Going Beyond 3-D Imaging: Automated 3-D Montaged image Analysis of Cytological Specimens

1996 ◽  
Vol 54 ◽  
pp. 282-283
Author(s):  
Badrinath Roysam ◽  
Hakan Ancin ◽  
Douglas E. Becker ◽  
Robert W. Mackin ◽  
Matthew M. Chestnut ◽  
...  
Keyword(s):  
Image Analysis ◽  
Structural Changes ◽  
Sampling Methods ◽  
Spatial Clustering ◽  
Three Dimensional ◽  
Field Of View ◽  
Cell Counting ◽  
Depth Of Field ◽  
Tissue Cell ◽  
Tissue Organization

This paper summarizes recent advances made by this group in the automated three-dimensional (3-D) image analysis of cytological specimens that are much thicker than the depth of field, and much wider than the field of view of the microscope. The imaging of thick samples is motivated by the need to sample large volumes of tissue rapidly, make more accurate measurements than possible with 2-D sampling, and also to perform analysis in a manner that preserves the relative locations and 3-D structures of the cells. The motivation to study specimens much wider than the field of view arises when measurements and insights at the tissue, rather than the cell level are needed.The term “analysis” indicates a activities ranging from cell counting, neuron tracing, cell morphometry, measurement of tracers, through characterization of large populations of cells with regard to higher-level tissue organization by detecting patterns such as 3-D spatial clustering, the presence of subpopulations, and their relationships to each other. Of even more interest are changes in these parameters as a function of development, and as a reaction to external stimuli. There is a widespread need to measure structural changes in tissue caused by toxins, physiologic states, biochemicals, aging, development, and electrochemical or physical stimuli. These agents could affect the number of cells per unit volume of tissue, cell volume and shape, and cause structural changes in individual cells, inter-connections, or subtle changes in higher-level tissue architecture. It is important to process large intact volumes of tissue to achieve adequate sampling and sensitivity to subtle changes. It is desirable to perform such studies rapidly, with utmost automation, and at minimal cost. Automated 3-D image analysis methods offer unique advantages and opportunities, without making simplifying assumptions of tissue uniformity, unlike random sampling methods such as stereology.12 Although stereological methods are known to be statistically unbiased, they may not be statistically efficient. Another disadvantage of sampling methods is the lack of full visual confirmation - an attractive feature of image analysis based methods.

From 3-D Light Microscopic Images to Quantitative Insight

1999 ◽  
Vol 5 (S2) ◽  
pp. 524-525
Author(s):  
B. Roysam ◽  
A. Can ◽  
H. Shen ◽  
K. Al-Kofahi ◽  
J.N. Turner
Keyword(s):  
Image Analysis ◽  
Three Dimensional ◽  
Image Data ◽  
Depth Of Field ◽  
Analysis Techniques ◽  
Core Set ◽  
Spatial Dimensions ◽  
Computational Resources ◽  
Image Analysis Techniques ◽  
Mouse Lymphoma

This presentation will describe a common core set of widely applicable image analysis techniques for automated quantitative analysis of volumetric microscope image data. Volumetric (as distinct from stereoscopic) three-dimensional (3-D) Microscopy is a rapidly maturing field offering the ability to image thick (compared to the depth of field) specimens using a variety of instrumentation techniques, and producing arrays of brightness values in three spatial dimensions. Also well developed are methods to correct the acquired images for a variety of physical effects including blur and attenuation.Commonly, what is of interest is the best-possible visualization of thick specimens. The next step, increasingly being considered in view of growing computational resources, and progress in image analysis techniques, seeks to quantify many of the processes and effects being studied. In some mainstream fields, such quantitation is essential. For instance, various assays for substance testing in pharmaceutical and chemical industries involve quantitative end points. As an illustration, the Draize assay for ocular irritancy testing of drugs and biochemical products for human use requires counting of live and dead cells that stain differently. Another example is the mouse lymphoma test that requires a 3-D counting of bacterial colonies. Neurobiological assays require morphometry, as well as quantification of changes in neurons as a function of time and various applied stimuli such as drugs, heat, and radiation. Angiogenesis assays require quantification of changes in vascular morphometry. Computerized image analysis is a powerful tool for extracting quantitative data from 3-D images for statistical analysis.

Automated counting and 3-D morphometry of nuclei in thick tissue slices from laser- scanning confocal microscopy data

1994 ◽  
Vol 52 ◽  
pp. 222-223
Author(s):  
H. Ancin ◽  
B. Roysam ◽  
M.H. Chestnut ◽  
T.E. Otte ◽  
D.H. Szarowski ◽  
...  
Keyword(s):  
Image Analysis ◽  
Laser Scanning ◽  
Three Dimensional ◽  
Laser Scanning Confocal Microscopy ◽  
Cell Counting ◽  
Image Analysis System ◽  
Tissue Slices ◽  
Scanning Confocal Microscopy ◽  
Nuclear Separation ◽  
Image Gradients

This paper presents recent advances in automated three-dimensional (3-D) image analysis methods for cell counting, and quantitative measurement of various nuclear properties in thick (30-120 μm) tissue sections that are imaged by a laser-scanning confocal microscope. The technical advances reported here are: (i) improved 3-D nuclear separation methods for analyzing samples containing large numbers of nuclei per unit volume, and large connected clusters; (ii) methods for adapting the image analysis system to handle a much larger variety of specimens with greater variability in image parameters, such as intensities, nuclear shapes and sizes; and (iii) methods for assisting a user in selecting parameter inputs to the counting system.Improved 3-D nuclear separation was achieved by computing image gradients from each optical slice. Perona and Malik’s algorithm was used to enhance the image gradient at true nuclear boundaries while suppressing the undesirable intra-nuclear image gradients. The result was used to compute a new proximity index for the partitional cluster analysis method.

Fast automated mosaic synthesis method for 2-D/3-D image analysis of specimens much wider than the field of view

1994 ◽  
Vol 52 ◽  
pp. 224-225
Author(s):  
D.E. Becker ◽  
H. Ancin ◽  
B. Roysam ◽  
J.N. Turner
Keyword(s):  
Image Analysis ◽  
Laser Scanning ◽  
Synthesis Method ◽  
Laser Scanning Confocal Microscopy ◽  
Field Of View ◽  
Cell Counting ◽  
Scanning Confocal Microscopy ◽  
Imaging Application ◽  
Partial View ◽  
The Individual

We present an efficient, robust, and widely-applicable technique for computational synthesis of wide-area images from a series of overlapping partial views. The synthesized image is the set union of the areas covered by the partial views, and is called the “mosaic”. One application is the laser-scanning confocal microscopy of specimens that are much wider than the field of view of the microscope. Another is imaging of the retinal periphery using a standard fundus imager. This technique can also be used to combine the results of various forms of image analysis, such as cell counting and neuron tracing, to generate large representations that are equivalent to processing the total mosaic, rather than the individual partial views.The synthesis begins by computing a concise set of landmark points for each partial view. The type of landmarks used can vary greatly depending on the application. For instance, in the retinal imaging application, the vascular branching and crossover points are a natural choice. Likewise, the locations of cells in Figs. 1 and 2 provide a natural set of landmarks for joining these images.

Perceptually Maximized Light Ray Synthesis with Only Two-Layered Light Field Display

2019 ◽  
Vol 63 (5) ◽  
pp. 50501-1-50501-11
Author(s):  
Ibragim R. Atadjanov ◽  
Seungkyu Lee
Keyword(s):  
Liquid Crystal ◽  
Light Field ◽  
Liquid Crystal Display ◽  
Three Dimensional ◽  
Field Of View ◽  
Depth Of Field ◽  
Perceptual Quality ◽  
Wide Field ◽  
Full Resolution ◽  
Small Depth

Abstract Multilayer light field three-dimensional displays are becoming popular due to their full resolution reconstruction and easy fabrication by utilizing existing display technologies such as liquid crystal display (LCD) panels. However, these displays still suffer from limited performance, achieving low angular resolution, narrow field of view, and small depth of field. One of the recent research ideas focusing on overcoming these limitations is perceptual quality improvement. But, currently introduced methods consider only specific issues/applications such as moiré fringe effect, and near-eye display technology. In this work, the authors propose a novel method of approximating light field data for dual-layered light field display considering the Human Visual and Perceptual System. The authors’ display configuration includes two liquid crystal panels with uniform backlight with no time multiplexing. It is not necessary for LCD panels to be parallel. For a wide field of view configuration, the authors introduce a quadratic penalization term to reduce ghost effects caused by neighboring views. This leads to an improved perceptual approximation of a given light field and increases the possibility of usage in design with a wider field of view configuration.

Computer Assisted Image Reconstruction of Oligomer Structure

1976 ◽  
Vol 34 ◽  
pp. 472-473
Author(s):  
A.M. Jones ◽  
A. Max Fiskin
Keyword(s):  
Three Dimensional ◽  
Depth Of Field ◽  
Computer Assisted ◽  
Projection Data ◽  
Two Dimensional ◽  
Single Particles ◽  
Dimensional Reconstruction ◽  
Computer Assisted Image ◽  
The Fourier Transform ◽  
Space Approach

If the tilt of a specimen can be varied either by the strategy of observing identical particles orientated randomly or by use of a eucentric goniometer stage, three dimensional reconstruction procedures are available (l). If the specimens, such as small protein aggregates, lack periodicity, direct space methods compete favorably in ease of implementation with reconstruction by the Fourier (transform) space approach (2). Regardless of method, reconstruction is possible because useful specimen thicknesses are always much less than the depth of field in an electron microscope. Thus electron images record the amount of stain in columns of the object normal to the recording plates. For single particles, practical considerations dictate that the specimen be tilted precisely about a single axis. In so doing a reconstructed image is achieved serially from two-dimensional sections which in turn are generated by a series of back-to-front lines of projection data.

Algorithms for automated montage synthesis of images from laser-scanning confocal microscopes

1995 ◽  
Vol 53 ◽  
pp. 650-651
Author(s):  
D. E. Becker
Keyword(s):  
Image Analysis ◽  
High Resolution ◽  
Laser Scanning ◽  
Cell Counting ◽  
Wide Area ◽  
Data Set ◽  
Computational Synthesis ◽  
Automated Cell Counting ◽  
Partial View ◽  
The Individual

An efficient, robust, and widely-applicable technique is presented for computational synthesis of high-resolution, wide-area images of a specimen from a series of overlapping partial views. This technique can also be used to combine the results of various forms of image analysis, such as segmentation, automated cell counting, deblurring, and neuron tracing, to generate representations that are equivalent to processing the large wide-area image, rather than the individual partial views. This can be a first step towards quantitation of the higher-level tissue architecture. The computational approach overcomes mechanical limitations, such as hysterisis and backlash, of microscope stages. It also automates a procedure that is currently done manually. One application is the high-resolution visualization and/or quantitation of large batches of specimens that are much wider than the field of view of the microscope.The automated montage synthesis begins by computing a concise set of landmark points for each partial view. The type of landmarks used can vary greatly depending on the images of interest. In many cases, image analysis performed on each data set can provide useful landmarks. Even when no such “natural” landmarks are available, image processing can often provide useful landmarks.

High-speed brightfield 3-D imaging and 3-D image analysis of thick and overlapped cell clusters in cytological preparations

1994 ◽  
Vol 52 ◽  
pp. 218-219
Author(s):  
Robert W. Mackin
Keyword(s):  
Image Analysis ◽  
High Speed ◽  
Three Dimensional ◽  
Image Data ◽  
Ccd Camera ◽  
Objective Lens ◽  
Array Processor ◽  
Vaginal Smears ◽  
Low Contrast ◽  
Computer Controlled

This paper presents two advances towards the automated three-dimensional (3-D) analysis of thick and heavily-overlapped regions in cytological preparations such as cervical/vaginal smears. First, a high speed 3-D brightfield microscope has been developed, allowing the acquisition of image data at speeds approaching 30 optical slices per second. Second, algorithms have been developed to detect and segment nuclei in spite of the extremely high image variability and low contrast typical of such regions. The analysis of such regions is inherently a 3-D problem that cannot be solved reliably with conventional 2-D imaging and image analysis methods.High-Speed 3-D imaging of the specimen is accomplished by moving the specimen axially relative to the objective lens of a standard microscope (Zeiss) at a speed of 30 steps per second, where the stepsize is adjustable from 0.2 - 5μm. The specimen is mounted on a computer-controlled, piezoelectric microstage (Burleigh PZS-100, 68/μm displacement). At each step, an optical slice is acquired using a CCD camera (SONY XC-11/71 IP, Dalsa CA-D1-0256, and CA-D2-0512 have been used) connected to a 4-node array processor system based on the Intel i860 chip.

Application of stereological analysis of cell volume to isolated myocytes in culture with and without adrenergic innervation

1990 ◽  
Vol 48 (3) ◽  
pp. 416-417
Author(s):  
Jane K. Rosenthal ◽  
Dianne L. Atkins ◽  
William J. Marvin ◽  
Penny A. Krumm
Keyword(s):  
Cardiac Myocytes ◽  
Structural Changes ◽  
Three Dimensional ◽  
Adrenergic Innervation ◽  
Culture Cell ◽  
Serial Sections ◽  
Newborn Rats ◽  
Primary Culture Cell ◽  
Biological Studies ◽  
Cell Volumes

To comprehend structural changes in cardiac myocytes accompanying adrenergic innervation, it is essential that a three dimensional analysis be performed. To date, biological studies which utilize stereological methods have been limited to cells in tissue and in organs. Our laboratory has utilized current stereological techniques for measuring absolute volumes of individual myocytes in primary culture. Cell volumes are calculated for two distinct groups of cells at 96 hours in culture: isolated myocytes and myocytes innervated with adrenergic neurons (Figure 1).Cardiac myocytes are cultured from the ventricular apices of newborn rats. Cells are plated directly onto tissue culture dishes with or without preplated explants from the paravertebral thoracolumbar sympathetic chain. On day four cultures are photographed and marked for one-to-one cell location. Following conventional fixation and embeddment in eponate-12, the cells are relocated and mounted for microtomy. The cells are completely sectioned at 120nm in their parallel orientation to the surface of the dish (Figure 2). Serial sections are collected on formvar coated slotted grids and are recorded in sequence.

Cryomicroscopy of crotoxin complex crystals at 400 KV

1989 ◽  
Vol 47 ◽  
pp. 826-827
Author(s):  
Jaap Brink ◽  
Wah Chiu
Keyword(s):  
Signal To Noise Ratio ◽  
3D Structure ◽  
Three Dimensional ◽  
Carbon Films ◽  
Depth Of Field ◽  
Basic Subunit ◽  
Ewald Sphere ◽  
Diffraction Patterns ◽  
Complex Crystals ◽  
Acidic Subunit

The crotoxin complex is a potent neurotoxin composed of a basic subunit (Mr = 12,000) and an acidic subunit (M = 10,000). The basic subunit possesses phospholipase activity whereas the acidic subunit shows no enzymatic activity at all. The complex's toxocity is expressed both pre- and post-synaptically. The crotoxin complex forms thin crystals suitable for electron crystallography. The crystals diffract up to 0.16 nm in the microscope, whereas images show reflections out to 0.39 nm2. Ultimate goal in this study is to obtain a three-dimensional (3D-) structure map of the protein around 0.3 nm resolution. Use of 100 keV electrons in this is limited; the unit cell's height c of 25.6 nm causes problems associated with multiple scattering, radiation damage, limited depth of field and a more pronounced Ewald sphere curvature. In general, they lead to projections of the unit cell, which at the desired resolution, cannot be interpreted following the weak-phase approximation. Circumventing this problem is possible through the use of 400 keV electrons. Although the overall contrast is lowered due to a smaller scattering cross-section, the signal-to-noise ratio of especially higher order reflections will improve due to a smaller contribution of inelastic scattering. We report here our preliminary results demonstrating the feasability of the data collection procedure at 400 kV.Crystals of crotoxin complex were prepared on carbon-covered holey-carbon films, quench frozen in liquid ethane, inserted into a Gatan 626 holder, transferred into a JEOL 4000EX electron microscope equipped with a pair of anticontaminators operating at −184°C and examined under low-dose conditions. Selected area electron diffraction patterns (EDP's) and images of the crystals were recorded at 400 kV and −167°C with dose levels of 5 and 9.5 electrons/Å, respectively.

Conformational characterization of nucleosome structure by Electron Microscopy

1993 ◽  
Vol 51 ◽  
pp. 194-195
Author(s):  
Gregory J. Czarnota
Keyword(s):  
Electron Microscopy ◽  
Structural Changes ◽  
Physiological State ◽  
Gene Activation ◽  
Three Dimensional ◽  
Macromolecular Complex ◽  
Ultrastructural Studies ◽  
Ionic Environment ◽  
Nucleosome Structure ◽  
Dimensional Reconstruction

Chromatin structure at the fundamental level of the nucleosome is important in vital cellular processes. Recent biochemical and genetic analyses show that nucleosome structure and structural changes are very active participants in gene expression, facilitating or inhibiting transcription and reflecting the physiological state of the cell. Structural states and transitions for this macromolecular complex, composed of DNA wound about a heterotypic octamer of variously modified histone proteins, have been measured by physico-chemical techniques and by enzyme-accessibility and are recognized to occur with various post-translational modifications, gene activation, transformation and with ionic-environment. In spite of studies which indicate various forms of nucleosome structure, all current x-ray and neutron diffraction studies have consistently resulted in only one structure, suggestive of a static conformation. In contrast, two-dimensional electron microscopy studies and three-dimensional reconstruction techniques have yielded different structures. These fundamental differences between EM and other ultrastructural studies have created a long standing quandary, which I have addressed and resolved using spectroscopic electron microscopy and statistical analyses of nucleosome images in a study of nucleosome structure with ionic environment.

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