Characterization of low‐light‐level cameras for digitized video microscopy

Tsay, Tsong-Tseh; Inman, Richard; Wray, Barnaby E.; Herman, Brian; Jacobson, Ken

doi:10.1111/j.1365-2818.1990.tb03054.x

Cited by 32 publications

(14 citation statements)

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“…Although use of a color camera reduced the detection efficiency, this could be compensated for by accumulating (integrating) the signal for greater periods of time on the faceplate of the camera. This also proved more precise than using a high gain intensifier tube (31). The output from the camera (red, green, and blue analog signals), was first converted to hue, saturation, and intensity (H, S, and I) and then by use of a n analog-to-digital convertor (A in Fig.…”

Section: Materials and Methods Automatic Fluorescence Image Cytometermentioning

confidence: 99%

“…Quantification is accurate because the intensity of the emitted fluorescence is directly proportional to the stain concentration in the specimen and because automatic image analysis is highly consistent in defining the boundaries of nuclei (16). Furthermore, automatic image analysis requires minimal manual effort and low levels of labeled markers can be detected using a low-light camera (31), making fluorescent stains at least as sensitive as lightabsorbing (colorimetric) stains.…”

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Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry

et al. 1994

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Automatic fluorescence image cytometry (AFIC) is a fast, sensitive, and reliable approach for screening slide-based clinical specimens. In this study, we applied AFIC to identify cancer-associated human papillomavirus (HPV) genotypes 16 and 18 in individual cells of cervical smears using a sensitive fluorescence based in situ hybridization (FISH) assay. HPV sequences were labeled by FISH and the cells imaged using an epi-fluorescence microscope coupled to a low-light color CCD camera. Before application to clinical specimens, AFIC was assessed using fluorescent calibration beads and cervical cancer cell lines containing known numbers of integrated HPV genomes per nucleus. Assessment showed that our AFIC had a linear response, was quantitatively accurate, and had the sensitivity to detect one HPV genome per nucleus. After acquisition of images, computer algorithms identified every cell nucleus (via a fluorescent DNA counterstain) and quantified the FISH signal per nucleus. AFIC was employed to screen 27 patient specimens for HPV 16/18, of which 12 were positive. The HPV status of the specimens positively correlated with the pathological diagnosis, and since AFIC automatically and correctly located every cell, it was possible to directly compare morphology and HPV status in the same cell. In conclusion, the combination of FISH and AFIC is a sensitive and quantitative method to detect high risk HPV sequences in cervical Smears. Q 1994 Wiley-Liss, Inc.Key terms: Fluorescent in situ hybridization, cervical smears, human papillomavirus, automated fluorescence image cytometry Cervical dysplasia and carcinoma are common among women throughout the world. Approximately 500,000 women worldwide are diagnosed annually as having cervical cancer (24) and the mortality rate associated with this malignancy is second to breast cancer (20). In the United States, it is estimated that 13,000 women develop invasive cervical cancer annually (25).Substantial evidence has accumulated associating specific human papillomaviruses (HPVs) with human anogenital disorders, most notably cervical cancer (3,11,13,35). To date, over 60 different types of HPV have been described, of which about 20 have been associated with anogenital lesions (8). These 20 HPVs have been divided into "high risk" and "low risk" groups based on their association with benign vs. ma-'Supported by grants from NIH (CA51323 and the Office of Research on Womens Health), the Whitaker Foundation, and a contract with American Innovision, Inc.

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Section: Materials and Methods Automatic Fluorescence Image Cytometermentioning

confidence: 99%

mentioning

confidence: 99%

Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry

et al. 1994

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“…By using interlace defeat circuitry, 16 msec temporal resolution might be possible. However, the half signal detector lag time of the ISIT camera employed was only slightly less than 33 msec (Tsay et al, 1989). This limitation might be overcome by using an intensified video-rate CCD camera, now commercially available, which has a much shorter detector lag.…”

Section: Discussionmentioning

confidence: 99%

Rescue of Injured Myocytes

Lemasters¹

1989

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“…Subsequent camera designs involve more sophisticated, "second or third generation" image intensifiers optically coupled to conventional video cameras or, better, to high-quality video-rate CCD cameras (Spring and Lowy 1989;Tsay et al 1990). While such optical coupling can be achieved using lenses, light transfer is more efficient using a bundle of optical fibres.…”

Section: Cameras For Low-light-level Fluorescence Imagingmentioning

confidence: 99%

Electronic light microscopy: present capabilities and future prospects

Shotton

1995

Histochem Cell Biol

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Electronic light microscopy involves the combination of microscopic techniques with electronic imaging and digital image processing, resulting in dramatic improvements in image quality and ease of quantitative analysis. In this review, after a brief definition of digital images and a discussion of the sampling requirements for the accurate digital recording of optical images, I discuss the three most important imaging modalities in electronic light microscopy--video-enhanced contrast microscopy, digital fluorescence microscopy and confocal scanning microscopy--considering their capabilities, their applications, and recent developments that will increase their potential. Video-enhanced contrast microscopy permits the clear visualisation and real-time dynamic recording of minute objects such as microtubules, vesicles and colloidal gold particles, an order of magnitude smaller than the resolution limit of the light microscope. It has revolutionised the study of cellular motility, and permits the quantitative tracking of organelles and gold-labelled membrane bound proteins. In combination with the technique of optical trapping (optical tweezers), it permits exquisitely sensitive force and distance measurements to be made on motor proteins. Digital fluorescence microscopy enables low-light-level imaging of fluorescently labelled specimens. Recent progress has involved improvements in cameras, fluorescent probes and fluorescent filter sets, particularly multiple bandpass dichroic mirrors, and developments in multiparameter imaging, which is becoming particularly important for in situ hybridisation studies and automated image cytometry, fluorescence ratio imaging, and time-resolved fluorescence. As software improves and small computers become more powerful, computational techniques for out-of-focus blur deconvolution and image restoration are becoming increasingly important. Confocal microscopy permits convenient, high-resolution, non-invasive, blur-free optical sectioning and 3D image acquisition, but suffers from a number of limitations. I discuss advances in confocal techniques that address the problems of temporal resolution, spherical and chromatic aberration, wavelength flexibility and cross-talk between fluorescent channels, and describe new optics to enhance axial resolution and the use of two-photon excitation to reduce photobleaching. Finally, I consider the desirability of establishing a digital image database, the BioImage database, which would permit the archival storage of, and public Internet access to, multidimensional image data from all forms of biological microscopy. Submission of images to the BioImage database would be made in coordination with the scientific publication of research results based upon these data.(ABSTRACT TRUNCATED AT 400 WORDS)

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Characterization of low‐light‐level cameras for digitized video microscopy

Cited by 32 publications

References 21 publications

Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry

Detection of human papillomavirus type 16/18 DNA in cervicovaginal cells by fluorescence based in situ hybridization and automated image cytometry

Rescue of Injured Myocytes

Electronic light microscopy: present capabilities and future prospects

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