Manual Focusing Research Articles

Color-encoded distance for interactive focus positioning in laser microsurgery

This paper presents a real-time method for interactive focus positioning in laser microsurgery. Registration of stereo vision and a surgical laser is performed in order to combine surgical scene and laser workspace information. In particular, stereo image data is processed to three-dimensionally reconstruct observed tissue surface as well as to compute and to highlight its intersection with the laser focal range. Regarding the surgical live view, three augmented reality concepts are presented providing visual feedback during manual focus positioning. A user study is performed and results are discussed with respect to accuracy and task completion time. Especially when using color-encoded distance superimposed to the live view, target positioning with sub-millimeter accuracy can be achieved in a few seconds. Finally, transfer to an intraoperative scenario with endoscopic human in vivo and cadaver images is discussed demonstrating the applicability of the image overlay in laser microsurgery.

Interaction on-the-go: a fine-grained exploration on wearable PROCAM interfaces and gestures in mobile situations

Wearable projector and camera (PROCAM) interfaces, which provide a natural, intuitive and spatial experience, have been studied for many years. However, existing hand input research into such systems revolved around investigations into stable settings such as sitting or standing, not fully satisfying interaction requirements in sophisticated real life, especially when people are moving. Besides, increasingly more mobile phone users use their phones while walking. As a mobile computing device, the wearable PROCAM system should allow for the fact that mobility could influence usability and user experience. This paper proposes a wearable PROCAM system, with which the user can interact by inputting with finger gestures like the hover gesture and the pinch gesture on projected surfaces. A lab-based evaluation was organized, which mainly compared two gestures (the pinch gesture and the hover gesture) in three situations (sitting, standing and walking) to find out: (1) How and to what degree does mobility influence different gesture inputs? Are there any significant differences between gesture inputs in different settings? (2) What reasons cause these differences? (3) What do people think about the configuration in such systems and to what extent does the manual focus impact such interactions? From qualitative and quantitative points of view, the main findings imply that mobility impacts gesture interactions in varying degrees. The pinch gesture undergoes less influence than the hover gesture in mobile settings. Both gestures were impacted more in walking state than in sitting and standing states by all four negative factors (lack of coordination, jittering hand effect, tired forearms and extra attention paid). Manual focus influenced mobile projection interaction. Based on the findings, implications are discussed for the design of a mobile projection interface with gestures.

Conversion of the Kowa RC-2 camera to a digital version, instructions for use, and postprocessing of the images.

To describe a means of converting the Kowa RC-2 camera to a digital version. The optic (OP) and the film portions (FP) of the Kowa RC-2 were separated. The lateral rails were unscrewed from the front plate of the FP. The front plate was detached from the FP. The plate was fixed to an anodized aluminum plate (AAP). The rails were screwed back onto the front plate. This system was reattached to the OP by means of the rails. A T2-E mount ring was screwed onto the AAP and fixed to a digital camera body (DCB). A flash sync extension cord was used to connect the flash cable of the Kowa to the DCB. The images obtained with the converted Kowa were inverted horizontally and vertically using image processing software. The images obtained with the converted Kowa RC-2 were identical in format to those using the film version with a circular image surrounded by a black rectangle. The imaged ocular structures were accurately depicted, appearing upside down and reversed on the LCD screen. The DCB was set to manual mode and focus. The shutter speed was initially set to 1/250 s, and the ISO was adjusted based on the ocular structure being photographed. An extra filter had to be placed over the light source to dim the light when imaging reflective fundi. The converted digital version of the Kowa RC-2 as described provides an affordable means of obtaining high-quality images of various ocular structures.

Robust particle outline extraction and its application to digital in-line holograms of marine organisms

Digital holography offers a method of high-resolution imaging of microscopic particles and organisms in their natural environment. Automated image extraction and data processing are essential for rapid interrogation and analysis of the vast amounts of information contained in a typical hologram. In this work, we describe a robust-automated particle focusing approach, which we have developed to extract outlines of all particles contained within the sampling volume of each hologram constituting a “holovideo.” The output data consists of ordered point-lists delineating polygons that match particle outlines and facilitate further processing such as extraction of focused images from the holograms themselves. The algorithm developed allows the reduction of, typically, a 2-GB holovideo to tens of megabytes, thereby greatly reducing analysis time by allowing rapid scanning of the contoured images without manual focusing. The algorithm has been demonstrated on synthetic and laboratory holograms and applied to holographic videos recorded in the North Sea. The algorithm output also lends itself to further automated analysis techniques like particle tracking or automated recognition.

Evaluation of a Corrected Cam for an Interchangeable Lens with a Distance Window

Recently, the number of camera companies that produce commercializing interchangeable lens systems such as digital single lens reflex (DSLR) and compact system camera (CSC) lenses has been gradually increasing. These interchangeable lenses have various kinds of lenses with distinct specifications. In particular, the distance window among these specifications is the function most preferred by customers. Mechanical manual zoom and manual focus in these high end camera lenses with a distance window are in particular desirable specifications and are required for product quality. However, the AF lens group is linked to the zoom cam and moves. Because the AF lens group moves along with the object distance, we can not realize the distance window with only zoom locus calculation. In this paper, in order to solve the problem, we suggest an optical calculation method for a corrected AF zoom cam for an interchangeable lens with a distance window to achieve product differentiation and analyze the error in the calculation.

Negative contrast Cerenkov luminescence imaging of blood vessels in a tumor mouse model using [68Ga]gallium chloride

BackgroundCerenkov luminescence imaging (CLI) is an emerging imaging technique where visible light emitted from injected beta-emitting radionuclides is detected with an optical imaging device. CLI research has mostly been focused on positive contrast imaging for ascertaining the distribution of the radiotracer in a way similar to other nuclear medicine techniques. Rather than using the conventional technique of measuring radiotracer distribution, we present a new approach of negative contrast imaging, where blood vessel attenuation of Cerenkov light emitted by [68Ga]GaCl3 is used to image vasculature.MethodsBALB/c nude mice were injected subcutaneously in the right flank with HT-1080 fibrosarcoma cells 14 to 21 days prior to imaging. On the imaging day, [68Ga]GaCl3 was injected and the mice were imaged from 45 to 90 min after injection using an IVIS Spectrum in vivo imaging system. The mice were imaged one at a time, and manual focus was used to bring the skin into focus. The smallest view with pixel size around 83 μm was used to achieve a sufficiently high image resolution for blood vessel imaging.ResultsThe blood vessels in the tumor were clearly visible, attenuating 7% to 18% of the light. Non-tumor side blood vessels had significantly reduced attenuation of 2% to 4%. The difference between the attenuation of light of tumor vessels (10% ± 4%) and the non-tumor vessels (3% ± 1%) was significant. Moreover, a necrotic core confirmed by histology was clearly visible in one of the tumors with a 21% reduction in radiance.ConclusionsThe negative contrast CLI technique is capable of imaging vasculature using [68Ga]GaCl3. Since blood vessels smaller than 50 μm in diameter could be imaged, CLI is able to image structures that conventional nuclear medicine techniques cannot. Thus, the negative contrast imaging technique shows the feasibility of using CLI to perform angiography on superficial blood vessels, demonstrating an advantage over conventional nuclear medicine techniques.

Fully automated analysis of chemically induced γH2AX foci in human peripheral blood mononuclear cells by indirect immunofluorescence

Analysis of phosphorylated histone protein H2AX (γH2AX) foci is currently the most sensitive method to detect DNA double-strand breaks (DSB). This protein modification has the potential to become an individual biomarker of cellular stress, especially in the diagnosis and monitoring of neoplastic diseases. To make γH2AX foci analysis available as a routine screening method, different software approaches for automated immunofluorescence pattern evaluation have recently been developed. In this study, we used novel pattern recognition algorithms on the AKLIDES® platform to automatically analyze immunofluorescence images of γH2AX foci and compared the results with visual assessments. Dose- and time-dependent γH2AX foci formation was investigated in human peripheral blood mononuclear cells (PBMCs) treated with the chemotherapeutic drug etoposide (ETP). Moreover, the AKLIDES system was used to analyze the impact of different immunomodulatory reagents on γH2AX foci formation in PBMCs. Apart from γH2AX foci counting the use of novel pattern recognition algorithms allowed the measurement of their fluorescence intensity and size, as well as the analysis of overlapping γH2AX foci. The comparison of automated and manual foci quantification showed overall a good correlation. After ETP exposure, a clear dose-dependent increase of γH2AX foci formation was evident using the AKLIDES as well as Western blot analysis. Kinetic experiments on PBMCs incubated with 5 μM ETP demonstrated a peak in γH2AX foci formation after 4 to 8 h, while a removal of ETP resulted in a strong reduction of γH2AX foci after 1 to 4 h. In summary, this study demonstrated that the AKLIDES system can be used as an efficient automatic screening tool for γH2AX foci analysis by providing new evaluation features and facilitating the identification of drugs which induce or modulate DNA damage.

Manual versus automated γ-H2AX foci analysis across five European laboratories: Can this assay be used for rapid biodosimetry in a large scale radiation accident?

The identification of severely exposed individuals and reassurance of the ‘worried well’ are of prime importance for initial triage following a large scale radiation accident. We aim to develop the γ-H2AX foci assay into a rapid biomarker tool for use in accidents. Here, five laboratories established a standard operating procedure and analysed 100 ex vivo γ-irradiated, 4 or 24h incubated and overnight-shipped lymphocyte samples from four donors to generate γ-H2AX reference data, using manual and/or automated foci scoring strategies. In addition to acute, homogeneous exposures to 0, 1, 2 and 4Gy, acute simulated partial body (4Gy to 50% of cells) and protracted exposures (4Gy over 24h) were analysed. Data from all laboratories could be satisfactorily fitted with linear dose response functions. Average yields observed at 4h post exposure were 2–4 times higher than at 24h and varied considerably between laboratories. Automated scoring caused larger uncertainties than manual scoring and was unable to identify partial exposures, which were detectable in manually scored samples due to their overdispersed foci distributions. Protracted exposures were detectable but doses could not be accurately estimated with the γ-H2AX assay. We conclude that the γ-H2AX assay may be useful for rapid triage following a recent acute radiation exposure. The potentially higher speed and convenience of automated relative to manual foci scoring needs to be balanced against its compromised accuracy and inability to detect partial body exposures. Regular re-calibration or inclusion of reference samples may be necessary to ensure consistent results between laboratories or over long time periods.

Semantic Focusing Allows Fully Automated Single-Layer Slide Scanning of Cervical Cytology Slides

Liquid-based cytology (LBC) in conjunction with Whole-Slide Imaging (WSI) enables the objective and sensitive and quantitative evaluation of biomarkers in cytology. However, the complex three-dimensional distribution of cells on LBC slides requires manual focusing, long scanning-times, and multi-layer scanning. Here, we present a solution that overcomes these limitations in two steps: first, we make sure that focus points are only set on cells. Secondly, we check the total slide focus quality. From a first analysis we detected that superficial dust can be separated from the cell layer (thin layer of cells on the glass slide) itself. Then we analyzed 2,295 individual focus points from 51 LBC slides stained for p16 and Ki67. Using the number of edges in a focus point image, specific color values and size-inclusion filters, focus points detecting cells could be distinguished from focus points on artifacts (accuracy 98.6%). Sharpness as total focus quality of a virtual LBC slide is computed from 5 sharpness features. We trained a multi-parameter SVM classifier on 1,600 images. On an independent validation set of 3,232 cell images we achieved an accuracy of 94.8% for classifying images as focused. Our results show that single-layer scanning of LBC slides is possible and how it can be achieved. We assembled focus point analysis and sharpness classification into a fully automatic, iterative workflow, free of user intervention, which performs repetitive slide scanning as necessary. On 400 LBC slides we achieved a scanning-time of 13.9±10.1 min with 29.1±15.5 focus points. In summary, the integration of semantic focus information into whole-slide imaging allows automatic high-quality imaging of LBC slides and subsequent biomarker analysis.

Comparison of image fusion and focus function-based techniques for autofocusing in fluorescence microscopy for tuberculosis screening

Manual screening of sputum smear slides for tuberculosis using microscopy is a time consuming exercise that strains laboratory resources in regions with high prevalence of the disease. A system to automatically screen smears would benefit such areas by reducing the reliance on technicians. The first step of such a system would be to ensure that the slide is optimally focused. In this paper, we compare the use of a focus measure and wavelet-based image fusion for automatic focusing of sputum smear slides for fluorescence microscopy to detect tuberculosis. Our objective is to obtain the sharpest image for input into further processing stages in the identification of Mycobacterium tuberculosis. We use manual focusing as ground truth and assess the performance of the two methods by comparing segmented bacillus borders in the autofocused images with those in the manually focused images. Image fusion-based focusing performs marginally better than focus measure-based focusing.

Colorimetric Focus-Forming Assay with Automated Focus Counting by Image Analysis for Quantification of Infectious Hepatitis C Virions

Hepatitis C virus (HCV) infection is the leading cause of liver transplantation in Western countries. Studies of HCV infection using cell culture-produced HCV (HCVcc) in vitro systems require quantification of infectious HCV virions, which has conventionally been performed by immunofluorescence-based focus-forming assay with manual foci counting; however, this is a laborious and time-consuming procedure with potentially biased results. In the present study, we established and optimized a method for convenient and objective quantification of HCV virions by colorimetric focus-forming assay with automated focus counting by image analysis. In testing different enzymes and chromogenic substrates, we obtained superior foci development using alkaline phosphatase-conjugated secondary antibody with BCIP/NBT chromogenic substrate. We additionally found that type I collagen coating minimized cell detachment during vigorous washing of the assay plate. After the colorimetric focus-forming assay, the foci number was determined using an ELISpot reader and image analysis software. The foci number and the calculated viral titer determined by this method strongly correlated with those determined by immunofluorescence-based focus-forming assay and manual foci counting. These results indicate that colorimetric focus-forming assay with automated focus counting by image analysis is applicable as a more-efficient and objective method for quantification of infectious HCV virions.

Anisotropy-based robust focus measure for non-mydriatic retinal imaging

Non-mydriatic retinal imaging is an important tool for diagnosis and progression assessment of ophthalmic diseases. Because it does not require pharmacological dilation of the patient's pupil, it is essential for screening programs performed by non-medical personnel. A typical camera is equipped with a manual focusing mechanism to compensate for the refractive errors in the eye. However, manual focusing is error prone, especially when performed by inexperienced photographers. In this work, we propose a new and robust focus measure based on a calculation of image anisotropy which, in turn, is evaluated from the directional variance of the normalized discrete cosine transform. Simulation and experimental results demonstrate the effectiveness of the proposed focus measure.

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.

Privacy and Confidentiality: What is the Difference?

When photography involved film, manual focusing, f stops and correct exposure, high-quality picture taking was the realm of professional photographers or serious amateurs. In the medical context, o...

γH2AX foci as a measure of DNA damage: A computational approach to automatic analysis

The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage – double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches.We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.

On the digital holographic interferometry of fibrous material, I: Optical properties of polymer and optical fibers

Digital holographic interferometry (DHI) was utilized for investigating the optical properties of polymer and optical fibers. The samples investigated here were polyvinylidene fluoride (PVDF) polymer fiber and graded-index (GRIN) optical fiber. The phase shifting Mach–Zehnder interferometer was used to obtain five phase-shifted holograms, in which the phase difference between two successive holograms is π/2, for each fiber sample. These holograms were recorded using a CCD camera and were combined to gain the complex wavefield, which was numerically reconstructed using the convolution approach into amplitude and phase distributions. The reconstructed phase distribution was used to determine the refractive index, birefringence and refractive index profile of the studied samples. The mean refractive index has been measured with an accuracy up to 4×10 −4. The main advantage of DHI is to overcome the manual focusing limitations by means of the numerical focusing. The results showed accurate measurements of the optical properties of fibers.

H2AX phosphorylation as a genotoxicity endpoint

The γH2AX focus assay, based on phosphorylation of the variant histone protein H2AX, was evaluated as a genotoxicity test in immortalised wild-type mouse embryonic fibroblasts (MEFs) treated for 4 h with a panel of reference compounds routinely used in genotoxicity testing. The topoisomerase II poison etoposide (0.006–60 μg/ml), the alkylating agent methyl methanesulfonate (1.3–65 μg/ml) and the direct DNA-damaging agent bleomycin (0.1–10 μg/ml) all produced a positive concentration–response relationship. The non-genotoxic compounds ampicillin (0.035–3500 μg/ml) and sodium chloride (0.058–580 μg/ml) showed no such response with increased concentrations. The H2AX phosphorylation results were compared with the outcome of two standard in vitro genotoxicity tests, namely the micronucleus and comet assays. Compounds that produced measurable DNA damage in the focus assay generated micronuclei at comparable concentrations. In this study, the focus assay identified genotoxic agents with the same specificity as the comet assay. These results were substantiated when H2AX phosphorylation was analysed using flow cytometry in the murine cell line L5178Y, growing in suspension. The data were in concordance with the manual scoring focus assay. To further this investigation, the γH2AX flow cytometry was compared to the in vitro micronucleus flow cytometry and mouse lymphoma assay using the same cell population after MMS treatment. The median γH2AX value increased significantly above the control at all four MMS concentrations tested. The percentage of micronucleus events in the in vitro micronucleus flow test and the mutation frequency in the mouse lymphoma assay were also significantly increased at each MMS concentration. The current data indicate that H2AX phosphorylation could be used as a biomarker of genotoxicity, which could predict the outcome of in vitro mammalian cell genotoxicity assays.

Balloon Focus: a Seamless Multi-Focus+Context Method for Treemaps

The treemap is one of the most popular methods for visualizing hierarchical data. When a treemap contains a large number of items, inspecting or comparing a few selected items in a greater level of detail becomes very challenging. In this paper, we present a seamless multi-focus and context technique, called Balloon Focus, that allows the user to smoothly enlarge multiple treemap items served as the foci, while maintaining a stable treemap layout as the context. Our method has several desirable features. First, this method is quite general and hence can be used with different treemap layout algorithms. Second, as the foci are enlarged, the relative positions among all items are preserved. Third, the foci are placed in a way that the remaining space is evenly distributed back to the non-focus treemap items. When Balloon Focus enlarges the focus items to a maximum degree, the above features ensure that the treemap will maintain a consistent appearance and avoid any abrupt layout changes. In our algorithm, a DAG (Directed Acyclic Graph) is used to maintain the positional constraints, and an elastic model is employed to govern the placement of the treemap items. We demonstrate a treemap visualization system that integrates data query, manual focus selection, and our novel multi-focus+context technique, Balloon Focus, together. A user study was conducted. Results show that with Balloon Focus, users can better perform the tasks of comparing the values and the distribution of the foci.

Dislocation tomography made easy: a reconstruction from ADF STEM images obtained using automated image shift correction

After previous work producing a successful 3D tomographic reconstruction of dislocations in GaN from conventional weak-beam dark-field (WBDF) images, we have reconstructed a cascade of dislocations in deformed and annealed silicon to a comparable standard using the more experimentally straightforward technique of STEM annular dark-field imaging (STEM ADF). In this mode, image contrast was much more consistent over the specimen tilt range than in conventional weak-beam dark-field imaging. Automatic acquisition software could thus restore the correct dislocation array to the field of view at each tilt angle, though manual focusing was still required. Reconstruction was carried out by sequential iterative reconstruction technique using FEI's Inspect3D software. Dislocations were distributed non-uniformly along cascades, with sparse areas between denser clumps in which individual dislocations of in-plane image width 24 nm could be distinguished in images and reconstruction. Denser areas showed more complicated stacking-fault contrast, hampering tomographic reconstruction. The general three-dimensional form of the denser areas was reproduced well, showing the dislocation array to be planar and not parallel to the foil surfaces.

Shoulder Pain and Holding Position of the Violin: A Case Report

A violinist with persistent shoulder pain, which had led to his inability to perform for 4 mos, was found to play regularly with extreme external rotation of his violin and left shoulder. Intensive manual and physiotherapy did not cure this problem. A combination of manual and music medicine focusing on analyzing and changing instrumental movement patterns was able to resolve this pain problem.