Design and implementation of algorithms for focus automation in digital imaging time-lapse microscopy

BACKGROUND: Digital time-lapse microscopy using Nomarski-DIC requires that an autofocusing system adapt to changes in cell shape, size, and position while contending with drift, noise, and hysteresis in the microscope and imager. We have designed and implemented an autofocusing system that tracks subjects under dynamic conditions and maintains focus within a threshold of discriminability. METHODS: With the use of proven and novel algorithms for autofocusing in Nomarski, we performed "virtual" experiments on recorded image stacks to simulate drift and sudden displacements and test the search algorithm response. RESULTS: We found that combining a simple [1, -1] contrast function with an adaptive "warmer-colder" focusing algorithm yields a reasonable compromise between focusing precision and noise tolerance. This method was implemented to record growth kinetics of yeast cells in single and multiple fields of view over several hours. CONCLUSIONS: We have implemented a robust digital autofocus that maintains focus on optically complex samples imaged at high resolution. The tolerance of this system of drift and vibration suggests that it is a practical system for time-lapse imaging in many biological applications.     

Comparative evaluation of autofocus algorithms for a real-time system for automatic detection of Mycobacterium tuberculosis

Microscopy images must be acquired at the optimal focal plane for the objects of interest in a scene. Although manual focusing is a standard task for a trained observer, automatic systems often fail to properly find the focal plane under different microscope imaging modalities such as bright field microscopy or phase contrast microscopy. This article assesses several autofocus algorithms applied in the study of fluorescence-labeled tuberculosis bacteria. The goal of this work was to find the optimal algorithm in order to build an automatic real-time system for diagnosing sputum smear samples, where both accuracy and computational time are important. We analyzed 13 focusing methods, ranging from well-known algorithms to the most recently proposed functions. We took into consideration criteria that are inherent to the autofocus function, such as accuracy, computational cost, and robustness to noise and to illumination changes. We also analyzed the additional benefit provided by preprocessing techniques based on morphological operators and image projection profiling.     

Comparison of autofocus methods for automated microscopy

Traditional autofocus methods were designed for microscopes driven by single processor computers. As computers are developed that exploit massive parallelism when acquiring and analyzing images, parallel cellular logic techniques became available to focus automatically. This paper introduces the reader to both cellular logic techniques for autofocus and a new spectral moment autofocus measure. It then compares these methods with more traditional autofocus methods. It is shown that traditional methods based on measurements of image power-give the best results when tested on one set of real images and two sets of synthetic images. The next best methods are the cellular logic and spectral moment techniques, while the worst are those based on the image probability density function or histogram.     

Passive autofocusing for digital endoscopic imaging systems.

An autofocus is a desirable feature of an endoscope, because it relieves the user from performing a task which can be automated and thereby prevents unnecessary interruptions in the work to be performed. Autofocusing is in general best achieved by an active system, i.e., on the basis of a distance measurement. Yet, in handheld medical endoscopes such a method is unsuited because of the added weight associated with the necessary electromechanical components. Autofocusing should rather be performed passively, furthermore, in applications which are particularly critical with respect to safety, e.g., in the eye, a stable and reliable operation in real time and without interruption is necessary. Passive autofocus strategies applied to date and known to the authors lead however to algorithms which are either too slow for a real time implementation and/or are influenced by the structure of the object which is to be brought into focus. Accordingly, a new autofocus procedure has been developed which exhibits a stable and reliable operation in real time under all circumstances of interest. It is based on the squared differences of the intensity of adjacent points in both dimensions of a plane image (Square Plane Sum Modulus Difference, SPSMD) and as such particularly suitable for digital camera systems and real-time needs (typically, 30 evaluations per second on an image of 1024 x 1024 pixels). The SPSMD criterion is more sensitive, has a larger SNR than other focus criteria known to the authors and exhibits in particular no secondary extrema which could adversely affect proper focusing. As it includes intensity differences in both (perpendicular) directions in the image plane, it is essentially independent of image structures.     

自动对焦算法评估线虫脂滴图像的研究

自动对焦是实现线虫自动化筛选的一个重要步骤．在光学显微镜系统中,通过采集同一个视野下不同焦面的图像,再通过清晰度评价函数对这些图像进行运算,得到的最大值被认为是最佳对焦位置．在本研究中,对16种常用的自动对焦算法以及最近提出的一些算法进行了评估,通过评估找出最适合线虫脂滴图像的自动对焦算法,从而搭建一套线虫脂滴自动化筛选系统．同时就对焦精度、运算时间、抗噪声能力、对焦曲线等特征进行了分析评价,结果表明,大多数算法对线虫脂滴图像都有较好的表现,特别是绝对Tenengrad算法在对焦精度上有最好的表现,我们将优选该算法应用到线虫脂滴自动化筛选系统中．     

siRNA cell arrays for high-content screening microscopy

RNA interference (RNAi) is a recent advance that provides the possibility to reduce the expression of specific target genes in cultured mammalian cells with potential applications on a genome-wide scale. However, to achieve this, robust methodologies that allow automated and efficient delivery of small interfering RNAs (siRNAs) into living cultured cells and reliable quality control of siRNA function must be in place. Here we describe the production of cell arrays for reverse transfection of tissue culture cells with siRNA and plasmid DNA suitable for subsequent high-content screening microscopy applications. All the necessary transfection components are mixed prior to the robotic spotting on noncoated chambered coverglass tissue culture dishes, which are ideally suited for time-lapse microscopy applications in living cells. The addition of fibronectin to the spotting solution improves cell adherence. After cell seeding, no further cell culture manipulations, such as medium changes or the addition of 7 serum, are needed. Adaptation of the cell density improves autofocus performance for high-quality data acquisition and cell recognition. The co-transfection of a nonspecific fluorescently labeled DNA oligomer with the specific siRNA helps to mark each successfully transfected cell and cell cluster. We demonstrate such an siRNA cell array in a microscope-based functional assay in living cells to determine the effect of various siRNA oligonucleotides against endogenous targets on cellular secretion.     

A Technique for Fluorescence Microscopy in Semithin Sections

We describe here a procedure to improve contrast and resolution in fluorescence microscopy of sectioned tissues. Tissue fragments were fixed in ethanol-glacial acetic acid, embedded in diethylene glycol distearate, and semithin sectioned. This method maintains tissue antigenicity while preserving the structure of cells and tissues. The thinness of the sections eliminates scattered and emitted light from tissue structures outside the plane of focus. The procedure is simple and quick, and works excellently with fluorescein-conjugated lectins and antibodies.     

A technique for fluorescence microscopy in semithin sections

We describe here a procedure to improve contrast and resolution in fluorescence microscopy of sectioned tissues. Tissue fragments were fixed in ethanol-glacial acetic acid, embedded in diethylene glycol distearate, and semithin sectioned. This method maintains tissue antigenicity while preserving the structure of cells and tissues. The thinness of the sections eliminates scattered and emitted light from tissue structures outside the plane of focus. The procedure is simple and quick, and works excellently with fluorescein-conjugated lectins and antibodies.     

Digital Inline Holographic Microscopy (DIHM) of Weakly-scattering Subjects

Weakly-scattering objects, such as small colloidal particles and most biological cells, are frequently encountered in microscopy. Indeed, a range of techniques have been developed to better visualize these phase objects; phase contrast and DIC are among the most popular methods for enhancing contrast. However, recording position and shape in the out-of-imaging-plane direction remains challenging. This report introduces a simple experimental method to accurately determine the location and geometry of objects in three dimensions, using digital inline holographic microscopy (DIHM). Broadly speaking, the accessible sample volume is defined by the camera sensor size in the lateral direction, and the illumination coherence in the axial direction. Typical sample volumes range from 200 µm x 200 µm x 200 µm using LED illumination, to 5 mm x 5 mm x 5 mm or larger using laser illumination. This illumination light is configured so that plane waves are incident on the sample. Objects in the sample volume then scatter light, which interferes with the unscattered light to form interference patterns perpendicular to the illumination direction. This image (the hologram) contains the depth information required for three-dimensional reconstruction, and can be captured on a standard imaging device such as a CMOS or CCD camera. The Rayleigh-Sommerfeld back propagation method is employed to numerically refocus microscope images, and a simple imaging heuristic based on the Gouy phase anomaly is used to identify scattering objects within the reconstructed volume. This simple but robust method results in an unambiguous, model-free measurement of the location and shape of objects in microscopic samples.     

Cooperative 4Pi excitation and detection yields sevenfold sharper optical sections in live-cell microscopy

Although the addition of just the excitation light field at the focus, or of just the fluorescence field at the detector is sufficient for a three- to fivefold resolution increase in 4Pi-fluorescence microscopy, substantial improvements of its optical properties are achieved by exploiting both effects simultaneously. They encompass not only an additional expansion of the optical bandwidth, but also an amplified transfer of the newly gained spatial frequencies to the image. Here we report on the realization and the imaging properties of this 4Pi microscopy mode of type C that also is the far-field microscope with the hitherto largest aperture. We show that in conjunction with two-photon excitation, the resulting optical transfer function displays a sevenfold improvement of axial three-dimensional resolution over confocal microscopy in aqueous samples, and more importantly, a marked transfer of all frequencies within its inner region of support. The latter is present also without the confocal pinhole. Thus, linear image deconvolution is possible both for confocalized and nonconfocalized live-cell 4Pi imaging. Realized in a state-of-the-art scanning microscope, this approach enables robust three-dimensional imaging of fixed and live cells at approximately 80 nm axial resolution.     

A new method for the study of lining-layer and pulmonary alveolar walls with electron microscope.

The authors describe an original method for the setting up of pulmonary parenchymal preparations to be observed with electron microscopy. The salient aspects of this method are: the material fixed with a supersaturated uranyl acetate solution and colored, after simple dehydration and inclusion in Epon 812, with bismuth subnitrate. With this method it is possible to obtain a good definition of the morphological aspects and a greater contrast of the phospholipidic structures of the pulmonary parenchyma.     

Microtubular origin of mitotic spindle form birefringence. Demonstration of the applicability of Wiener''s equation

Meiosis I metaphase spindles were isolated from oocytes of the sea-star Pisaster ochraceus by a method that produced no detectable net loss in spindle birefringence. Some of the spindles were fixed immediately and embedded and sectioned for electron microscopy. Others were laminated between gelatine pellicles in a perfusion chamber, then fixed and sequentially and reversibly imbibed with a series of media of increasing refractive indices. Electron microscopy showed little else besides microtubules in the isolates, and no other component present could account for the observed form birefringence. An Ambronn plot of the birefringent retardation measured during imbibition was a good least squares fit to a computer generated theoretical curve based on the Bragg-Pippard rederivation of the Wiener curve for form birefringence. The data were best fit by the curve for rodlet index (n1) = 1.512, rodlet volume fraction (f) = 0.0206, and coefficient of intrinsic birefringence = 4.7 X 10(-5). The value obtained for n1 is unequivocal and is virtually as good as the refractometer determinations of imbibing medium index on which it is based. The optically interactive volume of the microtubule subunit, calculated from our electron microscope determination of spindle microtubule distribution (106/mum2), 13 protofilaments per microtubules, an 8 nm repeat distance and our best value for f, is compatible with known subunit dimensions as determined by other means. We also report curves fitted to the results of Ambronn imbibition of Bouin's-fixed Lytechinus spindles and to the Noll and Weber muscle imbibition data.     

Quantitative Analysis of Dynamic Association in Live Biological Fluorescent Samples

Determining vesicle localization and association in live microscopy may be challenging due to non-simultaneous imaging of rapidly moving objects with two excitation channels. Besides errors due to movement of objects, imaging may also introduce shifting between the image channels, and traditional colocalization methods cannot handle such situations. Our approach to quantifying the association between tagged proteins is to use an object-based method where the exact match of object locations is not assumed. Point-pattern matching provides a measure of correspondence between two point-sets under various changes between the sets. Thus, it can be used for robust quantitative analysis of vesicle association between image channels. Results for a large set of synthetic images shows that the novel association method based on point-pattern matching demonstrates robust capability to detect association of closely located vesicles in live cell-microscopy where traditional colocalization methods fail to produce results. In addition, the method outperforms compared Iterated Closest Points registration method. Results for fixed and live experimental data shows the association method to perform comparably to traditional methods in colocalization studies for fixed cells and to perform favorably in association studies for live cells.     

X-ray scattering with momentum transfer in the plane of membrane. Application to gramicidin organization.

We demonstrate a technique for measuring x-ray (or neutron) scattering with the momentum transfer confined in the plane of membrane, for the purpose of studying lateral organization of proteins and peptides in membrane. Unlike freeze-fracture electron microscopy or atomic force microscopy which requires the membrane to be frozen or fixed, in-plane x-ray scattering can be performed with the membrane maintained in the liquid crystalline state. As an example, the controversial question of whether gramicidin forms aggregates in membrane was investigated. We used dilauroylphosphatidylcholine (DLPC) bilayers containing gramicidin in the molar ratio of 10:1. Very clear scattering curves reflecting gramicidin channel-channel correlation were obtained, even for the sample containing no heavy atoms. Thallium ions bound to gramicidin channels merely increase the magnitude of the scattering curve. Analysis of the data shows that the channels were randomly distributed in the membrane, similar to a computer simulation of freely moving disks in a plane. We suggest that oriented proteins may provide substantial x-ray contrast against the lipid background without requiring heavy-atom labeling. This should open up many possible new experiments.     

A user''s guide for avoiding errors in absorbance image cytometry: a review with original experimental observations

Summary The sources of errors which may occur when cytophotometric analysis is performed with video microscopy using a charged-coupled device (CCD) camera and image analysis are reviewed. The importance of these errors in practice has been tested, and ways of minimizing or avoiding them are described. Many of these sources of error are known from scanning and integrating cytophotometry; they include the use of white instead of monochromatic light, the distribution error, glare, diffraction, shading distortion, and inadequate depth of field. Sources of errors specifically linked with video microscopy or image analysis are highlighted as well; these errors include blooming, limited dynamic range of grey levels, non-linear responses of the camera, contrast transfer, photon noise, dark current, read-out noise, fixed scene noise and spatial calibration. Glare, contrast transfer, fixed scene noise, depth of field and spatial calibration seem to be the most serious sources of errors when measurements are not carried out correctly. We include a table summarizing all the errors discussed in this review and procedures for avoiding them. It can be concluded that if accurate calibration steps are performed and proper guidelines followed, image cytometry can be applied safely for quantifying amounts of chromophore per cell or per unit volume of tissue in sections, even when relatively simple and inexpensive instrumentation is being used.     

Fast, robust, and accurate determination of transmission electron microscopy contrast transfer function

Transmission electron microscopy, as most imaging devices, introduces optical aberrations that in the case of thin specimens are usually modeled in Fourier space by the so-called contrast transfer function (CTF). Accurate determination of the CTF is crucial for its posterior correction. Furthermore, the CTF estimation must be fast and robust if high-throughput three-dimensional electron microscopy (3DEM) studies are to be carried out. In this paper we present a robust algorithm that fits a theoretical CTF model to the power spectrum density (PSD) measured on a specific micrograph or micrograph area. Our algorithm is capable of estimating the envelope of the CTF which is absolutely needed for the correction of the CTF amplitude changes.     

Newly designed,simple relief phase contrast for microscopy of microorganisms

A new method providing a relief phase contrast for investigation of microorganisms by optical microscopy used a neutral filter Zeiss NG 10/1 that could be controllably slid at a certain azimuthal angle below the aperture condenser diaphragm of the microscope phase contrast. Two ways of application are described depending on the type of the microscope: (1) in a special holder, and (2) fixed on a rubber ring. The device enabled us to obtain excellent results in the area of both optical microscopy and microphotography. With the microorganisms visualized, a better resolution, higher contrast and a significant 3D effect were obtained; outer morphology and organelles (chloroplasts, nuclei, granules, oil reserve vacuoles, etc.) could also be investigated.     

Focal pair merging for contrast enhancement of single particles

A common technique in transmission electron microscopy is the collection of a focal pair, in which the first, close to focus image contains higher resolution information at lower contrast, and the second, far from focus image has high contrast but less reliable high-resolution information. Typically these second micrographs are used for purposes of particle selection, orientation estimate or micrograph evaluation. We introduce a technique for merging the information from both images, including signal to noise ratio weighting, contrast transfer function correction, and optional Weiner filtration. This produces a composite image with reduced contrast transfer function artifacts and optimized contrast. This technique is useful in numerous cases where low-contrast images are produced, such as small particles, proteins solubilized in detergent or projects with high-resolution goals when the first image is taken very close to focus.     

FogBank: a single cell segmentation across multiple cell lines and image modalities

Background

Many cell lines currently used in medical research, such as cancer cells or stem cells, grow in confluent sheets or colonies. The biology of individual cells provide valuable information, thus the separation of touching cells in these microscopy images is critical for counting, identification and measurement of individual cells. Over-segmentation of single cells continues to be a major problem for methods based on morphological watershed due to the high level of noise in microscopy cell images. There is a need for a new segmentation method that is robust over a wide variety of biological images and can accurately separate individual cells even in challenging datasets such as confluent sheets or colonies.

Results

We present a new automated segmentation method called FogBank that accurately separates cells when confluent and touching each other. This technique is successfully applied to phase contrast, bright field, fluorescence microscopy and binary images. The method is based on morphological watershed principles with two new features to improve accuracy and minimize over-segmentation.First, FogBank uses histogram binning to quantize pixel intensities which minimizes the image noise that causes over-segmentation. Second, FogBank uses a geodesic distance mask derived from raw images to detect the shapes of individual cells, in contrast to the more linear cell edges that other watershed-like algorithms produce.We evaluated the segmentation accuracy against manually segmented datasets using two metrics. FogBank achieved segmentation accuracy on the order of 0.75 (1 being a perfect match). We compared our method with other available segmentation techniques in term of achieved performance over the reference data sets. FogBank outperformed all related algorithms. The accuracy has also been visually verified on data sets with 14 cell lines across 3 imaging modalities leading to 876 segmentation evaluation images.

Conclusions

FogBank produces single cell segmentation from confluent cell sheets with high accuracy. It can be applied to microscopy images of multiple cell lines and a variety of imaging modalities. The code for the segmentation method is available as open-source and includes a Graphical User Interface for user friendly execution.

Electronic supplementary material

The online version of this article (doi:10.1186/s12859-014-0431-x) contains supplementary material, which is available to authorized users.     

Fabrication of a low-cost on-stage cell incubator with full automation

In recent years the possibility of observing by microscopy the dynamic activity of living cells has been largely pursued. We have developed a low-cost (~ 260 euros) on-stage cell incubator for inverted optical microscopes. This device allows to keep cells in good conditions for their survival and proliferation. The device is based on the use of the Arduino microprocessor interfaced with LabView. It can be connected to a computer via USB port allowing to monitor and register all the useful parameters of the measurements: temperature, CO₂ concentration and relative humidity. It consists of a closed metallic and plastic (PMMA) chassis which provides optical transparency to the petri dish in order to use interference contrast imaging techniques. The system exploits also a second Arduino microprocessor to perform autofocus of the images and to automatically acquire images at defined time intervals. Cell biology laboratories could easily construct this device to allow also students to follow dynamic processes of living cells and to practice with the DIY (Do-It-Yourself) approach to biology. At the same time, students could become familiar with the use of low-cost microprocessors like Arduino.