Bruno Jaggi

Cell motility measurements with an automated microscope system.

The motility of 3T3 cells has been studied using a newly developed automated microscope system which is capable of recognizing live unstained cells growing in tissue culture. A large number of individual cells can be rapidly identified and characterized and their precise positions recorded. All cells can be revisited automatically every few minutes, and the new cell positions can be determined. Quantitative data from up to 1 000 cells can then be obtained, and cell movement parameters like cell speed, distance travelled, direction of movement, etc., can be measured for individual cells and for the whole cell population. In addition, for any number of chosen cells, high-resolution digitized images can be taken for further morphological studies, including acquisition of images of individual cells.

Importance of image quality for computing texture features in biomedical specimens

Quantitative descriptions of features offer an objective cell classification. In addition, quantitative analysis is ideally suited for automated screening of samples for diseased (transformed) cells. More recently, it has been shown that subtle changes of DNA distribution in the nucleus can be detected by quantitative analysis which escape human detection . A key issue in quantitative analysis is the quality of the digital image which must be captured for such analysis . This paper focuses on the affect of image quality on the very important features--texture features--which have been shown to be the most powerful classifiers in quantitative pathology . Image quality affects these features in a direct and indirect way. The latter is the result of an effect on texture features by the segmentation process which in turn strongly depends on image quality.

Quantitative microscope for image cytometry

The acquisition of high resolution, large area, microscope images is a crucial aspect of image cytometry. A unique quantitative visible light microscope was developed for this purpose, by positioning a high density, charge-coupled device (CCD) in the primary image plane of a chromatic aberration free, flat field objective lens. The CCD clocking and output circuits of this device were designed to minimize dark currents, to allow 10 bit digitization, 500 kHz to 8 MHz readout rates and variable integration time. A circuit was also implemented to compress the CCD pixel array on the chip by a factor of four. This pixel additive circuit results in an approximate fourfold increase in signal strength, increased frame rates and smaller amounts of data while maintaining the large field of view. This is particularly useful in: (1) detecting images at the low light levels encountered during fluorescence imaging; and (2) searching for objects while screening a microscope slide. The geometric and spectral properties of the bright-field optics were also optimized for quantitative CCD imaging by providing homogeneous illumination over the entire field of view and minimizing chromatic and geometric aberrations. With this device, bright-field images of a 0.23 micron diatom striation pattern and fluorescence images of 7 X 103 molecules of equivalent soluble fluorochrome (MESF) fluorescein-labeled intensity beads have been acquired and digitized.

AUTOMATED LOW DOSE ASSAY SYSTEM FOR SURVIVAL MEASUREMENTS OF MAMMALIAN CELLS IN VITRO

To study the effects of low dose ionizing radiation on cells, we developed the automated low dose assay systems which is a semiautomatic computer aided system that permits efficient examination of individual cells. Specifically, it provides for systematic and reliable location of cell positions and it features the means to return to those positions in order to classify cell growth. The system allows for microscopic scanning of a 7 × 12 cm surface in 12.5-µm steps with a repeatability of 100 µm.

Hough spectrum and geometric texture feature analysis

Traditionally, statistical and structural method are the two major approaches in texture description. However, there are also texture patterns of other natures, such as those of breast tissue on a mammogram which manifest a geometric feature. This paper proposes a new approach to measure geometric texture feature of an image on its Hough spectrum. The preliminary application of this texture descriptor to mammographic image processing shows its potential in the detection of spiculated masses.

Design of a solid-state microscope

A solid-state microscope (SSM) for quantitative microscopy in image cytometry has been designed to optimize spatial, photometric, and spectral resolution. The SSM is an optoelectronic device for scanning and viewing microscopic objects in the visible light spectrum. Using Kohler illumination, pulsed light is transmitted through the microscope sample and focused by a single objective lens. The objective magnifies and projects the image onto a large-area charge-coupled device (CCD) located at the intermediate focal plane. Flexibility in scanning of the sample and optical considerations require a CCD array of >1000 X1000 picture elements, with each element having a sensing area of approximately 7 Am X7 um. The signals from the picture elements are directly digitized and mapped on a one-to-one basis into a large-frame memory at a rate of approximately 20 Mbytes/s. The entire digital image is continuously displayed in real time on a monochrome monitor at a rate of >60 Mbytes/s. The images stored in the frame memory can also be accessed by an external image processing system for quantitative measurements. The main features of the SSM are its simple optical path, high resolution, large field of view, image display of the image on a monitor with overlay graphics for object labeling, direct access to any part of the digital image, different scanning modes includ-ing full-frame scanning and time delay integration, spectral imaging, and large dynamic (sensitivity) range.

Performance evaluation of a 12-bit, 8Mpel/s digital camera

A new generation, large frame (1317 X 1035 pel) digital camera, based on the MicroImager 1400 (MI1400), has been developed. The new MicroImager 1400-12 (MI1400- 12) uses improved noise reduction circuits to allow for 12 bit digitization up to 8 Mpel/s. Methods of correlated double sampling were investigated and a clamp-and-sample design was chosen. The MicroImager is a full frame device with a 100% fill factor. Integration time, readout frequency and pixel additive mode (binning) are under computer control. The camera outputs digital, RS-422 compatible data to the host frame memory through a 50-pin high density connector. The system is compatible with various (E)ISA and VME based imaging boards. The applications are quantitative digital microscopy in brightfield and fluorescence and in numerous industrial and medical camera applications. A number of comparative performance measurements were defined and carried out including spatial resolution (impulse response), photometric response (linearity), sensitivity, dynamic range, signal-to-noise ratio and other noise parameters. The MI1400-12 has a slightly improved impulse response. When used with primary image plane optics, the MicroImager shows a 6% improvement in the spatial resolution over standard C-mount compensation optics. In further comparisons the linearity of the MI1400-12s response holds to a coefficient of regression larger than 0.999 and has a four-fold increase in sensitivity over the MI1400. A dynamic range of 61 dB was measured for single frames corresponding to an 8 dB increase. For a 30 frame average, 77 dB was achieved. Circuit noise parameters (random noise at dark levels) improved by a factor of 3 to 4.

Lung imaging fluorescence endoscope. Development and experimental prototype

A lung imaging fluorescence endoscope has been developed which can be used for detection and localization of early lung cancer. We exploited tissue autofluorescence alone or in combination with fluorescent tumor localizing drugs to create pseudo images which can clearly delineate the diseased sites from the surrounding normal tissues. With this technique it is possible to detect early lung cancer as well as pre-cancerous lesions of one to two millimeters in diameter and only a few cell layers thick.

Development Of A Solid State Microscope

A Solid State Microscope (SSM) has been designed and is being developed in order to improve and optimize spatial, photometric and spectral resolution for quantitative microscopy. The SSM is an opto-electronic device for scanning and viewing microscopic objects in the visible light spectrum. Using Kohler illumination, pulsed light is transmitted through the microscope sample and focused by a single objective lens. The objective magnifies and projects the image onto a large area charge-coupled device (CCD) located at the intermediate focal plane (PIP). Sample scanning procedures and optical considerations require a CCD array of greater than 1000 x 1000 picture elements, with each element having a sensing area of approximately 7 μm x 7 μm. The signals from the picture elements are directly digitized and mapped on a one-to-one basis into a large frame memory at a rate of 20 Mpixels/s. The full digital image is continuously displayed in real-time at a rate of 60 frames/s on a gray scale monitor. The images stored in frame memory are accessed by workstation for quantitative measurements. The rationale for these specifications and in particular, how CCD characteristics relate to the optics and image display are discussed. Of particular importance is the sampling density which has been experimentally determined. The data shows that oversampling the image 3 to 4 times will be optimal for this design and a spatial resolution of at least 0.4 μm can be expected at a field of view of 1.4 Mpixels.

Time lapse records of cells in vitro using optical memory disk and cell analyzer

A system has been developed for making automated cinematographic records, using the Cell Analyzer imaging apparatus and an optical memory disk recorder (OMDR). The system is particularly useful when numerous individual cells must be followed over an extended period in a single experiment where the cells are growing in a multichamber tissue culture vessel, or are sparsely distributed within a single flask. The system is programmed to revisit each cell location at a prescribed time interval and to record a video frame on the OMDR. Each frame is stored on a preselected position on the optical memory disk under computer control so that individual records of each cell can be played back in sequence at a selected speed immediately after the experiment. The system can also make use of the Cell Analyzer imaging functions, such as the tracking of cell positions and quantitative measurements of morphologic cell features.