Calibration of a resonance energy transfer imaging system.

A quantitative technique for the nondestructive visualization of nanometer scale intermolecular separations in a living system is described. A calibration procedure for the acquisition and analysis of resonance energy transfer (RET) image data is outlined. The factors limiting RET imaging of biological samples are discussed. Measurements required for the calibration include: (a) the spectral sensitivity of the image intensifier (or camera); (b) the transmission spectra of the emission filters; and (c) the quantum distribution functions of the energy transfer pair measured in situ. Resonance energy transfer imaging is demonstrated for two DNA specific dyes. The Förster critical distance for energy transfer between Hoechst 33342 (HO) and acridine orange (AO) is 4.5 +/- 0.7 nm. This distance is slightly greater than the distance of a single turn of the DNA helix (3.5 nm or approximately 10 base pairs), and is well below the optical diffraction limit. Timed sequences of intracellular energy transfer reveal nuclear structure, strikingly similar to that observed with confocal and electron microscopy, and may show the spatial distribution of eu- and hetero- chromatin in the interphase nuclei.     

Fluorescent image cytometry: from qualitative to quantitative measurements

Image analysis is being increasingly used in biology and medicine; however, in order to obtain truly quantitative data and thus avoid errors in interpretation, a certain number of precautions must be taken when the image is digitized, well before any attempt is made to analyse or interpret the data. This is particularly true for image microfluorometry. In this article we will examine an image analysis system for fluorescent images composed of a mercury lamp, a microscope, a high sensitivity video camera and an image analyser and evaluate the principal sources of random and non-random errors, various constraints, and their relative importance. A signal correction protocol is proposed to minimize non-random errors during digitalization. A few examples are given to illustrate its efficiency.     

Fast and Robust Stereo Vision Algorithm for Obstacle Detection

Binocular computer vision is based on bionics, after the calibration through the camera head by double-exposure image synchronization, access to the calculation of two-dimensional image pixels of the three-dimensional depth information. In this paper, a fast and robust stereo vision algorithm is described to perform in-vehicle obstacles detection and characterization. The stereo algorithm which provides a suitable representation of the geometric content of the road scene is described, and an in-vehicle embedded system is presented. We present the way in which the algorithm is used, and then report experiments on real situations which show that our solution is accurate, reliable and efficient. In particular, both processes are fast, generic, robust to noise and bad conditions, and work even with partial occlusion.     

Computer-assisted tracking of actin filament motility.

In vitro motility assays, in which fluorescently labeled actin filaments are propelled by myosin molecules adhered to a glass coverslip, require that actin filament velocity be determined. We have developed a computer-assisted filament tracking system that reduced the analysis time, minimized investigator bias, and provided greater accuracy in locating actin filaments in video images. The tracking routine successfully tracked filaments under experimental conditions where filament density, size, and extent of photobleaching varied dramatically. Videotaped images of actin filament motility were digitized and processed to enhance filament image contrast relative to background. Once processed, filament images were cross correlated between frames and a filament path was determined. The changes in filament centroid or center position between video frames were then used to calculate filament velocity. The tracking routine performance was evaluated and the sources of noise that contributed to errors in velocity were identified and quantified. Errors originated in algorithms for filament centroid determination and in the choice of sampling interval between video frames. With knowledge of these error sources, the investigator can maximize the accuracy of the velocity calculation through access to user-definable computer program parameters.     

Static and dynamic errors in particle tracking microrheology

Particle tracking techniques are often used to assess the local mechanical properties of cells and biological fluids. The extracted trajectories are exploited to compute the mean-squared displacement that characterizes the dynamics of the probe particles. Limited spatial resolution and statistical uncertainty are the limiting factors that alter the accuracy of the mean-squared displacement estimation. We precisely quantified the effect of localization errors in the determination of the mean-squared displacement by separating the sources of these errors into two separate contributions. A "static error" arises in the position measurements of immobilized particles. A "dynamic error" comes from the particle motion during the finite exposure time that is required for visualization. We calculated the propagation of these errors on the mean-squared displacement. We examined the impact of our error analysis on theoretical model fluids used in biorheology. These theoretical predictions were verified for purely viscous fluids using simulations and a multiple-particle tracking technique performed with video microscopy. We showed that the static contribution can be confidently corrected in dynamics studies by using static experiments performed at a similar noise-to-signal ratio. This groundwork allowed us to achieve higher resolution in the mean-squared displacement, and thus to increase the accuracy of microrheology studies.     

Imaging system for morphometric assessment of absorption or fluorescence in stained cells

An image acquisition and processing system has been developed for quantitative microscopy of absorption or fluorescence in stained cells. Three different light transducers are used in the system to exploit the best characteristics of these sensors for different biological measurements. A digital scanner, in the form of a linear array charge-coupled device (CCD), acquires data with high spatial and photometric resolution. A color (RGB) camera is employed when spectral information is required for the segmentation of cellular subcomponents. An image-intensified charged-injection device (CID) camera provides for very low light intensity measurements, primarily for fluorescence-labeled cells. Properties of these transducers, such as contrast transfer function, linearity, and photo-response nonuniformity, have been measured. Two dedicated image processing units were incorporated into the system. The front-end processor, based on a digital signal processor, provides functions such as object detection, raw image calibration, compression, artifact removal, and filtering. The second image processor is associated with the frame memory and includes a histogram processor, a dedicated arithmetic logic unit for image processing functions, and a graphics module for one-bit overlay functions. An interactive program was developed to acquire cell images and to experiment with a range of segmentation algorithms, feature extractions, and other image processing functions. The results of any image operation are displayed on the video monitor. Once a desired processing sequence is determined, the sequence may be stored to become part of a command library and can be executed thereafter as a single instruction.     

Applications of direct detection device in transmission electron microscopy

A prototype direct detection device (DDD) camera system has shown great promise in improving both the spatial resolution and the signal to noise ratio for electron microscopy at 120–400 keV beam energies (Xuong et al., 2007. Methods in Cell Biology, 79, 721–739). Without the need for a resolution-limiting scintillation screen as in the charge coupled device (CCD), the DDD camera can outperform CCD based systems in terms of spatial resolution, due to its small pixel size (5 μm). In this paper, the modulation transfer function (MTF) of the DDD prototype is measured and compared with the specifications of commercial scientific CCD camera systems. Combining the fast speed of the DDD with image mosaic techniques, fast wide-area imaging is now possible. In this paper, the first large area mosaic image and the first tomography dataset from the DDD camera are presented, along with an image processing algorithm to correct the specimen drift utilizing the fast readout of the DDD system.     

A new calibration method for 3-D position measurement in biomedical applications.

Aim of the study was to determine practicality and to test accuracy of a new calibration technique firstly introduced in 1998 by Schmid and Bess for biomechanical human tests. This technique enables three-dimensional calibration of camera positions as well as the calculation of internal and external camera parameters. It can be performed unlike other three-dimensional calibration techniques as the first with a planar calibration grid and only one single video image (of each camera) to calculate all 3-D reconstruction parameters. The tests were performed using two albavision ACAM G-Cameras with a resolution of 480 (h) by 420 (v) pixels. The achievable accuracy of distance measurements in recent commercially available motion measurement systems usually ranges from about 0.09% to 1.77% and higher. Accuracy of 0.0373% was determined with the new calibration technique. The 95% confidence interval ranged at +/- 0.02322 mm, the RMS (root mean square) error at 0.18776 mm. Better accuracy, easier and faster calibration are features of this new calibration technique. Required time for complete calibration ranged below one minute. Anticipating this new method will have good practicality in gait analysis or in research and industry due to increased accuracy and ease of use.     

A Virtual Clinical Trial of FDG-PET Imaging of Breast Cancer: Effect of Variability on Response Assessment

INTRODUCTION: There is growing interest in using positron emission tomography (PET) standardized uptake values (SUVs) to assess tumor response to therapy. However, many error sources compromise the ability to detect SUV changes. We explore relationships between these errors and overall SUV variability. METHODS: We used simulations in a virtual clinical trial framework to study impacts of error sources from scanning and analysis effects on assessment of SUV changes. We varied tumor diameter, scan duration, pretherapy SUV, magnitude of change in SUV, image reconstruction filter, and SUV metric. Poisson noise was added to the raw data before image reconstruction. Variance from global sources of error, e.g., scanner calibration, was incorporated. Two thousand independent noisy sinograms per scenario were generated and reconstructed. We used SUVs to create receiver operating characteristic (ROC) curves to quantify ability to assess response. Integrating area under the ROC curve summarized ability to detect SUV changes. RESULTS: Scan duration and image reconstruction method had relatively little impact on ability to measure response. SUV_MAX is nearly as effective as SUV_MEAN, especially with increased image smoothing and despite size-matched region of interest placement. For an effective variability of 15%, we found the Positron Emission Tomography Response Criteria in Solid Tumors criteria for measuring response (±30%) similar to the European Organization for Research and Treatment of Cancer criteria (±25%). CONCLUSIONS: For typical PET variance levels, tumor response must be 30% to 40% to be reliably determined using SUVs. PET scan duration and image reconstruction method had relatively little effect.     

Calibration characteristics of a video dimension analyser (VDA) system.

A comprehensive calibration and error analysis of a video dimension analyser (VDA) system was carried out to determine the effects of various parameters relevant to actual test conditions on the characteristics of the system. The parameters considered were: (1) varying distances or angles between the test specimen and the camera; (2) refractive effects of glass tank and physiologic solutions; and (3) dynamic response of the VDA system. The error analysis shows that, at low strain levels, the strain computed by the VDA is sensitive to camera placement and orientation as well as the media through which the object is observed. Very large errors can occur at small nominal strains, depending on the system parameters (e.g. 80% error at 1% nominal strain for one set of parameters). At higher strain levels, the sensitivity of the system is reduced, and the strain computed by the VDA system approaches the nominal strain (10% error at 10% nominal strain).     

Video intensification microscopy (VIM) as an aid in routine fluorescence microscopy

Summary Video intensification microscopy (VIM), in which a highly sensitive television camera is combined with fluorescence microscopy and a timelapse video tape recorder (Willingham and Pastan 1978), is becoming widely used for studying movements of fluorescent probes on living cells. In our experience, the VIM system is also extremely convenient for more conventional fluorescence microscopy of fixed tissues. The advantages of VIM are greatest in applications where convenience in handling and recording images from a large number of specimens is more important than achieving the best possible optical image quality.     

Localization Precision in Stepwise Photobleaching Experiments

The precise determination of the position of fluorescent labels is essential for the quantitative study of biomolecular structures by various localization microscopy techniques. Localization by stepwise photobleaching is especially suited for measuring nanometer-scale distances between two labels; however, the precision of this method has remained elusive. Here, we show that shot noise from other emitters and error propagation compromise the localization precision in stepwise photobleaching. Incorporation of point spread function-shaped shot noise into the variance term in the Fisher matrix yielded fundamental Cràmer-Rao lower bounds (CRLBs) that were in general anisotropic and depended on emitter intensity and position. We performed simulations to benchmark the extent to which different analysis procedures reached these ideal CRLBs. The accumulation of noise from several images accounted for the worse localization precision in image subtraction. Propagation of fitting errors compromised the CRLBs in sequential fitting using fixed parameters. Global fitting of all images was also governed by error propagation, but made optimal use of the available information. The precision of individual distance measurements depended critically on the exact bleaching kinetics and was correctly quantified by the CRLBs. The methods presented here provide a consistent framework for quantitatively analyzing stepwise photobleaching experiments and shed light on the localization precision in some other bleaching- or blinking-assisted techniques.     

Application of Composite Small Calibration Objects in Traffic Accident Scene Photogrammetry

In order to address the difficulty of arranging large calibration objects and the low measurement accuracy of small calibration objects in traffic accident scene photogrammetry, a photogrammetric method based on a composite of small calibration objects is proposed. Several small calibration objects are placed around the traffic accident scene, and the coordinate system of the composite calibration object is given based on one of them. By maintaining the relative position and coplanar relationship of the small calibration objects, the local coordinate system of each small calibration object is transformed into the coordinate system of the composite calibration object. The two-dimensional direct linear transformation method is improved based on minimizing the reprojection error of the calibration points of all objects. A rectified image is obtained using the nonlinear optimization method. The increased accuracy of traffic accident scene photogrammetry using a composite small calibration object is demonstrated through the analysis of field experiments and case studies.     

Verification of biomechanical methods employed in a comprehensive study of mild traumatic brain injury and the effectiveness of American football helmets

Concussion, or mild traumatic brain injury, occurs in many activities, mostly as a result of the head being accelerated. A comprehensive study has been conducted to understand better the mechanics of the impacts associated with concussion in American football. This study involves a sequence of techniques to analyse and reconstruct many different head impact scenarios. It is important to understand the validity and accuracy of these techniques in order to be able to use the results of the study to improve helmets and helmet standards. Two major categories of potential errors have been investigated. The first category concerns error sources specific to the use of crash test dummy instrumentation (accelerometers) and associated data processing techniques. These are relied upon to establish both linear and angular head acceleration responses. The second category concerns the use of broadcast video data and crash test dummy head-neck-torso systems. These are used to replicate the complex head impact scenarios of whole body collisions that occur on the football field between two living human beings. All acceleration measurement and processing techniques were based on well-established practices and standards. These proved to be reliable and reproducible. Potential errors in the linear accelerations due to electrical or mechanical noise did not exceed 2% for the three different noise sources investigated. Potential errors in the angular accelerations due to noise could be as high as 6.7%, due to error accumulation of multiple linear acceleration measurements. The potential error in the relative impact velocity between colliding heads could be as high as 11%, and was found to be the largest error source in the sequence of techniques to reconstruct the game impacts. Full-scale experiments with complete crash test dummies in staged head impacts showed maximum errors of 17% for resultant linear accelerations and 25% for resultant angular accelerations.     

Efficient Illumination for Microsecond Tracking Microscopy

The possibility to observe microsecond dynamics at the sub-micron scale, opened by recent technological advances in fast camera sensors, will affect many biophysical studies based on particle tracking in optical microscopy. A main limiting factor for further development of fast video microscopy remains the illumination of the sample, which must deliver sufficient light to the camera to allow microsecond exposure times. Here we systematically compare the main illumination systems employed in holographic tracking microscopy, and we show that a superluminescent diode and a modulated laser diode perform the best in terms of image quality and acquisition speed, respectively. In particular, we show that the simple and inexpensive laser illumination enables less than s camera exposure time at high magnification on a large field of view without coherence image artifacts, together with a good hologram quality that allows nm-tracking of microscopic beads to be performed. This comparison of sources can guide in choosing the most efficient illumination system with respect to the specific application.     

A method to minimise error in 2D-DLT reconstruction of non-planar markers filmed with a moving camera

This article describes a method that allows estimating, with the 2D version of the direct linear transformation (DLT), the actual 2D coordinates of a point when the latter is not strictly in the calibration plane. Markers placed in vertical line, above, below and in the centre of a horizontal calibration plane were filmed by a moving camera. Without correction, strong errors (up to 64.5%) were noticed for markers out of the calibration plane. After correction, calculated coordinates were consistent with actual values (error < 0.55%). The method was then applied to slip distance measurement, using a marker fixed on the hoof of a horse trotting on a calibrated track while being followed with a camera. The correction effect represented 6.6% of slip distance. Combined with the 2D-DLT transformation, the proposed corrective method allows an accurate measurement of slip distances, for high-speed outdoor locomotion analysis, using a moving camera.     

Localization Precision in Stepwise Photobleaching Experiments

The precise determination of the position of fluorescent labels is essential for the quantitative study of biomolecular structures by various localization microscopy techniques. Localization by stepwise photobleaching is especially suited for measuring nanometer-scale distances between two labels; however, the precision of this method has remained elusive. Here, we show that shot noise from other emitters and error propagation compromise the localization precision in stepwise photobleaching. Incorporation of point spread function-shaped shot noise into the variance term in the Fisher matrix yielded fundamental Cràmer-Rao lower bounds (CRLBs) that were in general anisotropic and depended on emitter intensity and position. We performed simulations to benchmark the extent to which different analysis procedures reached these ideal CRLBs. The accumulation of noise from several images accounted for the worse localization precision in image subtraction. Propagation of fitting errors compromised the CRLBs in sequential fitting using fixed parameters. Global fitting of all images was also governed by error propagation, but made optimal use of the available information. The precision of individual distance measurements depended critically on the exact bleaching kinetics and was correctly quantified by the CRLBs. The methods presented here provide a consistent framework for quantitatively analyzing stepwise photobleaching experiments and shed light on the localization precision in some other bleaching- or blinking-assisted techniques.     

Video fluorescence microscopy as a tool for the study of cellular heterogeneity in epithelia

The use of functional fluorescent dyes has allowed us to monitor intracellular pH in individually identified cells in renal epithelia. Using video microscopy we simultaneously measured the change in intracellular pH in several contiguous cells in response to various maneuvers. The video equipment included a silicon intensified target camera, a VHS videocassette recorder, a high resolution monochrome monitor, a video photometric analyzer and a 2-channel chart recorder. This equipment had a spatial resolution of 1 micron by light microscopy and a response time of less than 200 ms; it allowed us to perform double fluorescent labeling and obtain reliable measurements of intracellular pH, independent of gain, regardless of the location of the image on the screen. Using this video system we have shown that there is substantial heterogeneity in activity of H+/HCO3- transport pathways among adjacent cells in a monolayer of cells cultured from the rat renal inner medullary collecting duct. In isolated perfused rabbit renal cortical collecting ducts, video microscopy allowed us to show that there are two different types of intercalated cells: one that exhibits apical Cl-/HCO3- exchange and one that does not. Both show alkaline intracellular pH with respect to non-acid-base transporting epithelia. Video microscopy has several advantages over conventional microspectrophotometry. It provides rapid data acquisition along with increased sensitivity and the capacity for some subcellular analyses. One is able to analyze several individually identified cells during an experimental maneuver. The present video system was assembled for less than $15,000 and permits a more complete analysis of an epithelium than either single-cell photometry or spectrophotometric analysis of thousands of cells in suspension or monolayers.     

Automatic Determination of Bacterioplankton Biomass by Image Analysis

Image analysis was applied to epifluorescense microscopy of acridine orange-stained plankton samples. A program was developed for discrimination and binary segmentation of digitized video images, taken by an ultrasensitive video camera mounted on the microscope. Cell volumes were estimated from area and perimeter of the objects in the binary image. The program was tested on fluorescent latex beads of known diameters. Biovolumes measured by image analysis were compared with directly determined carbon biomasses in batch cultures of estuarine and freshwater bacterioplankton. This calibration revealed an empirical conversion factor from biovolume to biomass of 0.35 pg of C μm⁻³ (± 0.03 95% confidence limit). The deviation of this value from the normally used conversion factors of 0.086 to 0.121 pg of C μm⁻³ is discussed. The described system was capable of measuring 250 cells within 10 min, providing estimates of cell number, mean cell volume, and biovolume with a precision of 5%.     

Self-calibrated collinearity detector

The human visual system can make remarkably precise spatial judgements. There are reasons to believe that this accuracy is achieved and maintained by using processes that calibrate and correct errors in the system. This work investigate this problem of self-calibration and describes an adaptive system for detecting the collinearity of points and the straightness of lines. The system is initially inaccurate, but, by using an error correction mechanism, it eventually becomes highly accurate. The error correction is performed by a simple self calibration process named proportional multi-gain adjustment. The calibration process adjusts the gain values of the system input units. The process utilizes statistical regularities in the input stimuli. It compensate for errors due to noise in the input units receptive fields location and response functions by ensuring that the average deviation from collinearity offset detected by the system is zero. As a by product of the error correction, the system exhibits also adaptation and aftereffect phenomena, similar to those observed in the human visual system.