging result with enhanced resolution can be achieved with the method of fluorescence emission difference. Two specific methods are proposed in this paper, showing that the difference between an image scanning microscopy image and a confocal image will achieve an improvement of transverse resolution by approximately 43% compared with that in confocal microscopy, and the axial resolution can also be enhanced by at least 22% experimentally and 35% theoretically. Moreover, the methods presented in this paper can improve the lateral resolution by around 10% than fluorescence emission difference and 15% than Airyscan. The mechanism of our methods is verified by numerical simulations and experimental results, and it has significant potential in biomedical applications.     

Imaging enzyme activity with polarization-sensitive confocal fluorescence microscopy

We describe a technique for imaging enzyme activity through steady‐state fluorescence anisotropy measurements on a per‐pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 µm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy‐FL‐labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five‐fold. Fluorescence lifetime and time‐resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy‐FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy‐containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs.     

A comparison of image restoration approaches applied to three-dimensional confocal and wide-field fluorescence microscopy

We have compared different image restoration approaches for fluorescence microscopy. The most widely used algorithms were classified with a Bayesian theory according to the assumed noise model and the type of regularization imposed. We considered both Gaussian and Poisson models for the noise in combination with Tikhonov regularization, entropy regularization, Good's roughness and without regularization (maximum likelihood estimation). Simulations of fluorescence confocal imaging were used to examine the different noise models and regularization approaches using the mean squared error criterion. The assumption of a Gaussian noise model yielded only slightly higher errors than the Poisson model. Good's roughness was the best choice for the regularization. Furthermore, we compared simulated confocal and wide-field data. In general, restored confocal data are superior to restored wide-field data, but given sufficient higher signal level for the wide-field data the restoration result may rival confocal data in quality. Finally, a visual comparison of experimental confocal and wide-field data is presented.     

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.     

Compression of fluorescence microscopy images based on the signal-to-noise estimation

Modern microscopic techniques like high-content screening (HCS), high-throughput screening, 4D imaging, and multispectral imaging may involve collection of thousands of images per experiment. Efficient image-compression techniques are indispensable to manage these vast amounts of data. This goal is frequently achieved using lossy compression algorithms such as JPEG and JPEG2000. However, these algorithms are optimized to preserve visual quality but not necessarily the integrity of the scientific data, which are often analyzed in an automated manner. Here, we propose three observer-independent compression algorithms, designed to preserve information contained in the images. These algorithms were constructed using signal-to-noise ratio (SNR) computed from a single image as a quality measure to establish which image components may be discarded. The compression efficiency was measured as a function of image brightness and SNR. The alterations introduced by compression in biological images were estimated using brightness histograms (earth's mover distance (EMD) algorithm) and textures (Haralick parameters). Furthermore, a microscope test pattern was used to assess the effect of compression on the effective resolution of microscope images.     

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.     

Low-cost, frequency-domain, fluorescence lifetime confocal microscopy

We describe the theory and implementation of a frequency‐domain fluorescence lifetime confocal microscope using switched diode laser illumination. Standard, communications‐type, radio‐frequency electronics are used to provide inexpensive modulation references and to perform phase‐sensitive detection. This allows the rapid acquisition of fluorescence intensity and lifetime images and their display in real time. We show fluorescence lifetime images of bead objects and fluorescence lifetime images of biological specimens from a single confocal scan.     

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.     

Structure-orientated fluorescence photobleaching analysis: a method for double fluorescent labelling studies

Differences in the degree of photodegradation can be used for fluorophore identification in double fluorescently labelled specimens. Based on the use of morphological information, a noise-insensitive method is presented for discriminating between the fluorophores, assuming spatially uniform photodegradation. Separate images of the labelled structures can be obtained. Alternatively, with spatially nonuniform photodegradation, the photodynamics of one fluorophore — i.e. photodegradation, concentration associated quenching, etc. — in relation to its microenvironment can be investigated.     

Digital image acquisition in in vivo confocal microscopy

A flexible system for the real-time acquisition of in vivo images has been developed. Images are generated using a tandem scanning confocal microscope interfaced to a low-light-level camera. The video signal from the camera is digitized and stored using a Gould image processing system with a real-time digital disk (RTDD). The RTDD can store up to 3200 512 times 512 pixel images at video rates (30 images s^?1). Images can be input directly from the camera during the study, or off-line from a Super VHS video recorder. Once a segment of experimental interest is digitized onto the RTDD, the user can interactively step through the images, average stable sequences, and identify candidates for further processing and analysis. Examples of how this system can be used to study the physiology of various organ systems in vivo are presented.