Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots

Karger, Achim E.; Weiss, Robert B.; Gesteland, Raymond F.

doi:10.1021/ac00061a024

Cited by 14 publications

(11 citation statements)

References 22 publications

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“…The number of exposures can also be reduced through the use of time-delayed integration (TDI) CCD imaging. Karger et al (6) detected chemiluminescent DNA sequence information at the rate of 52cm/h in the vertical direction using this technique. Finally, we believe that further development and improvement of CCD camera systems with concomitant cost lowering will enable rapid data acquisition and permit automated chemiluminescent imaging.…”

Section: Discussionmentioning

confidence: 99%

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Imaging of chemiluminescent signals with cooled CCD camera systems

Martin¹,

Bronstein²

1994

J. Biolumin. Chemilumin.

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We investigated imaging of chemiluminescent signals from 1,2-dioxetanes with cooled CCD cameras. Non-radioactive detection methods for biomolecules utilizing these chemiluminescent substrates for alkaline phosphatase have been developed. Applications which have been successfully adapted to this technology include Southern and Northern blotting, immunoblotting, ELISA methods and DNA sequencing. Dephosphorylation of the dioxetane CSPD by alkaline phosphatase generates an unstable anion that decomposes resulting in light production. The wavelength of the emitted light is approximately 460 nm. We have utilized Photometrics Star and MXC 200L cooled CCD cameras for direct imaging of chemiluminescent signals. Benefits of utilizing a CCD detector include rapid data digitization and more accurate quantitation of chemiluminescent signals compared to film-based densitometry owing to the significantly greater dynamic range. Chemiluminescent images from dot blots of biotinylated DNA, Southern blots and DNA sequencing gel blots were obtained. In a chemiluminescent microtitre plate assay, serial dilutions of alkaline phosphatase spanning four orders of magnitude can be detected. Our results indicate that the digitization of chemiluminescent signal data with cooled CCD cameras is an excellent alternative to 32P detection methods utilizing storage phosphor screen imaging systems.

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Section: Discussionmentioning

confidence: 99%

“…Similar, dedicated instrumentation for imaging chemiluminescent signals has not been available. Several studies have described the use of cooled and intensified CCD cameras to image chemiluminescent signals from a variety of applications, including dot blots (3), immunoblotting (3,4) and DNA sequencing (5,6).…”

Section: Introductionmentioning

confidence: 99%

Imaging of chemiluminescent signals with cooled CCD camera systems

Martin¹,

Bronstein²

1994

J. Biolumin. Chemilumin.

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“…The group III eigenvectors are discarded, and all the group II eigenvectors are processed using the MF cosmic spike suppression procedure. 3,4,12 The group IIa eigenvectors are processed using a 7 point MF algorithm, while group IIb eigenvectors are processed with a 5 point MF. Two different lter widths are chosen because the IIa eigenvectors are m ore likely to contain cosmic spikes of m ultiple pixel width.…”

Section: Ax(s )mentioning

confidence: 99%

“…Although this method is quite general and very effective, the present work describes a variant of the UBS method that is speci cally designed to remove cosmic spikes from large hyper-spectral data matrices (DM) but does not require performing sequential measurem ents on the same sample. The new UBS-DM method is a hybrid of the UBS, principle component analysis (PCA), 2 and median lter (MF) 3,4 algorithms. The UBS-DM method differs signi cantly from previous applications of MF and PCA for cosm ic-spike suppression 3,4 and/or denoising 5 and better preserves the full spectral feature information content of the input data.…”

Section: Introductionmentioning

confidence: 99%

Removal of Cosmic Spikes from Hyper-Spectral Images Using a Hybrid Upper-Bound Spectrum Method

Zhang

Ben‐Amotz

2002

Appl Spectrosc

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A hybrid of the upper-bound spectrum (UBS), principle component analysis (PCA), and median filter (MF) algorithms is used to efficiently remove cosmic spike spectral artifacts from hyper-spectral data matrices (DM). The resulting UBS-DM method is shown to introduce less spectral distortion than the widely used MF cosmic spike suppression algorithm alone. The new method relies on a truncated series of PCA eigenvectors to produce an upper-bound spectrum, which is in turn used to detect and remove cosmic spikes. The PCA eigenvectors are separated into three groups, each of which is treated in a different manner. The first group of eigenvectors, which contain most of the spectral correlation of interest, are not preprocessed prior to applying the UBS-DM method. The second group, which are more severely contaminated by cosmic spikes (but may also contain important spectral correlations), are MF preprocessed before applying UBS. The third group of eigenvectors, which contain virtually no spectral information, are ignored in regenerating the upper-bound spectra used for spike detection and removal. The UBS-DM algorithm is tested using both synthetic and experimental (iron oxide) Raman hyper-spectral data sets, each containing several thousand spectra, with about 1000 wavelength pixels.

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“…The main limitation concerns the size of the sample to be analysed, which is limited to the size of a microplate (10 x 13 cm), and it is not possible to analyse the whole blot corresponding to the sequencing gel with as good resolution as with X-ray films. Sequencing can be automated using direct transfer electrophoresis and a cooled CCD camera (1 6) or a line scanning system producing digital images (17).…”

Section: Nucleic Acid Probesmentioning

confidence: 99%

Applications of low‐light imaging to life sciences

Nicolas

1994

J. Biolumin. Chemilumin.

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Photon imaging is a new technique for the quantitative analysis of bioluminescence and chemiluminescence and can be performed both at the macro and micro levels. The high sensitivity and spatial resolution of photon-counting cameras have resulted in the development of new applications in the life sciences. At the macro level, imaging is a valuable tool for the rapid identification of biological samples emitting long-lasting glows in assays using microtitre plates or filter formats (immunoassays, DNA probes, phagocytosis, gene expression, metabolite and drug analysis) and also for in vivo studies of promoter activity. At the micro level, low-light imaging can be used for analysing multiple analytes on micro sensors) and for advanced cell analysis (immunocytology, in situ hybridization, identification of cells or tissues expressing the luciferase gene, intracellular or intercellular protein traffic, metabolite analysis and imaging of Ca2+ flux and phosphorylation reactions). Two-dimensional photon-counting instrumentation is a versatile and powerful research tool for imaging and is complementary to conventional luminometers. The main applications to the life sciences involve many types of luminescence assays and can be performed on multiple samples in standard and non-standard formats. Photon-counting coupled to imaging is very helpful in selecting microorganisms or cells expressing bioluminescent genes. Measurements can be made in vitro and in vivo with a sensitivity comparable to that of phototube luminometers.

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Line scanning system for direct digital chemiluminescence imaging of DNA sequencing blots

Cited by 14 publications

References 22 publications

Imaging of chemiluminescent signals with cooled CCD camera systems

Imaging of chemiluminescent signals with cooled CCD camera systems

Removal of Cosmic Spikes from Hyper-Spectral Images Using a Hybrid Upper-Bound Spectrum Method

Applications of low‐light imaging to life sciences

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