Improved quantification in CEST-MRI by joint spatial total generalized variation.

PURPOSE: In this work, the use of joint Total Generalized Variation (TGV) regularization to improve Multipool-Lorentzian fitting of chemical exchange saturation transfer (CEST) Spectra in terms of stability and parameter signal-to-noise ratio (SNR) was investigated. THEORY AND METHODS: The joint TGV term was integrated into the nonlinear parameter fitting problem. To increase convergence and weight the gradients, preconditioning using a voxel-wise singular value decomposition was applied to the problem, which was then solved using the iteratively regularized Gauss-Newton method combined with a Primal-Dual splitting algorithm. The TGV method was evaluated on simulated numerical phantoms, 3T phantom data and 7T in vivo data with respect to systematic errors and robustness. Three reference methods were also implemented: The standard nonlinear fitting, a method using a nonlocal-means filter for denoising and the pyramid scheme, which uses downsampled images to acquire accurate start values. RESULTS: The proposed regularized fitting method showed significantly improved robustness (compared to the reference methods). In testing, over a range of SNR values the TGV fit outperformed the other methods and showed accurate results even for large amounts of added noise. Parameter values found were closer or comparable to the ground truth. For in vivo datasets, the added regularization increased the parameter map SNR and prevented instabilities. CONCLUSION: The proposed fitting method using TGV regularization leads to improved results over a range of different data-sets and noise levels. Furthermore, it can be applied to all Z-spectrum data, with different amounts of pools, where the improved SNR and stability can increase diagnostic confidence.

An ultrastructural 3D reconstruction method for observing the arrangement of collagen fibrils and proteoglycans in the human aortic wall under mechanical load.

An insight into changes of soft biological tissue ultrastructures under loading conditions is essential to understand their response to mechanical stimuli. Therefore, this study offers an approach to investigate the arrangement of collagen fibrils and proteoglycans (PGs), which are located within the mechanically loaded aortic wall. The human aortic samples were either fixed directly with glutaraldehyde in the load-free state or subjected to a planar biaxial extension test prior to fixation. The aortic ultrastructure was recorded using electron tomography. Collagen fibrils and PGs were segmented using convolutional neural networks, particularly the ESPNet model. The 3D ultrastructural reconstructions revealed a complex organization of collagen fibrils and PGs. In particular, we observed that not all PGs are attached to the collagen fibrils, but some fill the spaces between the fibrils with a clear distance to the collagen. The complex organization cannot be fully captured or can be severely misinterpreted in 2D. The approach developed opens up practical possibilities, including the quantification of the spatial relationship between collagen fibrils and PGs as a function of the mechanical load. Such quantification can also be used to compare tissues under different conditions, e.g., healthy and diseased, to improve or develop new material models. STATEMENT OF SIGNIFICANCE: The developed approach enables the 3D reconstruction of collagen fibrils and proteoglycans as they are embedded in the loaded human aortic wall. This methodological pipeline comprises the knowledge of arterial mechanics, imaging with transmission electron microscopy and electron tomography, segmentation of 3D image data sets with convolutional neural networks and finally offers a unique insight into the ultrastructural changes in the aortic tissue caused by mechanical stimuli.

Streaking artifact suppression of quantitative susceptibility mapping reconstructions via L1-norm data fidelity optimization (L1-QSM).

PURPOSE: The presence of dipole-inconsistent data due to substantial noise or artifacts causes streaking artifacts in quantitative susceptibility mapping (QSM) reconstructions. Often used Bayesian approaches rely on regularizers, which in turn yield reduced sharpness. To overcome this problem, we present a novel L1-norm data fidelity approach that is robust with respect to outliers, and therefore prevents streaking artifacts. METHODS: QSM functionals are solved with linear and nonlinear L1-norm data fidelity terms using functional augmentation, and are compared with equivalent L2-norm methods. Algorithms were tested on synthetic data, with phase inconsistencies added to mimic lesions, QSM Challenge 2.0 data, and in vivo brain images with hemorrhages. RESULTS: The nonlinear L1-norm-based approach achieved the best overall error metric scores and better streaking artifact suppression. Notably, L1-norm methods could reconstruct QSM images without using a brain mask, with similar regularization weights for different data fidelity weighting or masking setups. CONCLUSION: The proposed L1-approach provides a robust method to prevent streaking artifacts generated by dipole-inconsistent data, renders brain mask calculation unessential, and opens novel challenging clinical applications such asassessing brain hemorrhages and cortical layers.

Non-linear fitting with joint spatial regularization in arterial spin labeling.

Multi-Delay single-shot arterial spin labeling (ASL) imaging provides accurate cerebral blood flow (CBF) and, in addition, arterial transit time (ATT) maps but the inherent low SNR can be challenging. Especially standard fitting using non-linear least squares often fails in regions with poor SNR, resulting in noisy estimates of the quantitative maps. State-of-the-art fitting techniques improve the SNR by incorporating prior knowledge in the estimation process which typically leads to spatial blurring. To this end, we propose a new estimation method with a joint spatial total generalized variation regularization on CBF and ATT. This joint regularization approach utilizes shared spatial features across maps to enhance sharpness and simultaneously improves noise suppression in the final estimates. The proposed method is evaluated at three levels, first on synthetic phantom data including pathologies, followed by in vivo acquisitions of healthy volunteers, and finally on patient data following an ischemic stroke. The quantitative estimates are compared to two reference methods, non-linear least squares fitting and a state-of-the-art ASL quantification algorithm based on Bayesian inference. The proposed joint regularization approach outperforms the reference implementations, substantially increasing the SNR in CBF and ATT while maintaining sharpness and quantitative accuracy in the estimates.

TGV-regularized inversion of the Radon transform for photoacoustic tomography.

We propose and study a reconstruction method for photoacoustic tomography (PAT) based on total generalized variation (TGV) regularization for the inversion of the slice-wise 2D-Radon transform in 3D. The latter problem occurs for recently-developed PAT imaging techniques with parallelized integrating ultrasound detection where projection data from various directions is sequentially acquired. As the imaging speed is presently limited to 20 seconds per 3D image, the reconstruction of temporally-resolved 3D sequences of, e.g., one heartbeat or breathing cycle, is very challenging and currently, the presence of motion artifacts in the reconstructions obstructs the applicability for biomedical research. In order to push these techniques forward towards real time, it thus becomes necessary to reconstruct from less measured data such as few-projection data and consequently, to employ sophisticated reconstruction methods in order to avoid typical artifacts. The proposed TGV-regularized Radon inversion is a variational method that is shown to be capable of such artifact-free inversion. It is validated by numerical simulations, compared to filtered back projection (FBP), and performance-tested on real data from phantom as well as in-vivo mouse experiments. The results indicate that a speed-up factor of four is possible without compromising reconstruction quality.

Robust single-shot acquisition of high resolution whole brain ASL images by combining time-dependent 2D CAPIRINHA sampling with spatio-temporal TGV reconstruction.

For ASL perfusion imaging in clinical settings the current guidelines recommends pseudo-continuous arterial spin labeling with segmented 3D readout. This combination achieves the best signal to noise ratio with reasonable resolution but is prone to motion artifacts due to the segmented readout. Motion robust single-shot 3D acquisitions suffer from image blurring due to the T2 decay of the sampled signals during the long readout. To tackle this problem, we propose an accelerated 3D-GRASE sequence with a time-dependent 2D-CAIPIRINHA sampling pattern. This has several advantages: First, the single-shot echo trains are shortened by the acceleration factor; Second, the temporal incoherence between measurements is increased; And third, the coil sensitivity maps can be estimated directly from the averaged k-space data. To obtain improved perfusion images from the undersampled time series, we developed a variational image reconstruction approach employing spatio-temporal total-generalized-variation (TGV) regularization. The proposed ASL-TGV method reduced the total acquisition time, improved the motion robustness of 3D ASL data, and the image quality of the cerebral blood flow (CBF) maps compared to those by a standard segmented approach. An evaluation was performed on 5 healthy subjects including intentional movement for 2 subjects. Single-shot whole brain CBF-maps with high resolution 3.1 × 3.1 × 3 mm and image quality can be acquired in 1min 46sec. Additionally high quality CBF- and arterial transit time (ATT) -maps from single-shot multi-post-labeling delay (PLD) data can be gained with the proposed method. This method may improve the robustness of 3D ASL in clinical settings, and may be applied for perfusion fMRI.

Total generalized variation regularization for multi-modal electron tomography.

In multi-modal electron tomography, tilt series of several signals such as X-ray spectra, electron energy-loss spectra, annular dark-field, or bright-field data are acquired at the same time in a transmission electron microscope and subsequently reconstructed in three dimensions. However, the acquired data are often incomplete and suffer from noise, and generally each signal is reconstructed independently of all other signals, not taking advantage of correlation between different datasets. This severely limits both the resolution and validity of the reconstructed images. In this paper, we show how image quality in multi-modal electron tomography can be greatly improved by employing variational modeling and multi-channel regularization techniques. To achieve this aim, we employ a coupled Total Generalized Variation (TGV) regularization that exploits correlation between different channels. In contrast to other regularization methods, coupled TGV regularization allows to reconstruct both hard transitions and gradual changes inside each sample, and links different channels at the level of first and higher order derivatives. This favors similar interface positions for all reconstructions, thereby improving the image quality for all data, in particular, for 3D elemental maps. We demonstrate the joint multi-channel TGV reconstruction on tomographic energy-dispersive X-ray spectroscopy (EDXS) and high-angle annular dark field (HAADF) data, but the reconstruction method is generally applicable to all types of signals used in electron tomography, as well as all other types of projection-based tomographies.

Ultrafast 3D Bloch-Siegert B 1 + -mapping using variational modeling.

PURPOSE: Highly accelerated B 1 + -mapping based on the Bloch-Siegert shift to allow 3D acquisitions even within a brief period of a single breath-hold. THEORY AND METHODS: The B 1 + dependent Bloch-Siegert phase shift is measured within a highly subsampled 3D-volume and reconstructed using a two-step variational approach, exploiting the different spatial distribution of morphology and B 1 + -field. By appropriate variable substitution the basic non-convex optimization problem is transformed in a sequential solution of two convex optimization problems with a total generalized variation (TGV) regularization for the morphology part and a smoothness constraint for the B 1 + -field. The method is evaluated on 3D in vivo data with retro- and prospective subsampling. The reconstructed B 1 + -maps are compared to a zero-padded low resolution reconstruction and a fully sampled reference. RESULTS: The reconstructed B 1 + -field maps are in high accordance to the reference for all measurements with a mean error below 1% and a maximum of about 4% for acceleration factors up to 100. The minimal error for different sampling patterns was achieved by sampling a dense region in k-space center with acquisition times of around 10-12 s for 3D-acquistions. CONCLUSIONS: The proposed variational approach enables highly accelerated 3D acquisitions of Bloch-Siegert data and thus full liver coverage in a single breath hold.

Rapid T1 quantification from high resolution 3D data with model-based reconstruction.

PURPOSE: Magnetic resonance imaging protocols for the assessment of quantitative information suffer from long acquisition times since multiple measurements in a parametric dimension are required. To facilitate the clinical applicability, accelerating the acquisition is of high importance. To this end, we propose a model-based optimization framework in conjunction with undersampling 3D radial stack-of-stars data. THEORY AND METHODS: High resolution 3D T1 maps are generated from subsampled data by employing model-based reconstruction combined with a regularization functional, coupling information from the spatial and parametric dimension, to exploit redundancies in the acquired parameter encodings and across parameter maps. To cope with the resulting non-linear, non-differentiable optimization problem, we propose a solution strategy based on the iteratively regularized Gauss-Newton method. The importance of 3D-spectral regularization is demonstrated by a comparison to 2D-spectral regularized results. The algorithm is validated for the variable flip angle (VFA) and inversion recovery Look-Locker (IRLL) method on numerical simulated data, MRI phantoms, and in vivo data. RESULTS: Evaluation of the proposed method using numerical simulations and phantom scans shows excellent quantitative agreement and image quality. T1 maps from accelerated 3D in vivo measurements, e.g. 1.8 s/slice with the VFA method, are in high accordance with fully sampled reference reconstructions. CONCLUSIONS: The proposed algorithm is able to recover T1 maps with an isotropic resolution of 1 mm3 from highly undersampled radial data by exploiting structural similarities in the imaging volume and across parameter maps.

Evaluation of Parallel Level Sets and Bowsher's Method as Segmentation-Free Anatomical Priors for Time-of-Flight PET Reconstruction.

In this article, we evaluate Parallel Level Sets (PLS) and Bowsher's method as segmentation-free anatomical priors for regularized brain positron emission tomography (PET) reconstruction. We derive the proximity operators for two PLS priors and use the EM-TV algorithm in combination with the first order primal-dual algorithm by Chambolle and Pock to solve the non-smooth optimization problem for PET reconstruction with PLS regularization. In addition, we compare the performance of two PLS versions against the symmetric and asymmetric Bowsher priors with quadratic and relative difference penalty function. For this aim, we first evaluate reconstructions of 30 noise realizations of simulated PET data derived from a real hybrid positron emission tomography/magnetic resonance imaging (PET/MR) acquisition in terms of regional bias and noise. Second, we evaluate reconstructions of a real brain PET/MR data set acquired on a GE Signa time-of-flight PET/MR in a similar way. The reconstructions of simulated and real 3D PET/MR data show that all priors were superior to post-smoothed maximum likelihood expectation maximization with ordered subsets (OSEM) in terms of bias-noise characteristics in different regions of interest where the PET uptake follows anatomical boundaries. Our implementation of the asymmetric Bowsher prior showed slightly superior performance compared with the two versions of PLS and the symmetric Bowsher prior. At very high regularization weights, all investigated anatomical priors suffer from the transfer of non-shared gradients.

Quantitative susceptibility mapping: Report from the 2016 reconstruction challenge.

PURPOSE: The aim of the 2016 quantitative susceptibility mapping (QSM) reconstruction challenge was to test the ability of various QSM algorithms to recover the underlying susceptibility from phase data faithfully. METHODS: Gradient-echo images of a healthy volunteer acquired at 3T in a single orientation with 1.06 mm isotropic resolution. A reference susceptibility map was provided, which was computed using the susceptibility tensor imaging algorithm on data acquired at 12 head orientations. Susceptibility maps calculated from the single orientation data were compared against the reference susceptibility map. Deviations were quantified using the following metrics: root mean squared error (RMSE), structure similarity index (SSIM), high-frequency error norm (HFEN), and the error in selected white and gray matter regions. RESULTS: Twenty-seven submissions were evaluated. Most of the best scoring approaches estimated the spatial frequency content in the ill-conditioned domain of the dipole kernel using compressed sensing strategies. The top 10 maps in each category had similar error metrics but substantially different visual appearance. CONCLUSION: Because QSM algorithms were optimized to minimize error metrics, the resulting susceptibility maps suffered from over-smoothing and conspicuity loss in fine features such as vessels. As such, the challenge highlighted the need for better numerical image quality criteria. Magn Reson Med 79:1661-1673, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Noise reduction in FLAIR2 images using total generalized variation, Gaussian and Wiener filtering.

PURPOSE: Multiplication of FLAIR and T2-weighted MRI scans results in images (called FLAIR2) with an improved contrast-to-noise ratio (CNR) for multiple sclerosis (MS) lesions but with a reduced signal-to-noise ratio (SNR). Denoising of these images may therefore further improve FLAIR2 image quality. The purpose of this work is to present a systematic investigation of FLAIR2 image denoising methods using Gaussian, Wiener and Total Generalized Variation (TGV) filtering approaches. MATERIALS AND METHODS: T2-weighted and FLAIR data of four MS patients were used. For CNR and SNR measurements, each scan was performed up to three times. TGV, Gaussian and Wiener filtering was applied to T2, FLAIR and the FLAIR2 data. FLAIR2 images were afterwards additionally created using all combinations of input data (native, filtered T2 and filtered FLAIR). SNR and CNR measurements were performed using the subtraction method for all FLAIR2 approaches (native and filtered input data) and for twenty MS lesions. Additionally, quantitative analysis of filtering based image blurring was performed on all data sets. RESULTS: FLAIR2 images denoised with TGV showed the highest SNR and CNR, while SNR values were similar for Gaussian and Wiener filtered images. The average CNR over 20 MS lesions within the native FLAIR2 (32.99) achieved an improvement to 91.17, 82.33 and 56.07 corresponding to TGV, Wiener and Gaussian filtering. FLAIR multiplied with T2.denoised showed no improvement, while FLAIR.denoised multiplied with T2 showed an increase by a factor of two to the native, not filtered FLAIR2. Blurring was most pronounced in Gaussian filtered images and similar in TGV and Wiener filtered images. CONCLUSION: FLAIR images filtered with Wiener or TGV multiplied with the unfiltered T2 results in FLAIR2 images with increased SNR and CNR and with minimal edge blurring.

Spatio-temporal TGV denoising for ASL perfusion imaging.

In arterial spin labeling (ASL) a perfusion weighted image is achieved by subtracting a label image from a control image. This perfusion weighted image has an intrinsically low signal to noise ratio and numerous measurements are required to achieve reliable image quality, especially at higher spatial resolutions. To overcome this limitation various denoising approaches have been published using the perfusion weighted image as input for denoising. In this study we propose a new spatio-temporal filtering approach based on total generalized variation (TGV) regularization which exploits the inherent information of control and label pairs simultaneously. In this way, the temporal and spatial similarities of all images are used to jointly denoise the control and label images. To assess the effect of denoising, virtual ground truth data were produced at different SNR levels. Furthermore, high-resolution in-vivo pulsed ASL data sets were acquired and processed. The results show improved image quality, quantitative accuracy and robustness against outliers compared to seven state of the art denoising approaches.

Joint MR-PET Reconstruction Using a Multi-Channel Image Regularizer.

While current state of the art MR-PET scanners enable simultaneous MR and PET measurements, the acquired data sets are still usually reconstructed separately. We propose a new multi-modality reconstruction framework using second order Total Generalized Variation (TGV) as a dedicated multi-channel regularization functional that jointly reconstructs images from both modalities. In this way, information about the underlying anatomy is shared during the image reconstruction process while unique differences are preserved. Results from numerical simulations and in-vivo experiments using a range of accelerated MR acquisitions and different MR image contrasts demonstrate improved PET image quality, resolution, and quantitative accuracy.

An illustrated comparison of processing methods for phase MRI and QSM: removal of background field contributions from sources outside the region of interest.

The elimination of so-called background fields is an essential step in phase MRI and quantitative susceptibility mapping (QSM). Background fields, which are caused by sources outside the region of interest (ROI), are often one to two orders of magnitude stronger than tissue-related field variations from within the ROI, hampering quantitative interpretation of field maps. This paper reviews the current literature on background elimination algorithms for QSM and provides insights into similarities and differences between the many algorithms proposed. We discuss the basic theoretical foundations and derive fundamental limitations of background field elimination. Copyright © 2016 John Wiley & Sons, Ltd.

An illustrated comparison of processing methods for MR phase imaging and QSM: combining array coil signals and phase unwrapping.

Phase imaging benefits from strong susceptibility effects at very high field and the high signal-to-noise ratio (SNR) afforded by multi-channel coils. Combining the information from coils is not trivial, however, as the phase that originates in local field effects (the source of interesting contrast) is modified by the inhomogeneous sensitivity of each coil. This has historically been addressed by referencing individual coil sensitivities to that of a volume coil, but alternative approaches are required for ultra-high field systems in which no such coil is available. An additional challenge in phase imaging is that the phase that develops up to the echo time is "wrapped" into a range of 2π radians. Phase wraps need to be removed in order to reveal the underlying phase distribution of interest. Beginning with a coil combination using a homogeneous reference volume coil - the Roemer approach - which can be applied at 3 T and lower field strengths, we review alternative methods for combining single-echo and multi-echo phase images where no such reference coil is available. These are applied to high-resolution data acquired at 7 T and their effectiveness assessed via an index of agreement between phase values over channels and the contrast-to-noise ratio in combined images. The virtual receiver coil and COMPOSER approaches were both found to be computationally efficient and effective. The main features of spatial and temporal phase unwrapping methods are reviewed, placing particular emphasis on recent developments in temporal phase unwrapping and Laplacian approaches. The features and performance of these are illustrated in application to simulated and high-resolution in vivo data. Temporal unwrapping was the fastest of the methods tested and the Laplacian the most robust in images with low SNR. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd.

Accelerated mapping of magnetic susceptibility using 3D planes-on-a-paddlewheel (POP) EPI at ultra-high field strength.

With the advent of ultra-high field MRI scanners in clinical research, susceptibility based MRI has recently gained increasing interest because of its potential to assess subtle tissue changes underlying neurological pathologies/disorders. Conventional, but rather slow, three-dimensional (3D) spoiled gradient-echo (GRE) sequences are typically employed to assess the susceptibility of tissue. 3D echo-planar imaging (EPI) represents a fast alternative but generally comes with echo-time restrictions, geometrical distortions and signal dropouts that can become severe at ultra-high fields. In this work we assess quantitative susceptibility mapping (QSM) at 7 T using non-Cartesian 3D EPI with a planes-on-a-paddlewheel (POP) trajectory, which is created by rotating a standard EPI readout train around its own phase encoding axis. We show that the threefold accelerated non-Cartesian 3D POP EPI sequence enables very fast, whole brain susceptibility mapping at an isotropic resolution of 1 mm and that the high image quality has sufficient signal-to-noise ratio in the phase data for reliable QSM processing. The susceptibility maps obtained were comparable with regard to QSM values and geometric distortions to those calculated from a conventional 4 min 3D GRE scan using the same QSM processing pipeline. Copyright © 2016 John Wiley & Sons, Ltd.

Infimal convolution of total generalized variation functionals for dynamic MRI.

PURPOSE: To accelerate dynamic MR applications using infimal convolution of total generalized variation functionals (ICTGV) as spatio-temporal regularization for image reconstruction. THEORY AND METHODS: ICTGV comprises a new image prior tailored to dynamic data that achieves regularization via optimal local balancing between spatial and temporal regularity. Here it is applied for the first time to the reconstruction of dynamic MRI data. CINE and perfusion scans were investigated to study the influence of time dependent morphology and temporal contrast changes. ICTGV regularized reconstruction from subsampled MR data is formulated as a convex optimization problem. Global solutions are obtained by employing a duality based non-smooth optimization algorithm. RESULTS: The reconstruction error remains on a low level with acceleration factors up to 16 for both CINE and dynamic contrast-enhanced MRI data. The GPU implementation of the algorithm suites clinical demands by reducing reconstruction times of one dataset to less than 4 min. CONCLUSION: ICTGV based dynamic magnetic resonance imaging reconstruction allows for vast undersampling and therefore enables for very high spatial and temporal resolutions, spatial coverage and reduced scan time. With the proposed distinction of model and regularization parameters it offers a new and robust method of flexible decomposition into components with different degrees of temporal regularity. Magn Reson Med 78:142-155, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

Fast quantitative susceptibility mapping using 3D EPI and total generalized variation.

Quantitative susceptibility mapping (QSM) allows new insights into tissue composition and organization by assessing its magnetic property. Previous QSM studies have already demonstrated that magnetic susceptibility is highly sensitive to myelin density and fiber orientation as well as to para- and diamagnetic trace elements. Image resolution in QSM with current approaches is limited by the long acquisition time of 3D scans and the need for high signal to noise ratio (SNR) to solve the dipole inversion problem. We here propose a new total-generalized-variation (TGV) based method for QSM reconstruction, which incorporates individual steps of phase unwrapping, background field removal and dipole inversion in a single iteration, thus yielding a robust solution to the reconstruction problem. This approach has beneficial characteristics for low SNR data, allowing for phase data to be rapidly acquired with a 3D echo planar imaging (EPI) sequence. The proposed method was evaluated with a numerical phantom and in vivo at 3 and 7 T. Compared to total variation (TV), TGV-QSM enforced higher order smoothness which yielded solutions closer to the ground truth and prevented stair-casing artifacts. The acquisition time for images with 1mm isotropic resolution and whole brain coverage was 10s on a clinical 3 Tesla scanner. In conclusion, 3D EPI acquisition combined with single-step TGV reconstruction yields reliable QSM images of the entire brain with 1mm isotropic resolution in seconds. The short acquisition time combined with the robust reconstruction may enable new QSM applications in less compliant populations, clinical susceptibility tensor imaging, and functional resting state examinations.

Computer-assisted counting of retinal cells by automatic segmentation after TV denoising.

BACKGROUND: Quantitative evaluation of mosaics of photoreceptors and neurons is essential in studies on development, aging and degeneration of the retina. Manual counting of samples is a time consuming procedure while attempts to automatization are subject to various restrictions from biological and preparation variability leading to both over- and underestimation of cell numbers. Here we present an adaptive algorithm to overcome many of these problems.Digital micrographs were obtained from cone photoreceptor mosaics visualized by anti-opsin immuno-cytochemistry in retinal wholemounts from a variety of mammalian species including primates. Segmentation of photoreceptors (from background, debris, blood vessels, other cell types) was performed by a procedure based on Rudin-Osher-Fatemi total variation (TV) denoising. Once 3 parameters are manually adjusted based on a sample, similarly structured images can be batch processed. The module is implemented in MATLAB and fully documented online. RESULTS: The object recognition procedure was tested on samples with a typical range of signal and background variations. We obtained results with error ratios of less than 10% in 16 of 18 samples and a mean error of less than 6% compared to manual counts. CONCLUSIONS: The presented method provides a traceable module for automated acquisition of retinal cell density data. Remaining errors, including addition of background items, splitting or merging of objects might be further reduced by introduction of additional parameters. The module may be integrated into extended environments with features such as 3D-acquisition and recognition.