Low-cost conversion of the Polaroid MD-4 land camera to a digital gel documentation system

A simple, inexpensive design is presented for the rapid conversion of the popular MD-4 Polaroid land camera to a high quality digital gel documentation system. Images of ethidium bromide stained DNA gels captured using the digital system were compared to images captured on Polaroid instant film. Resolution and sensitivity were enhanced using the digital system. In addition to the low cost and superior image quality of the digital system, there is also the added convenience of real-time image viewing through the swivel LCD of the digital camera, wide flexibility of gel sizes, accurate automatic focusing, variable image resolution, and consistent ease of use and quality. Images can be directly imported to a computer by using the USB port on the digital camera, further enhancing the potential of the digital system for documentation, analysis, and archiving. The system is appropriate for use as a start-up gel documentation system and for routine gel analysis.     

Basic strategies for valid cytometry using image analysis

The present review provides a starting point for setting up an image analysis system for quantitative densitometry and absorbance or fluorescence measurements in cell preparations, tissue sections or gels. Guidelines for instrumental settings that are essential for the valid application of image analysis in cytophotometry and cytofluorometry are described. The general principles of the working mechanism of CCD cameras in combination with general methods to improve the behaviour of the cameras are presented. Optimization of illumination of microscopical and macroscopical objects receives special attention because of its importance for valid cytometry. Sources of errors in quantitative measurements are listed and step-by-step charts for tuning the CCD camera, frame grabber and illumination for the optimal use of the systems are described. Suggestions are given for improvement of image arithmetics in difficult imaging situations, such as low fluorescence signals and high absorbance signals. This revised version was published online in November 2006 with corrections to the Cover Date.     

Measurement of fluorescence using digital integration of video images

The authors describe a system for the quantitation of fluorescence light output by individual cells using the signal obtained from a silicon intensifier target video camera. The video image is digitized to 4 bits (16 levels), and a 512 X 512 matrix is constructed and stored in 128K of video memory. Areas to be measured are user-specified by means of a light pen entry system. Recorded intensities are integrated under microprocessor control to provide a measure of total fluorescence in the selected areas. The distribution of light intensities per pixel over the 16-level gray scale as well as morphometric data are also obtainable. Linear response to transmission and fluorescence standards was verified. Reproducibility of the system was evaluated using fluorescent beads and glutaraldehyde-fixed chick red blood cells, which gave coefficients of variation comparable to those obtainable from other systems. Measurements of DNA per nucleus of human diploid fibroblasts using Hoechst dye 33258 yielded the two sharp peaks corresponding to the 2C and 4C values of DNA expected from such cells. We conclude that digital video measurement of fluorescence provides meaningful data and has considerable promise as a sensitive tool for the quantitation of fluorescence at the cellular level.     

High-sensitivity DNA detection with a laser-excited confocal fluorescence gel scanner

A high-sensitivity, laser-excited confocal fluorescence gel scanner has been developed and applied to the detection of fluorescently labeled DNA. An argon ion laser (1-10 mW at 488 nm) is focused in the gel with a high-numerical aperture microscope objective. The laser-excited fluorescence is gathered by the objective and focused on a confocal spatial filter, followed by a spectral filter and photodetector. The gel is placed on a computer-controlled scan stage, and the scanned image of the gel fluorescence is stored and analyzed in a computer. This scanner has been used to detect DNA separated on sequencing gels, agarose mapping gels and pulsed field gels. Sanger sequencing gels were run on M13mp18 DNA using a fluoresceinated primer. The 400-microns-thick gels, loaded with 30 fmol of DNA fragments in 3-mm lanes, were scanned at 78-microns resolution. The high resolution of our scanner coupled with image processing allows us to read up to approximately 300 bases in four adjacent sequencing lanes. The minimum band size that could be detected and read was approximately 200 microns. This instrument has a limiting detection sensitivity of approximately 10 amol of fluorescein-labeled DNA in a 1 x 3-mm band. In applications to agarose mapping gels, we have exploited the fact that DNA can be prestained with ethidium homodimer, followed by electrophoresis and fluorescence detection to achieve picogram sensitivity. We have also developed methods using both ethidium homodimer and thiazole orange staining which permit two-color detection of DNA in one lane.(ABSTRACT TRUNCATED AT 250 WORDS)     

Objective quality evaluation of fluorescence images to optimize automatic image acquisition.

BACKGROUND: Because different spectral sensitivities of human eye and image sensor lead to different perception of fluorescence signals, data generation is an important step in image analysis, because following work steps depend on it. METHODS: We developed a method to determine image parameters allowing an objective appraisal of quality of image data as well as a separation of object and background. RESULTS: Calculated parameters can be used for an automated adjustment of camera parameters in image analysis systems. DISCUSSION: Our approach for objective adjusted data generation achieves an improvement of analysis quality.     

High resolution scanning of absorbing and fluorescent electrophoresis gels using video image analysis

A low cost, microcomputer-controlled image analysis system isdescribed which scans electrophoresis gels or photographic negativeswith high resolution. Absorbing gels (e.g. with stained proteins)are analyzed using broadband or monochromatic visible light.The gel is scanned by a video camera with a macro objective;the gray level of each pixel is digitized sequentially and thevalues are stored in the computer memory. Repetitive scanningis used to average the absorption values before plotting ona digital plotter. Photographic negatives of gels and autoradiogramsare scanned using the same technique. Fluorescent, non-absorbinggels (e.g. nucleic acids stained with ethidium bromide) areanalyzed on a transilluminator with u. v. excitation radiation.The fluorescence intensity of each pixel is digitized and processedas described above. Received on June 4, 1987; accepted on July 21, 1987     

SYBR Green I staining of pulsed field agarose gels is a sensitive and inexpensive way of quantitating DNA double-strand breaks in mammalian cells.

Pulsed field gel electrophoresis (PFGE) is widely used to measure DNA double strand breaks (dsb). The DNA of cultured cells can be prelabelled with radioactivity, which helps greatly in detection and quantitation of DNA dsb. However, this approach cannot be used with non-cycling cells from biopsy material. We describe a method which uses SYBR Green I to stain DNA in dried agarose gels. DNA is detected and analysed using readily available camera equipment and image analysis software. This method is as sensitive as [3H]thymidine prelabelling of cells and allows DNA dsb to be measured simply and economically in non-cycling cells.     

Practical applications in molecular biology of sensitive fluorescence detection by a laser-excited fluorescence image analyzer.

A new kind of fluorescence image analyzer was developed for a variety of uses, especially in molecular biology. Compounds labeled with fluorescent groups on a gel or nitrocellulose membrane are excited with 532 nm of light from a green laser. The fluorescence emitted passes through light-collecting fibers to a photomultiplier. Imaging data converted from the emitted light are analyzed by a microcomputer and stored on a magnetic optical disk. Dideoxy DNA sequencing was done with the same amount of DNA used for autoradiography, and the sequencing ladders obtained from gel scanning were automatically converted to sequence data by the analyzer. When an agarose gel was analyzed after electrophoresis, DNA stained with ethidium bromide was detected by the analyzer with higher sensitivity rather than by the conventional photographic method. Nylon and nitrocellulose membranes could be read by the analyzer, so blot hybridization experiments can be done without radioisotopes. High-quality computer storage of the imaging data from gel electrophoresis and hybridized membranes, including pulsed-field gels, make it possible to quantify image intensity and to construct many kinds of databases.     

Computer video acquisition and analysis system for biological data

The BIAS (Biological Image Analysis System) was developed to:(i) permit accurate entry and image processing of biologicaldata; (ii) minimize the need for specialized hardware; and (iii)aid in the human genome mapping and other projects. The firstmouse/cursor key-driven module was designed to be user interactiveand readily accessible to many laboratories. It contains theDRSNDS programs which automate the entering of data in a systematicformat. The types of data that can be entered utilizing thismodule are DNA — RNA gels from either a positive or negativePolaroid^TM image, autoradiograms or biotinylated images fromSouthern, Northern and dot or slot blot hybridization analyses.The image is acquired using a video camera and then digitizedfor subsequent analysis. During the analysis graphical representationsof the intermediate results are provided to assure user confidence.At any point within the program the user may obtain on-linehelp with the current task. The output displays the mol. wtof each individual component in the appropriate context. Thepresent version of the program produces results comparable witha human interpreter for some data. Band shifting and opticaldensity calculations are in a prototype form to permit evaluationof various techniques. Future work is directed at expandingthe system's capabilities to interpret data from other biologicalanalyses including DNA sequencing gels. Received on December 1, 1987; accepted on December 12, 1987     

Simultaneous intracellular recording and calcium imaging in single neurons of hippocampal slices

Fluorescent Ca2+ indicator dyes can be introduced into cells through the same microelectrode used for intracellular voltage recording. Simultaneous measurement of cell membrane potential and intracellular Ca2+ concentration can be very helpful in interpreting the mechanisms of Ca2+ increases. This chapter describes fluorescence image acquisition using a CCD camera and a computer program that also records a synchronized membrane potential trace. The same program allows for preliminary data analysis. More elaborate analyses can be accomplished with commercial programs. We also describe quantitative evaluations of sources of error in the use of the statistic deltaF/F as an indicator of Ca2+ concentration. Especially important errors to minimize are changes in background fluorescence and inappropriate autofluorescence corrections. Some improvement of fluorescence images of cells deep within slices may be accomplished by masking. One method is described for making a mask based on the raw fluorescence image. With another method, highly detailed cell morphologies may be conveyed by using masks based on neurobiotin injections and camera lucida drawings.     

Using a CCD camera imaging system as a recording device to quantify human DNA by slot blot hybridization

Slot blot hybridization of membrane-immobilized, single-stranded human DNA with the higher primate-specific alphoid probe D17Z1 is routinely used in forensic science to estimate the amount of DNA in biological samples. Typically, a chemiluminescent signal captured on film records the hybridization, and the quantity of the signal is related to the amount of immobilized DNA. Digital imaging using a cooled CCD camera offers an alternate non-film-based method for image acquisition with comparable sensitivity of detection, a greater dynamic range, enhanced capability of data interpretation, and often faster results than film. In addition, the data support the premise that more accurate and precise human DNA quantification should be obtained by not assuming a linear response of signal to known standards. Instead, quantity should be estimated using a second-order standard curve (R2 = 0.999). Finally, a CCD camera imaging system offers versatility for image capture of different signal sources and analysis of samples on a variety of support media.     

A simple computer-based video image analysis system and potential applications to microbiology

An inexpensive microcomputer-based image analysis system is described in which an Apple microcomputer acquires data from a video camera or video cassette recorder and measures the brightness of the image received at specified points or areas. Suggested uses for this apparatus include measurements of chlorophyll fluorescence in algal cells, determination of the effects of ultraviolet illumination on chlorophyll fluorescence, estimation of total amounts of chlorophyll in a microscope field, and microspectrophotometic and microdensitometic measurements. A similar ssytem using the IBM personal computer with a different interface is also described.     

A fast,low cost,and highly efficient fluorescent DNA labeling method using methyl green

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.     

A graphical user interface for quantitative imaging and analysis of electrophoretic gels and autoradiograms.

DNA/GUI (DNA Graphical User Interface) is an interactive software system for rapid and efficient analysis of images of the types used in genome mapping, such as autoradiograms and electrophoretic gels. Images are digitized using a commercially available charge-coupled-device (CCD) camera system and analyzed on a graphics workstation using a menu-driven user interface. DNA/GUI features automatic lane and band detection, simultaneous display of multiple images and a unique spatial-normalization algorithm. Images and their associated data are archived and easily available for later recall. Preliminary results indicate that DNA/GUI is a useful tool in the analysis and comparison of images used in a variety of applications such as genetic-linkage analysis and DNA restriction mapping. The interactive display software is based on the X Window System and is therefore readily portable to a variety of graphics workstations.     

Time resolved imaging microscopy. Phosphorescence and delayed fluorescence imaging.

An optical microscope capable of measuring time resolved luminescence (phosphorescence and delayed fluorescence) images has been developed. The technique employs two phase-locked mechanical choppers and a slow-scan scientific CCD camera attached to a normal fluorescence microscope. The sample is illuminated by a periodic train of light pulses and the image is recorded within a defined time interval after the end of each excitation period. The time resolution discriminates completely against light scattering, reflection, autofluorescence, and extraneous prompt fluorescence, which ordinarily decrease contrast in normal fluorescence microscopy measurements. Time resolved image microscopy produces a high contrast image and particular structures can be emphasized by displaying a new parameter, the ratio of the phosphorescence to fluorescence. Objects differing in luminescence decay rates are easily resolved. The lifetime of the long lived luminescence can be measured at each pixel of the microscope image by analyzing a series of images that differ by a variable time delay. The distribution of luminescence decay rates is displayed directly as an image. Several examples demonstrate the utility of the instrument and the complementarity it offers to conventional fluorescence microscopy.     

Imaging technologies for the detection of multiple stains in proteomics

Laser-based scanners and charge-coupled device (CCD) camera systems are evolving to have greater functional capabilities for capturing images from a range of staining technologies used in gel electrophoresis and electroblotting. Digitizing Coomassie Brilliant Blue (CBB) stained gels and silver stained gels has now become possible using a laser-based gel scanner, the FLA-5000 fluorescent image analyzer system. Also, a simultaneous dual fluorescent imaging function has been incorporated into the FLA-5000 system, utilizing dichroic mirrors with both the optical system and the emission filter. In the workflow of routine proteomics research, the relationship between SYPRO dye staining and fluorescent detection using the FLA-5000 system have become symbiotic. Additionally in many cases, subsequent staining of the gel with CBB is useful for future research, and thus imaging instruments should be able to handle both staining formats. Digitizing the CBB stained gel can now be easily performed by the FLA-5000 fluorescent image analyzer system using a fluorescent board as an epi-illumination background. A cooled CCD camera system has the potential of imaging not only chemiluminescent membranes but also digitizing molecular weight markers and fluorescent detection of SYPRO dye-stained gels. With Multi Gauge software version 2.0 it is now a simple task to combine two images into one, as commonly required in dual detection experiments. The LAS-3000 system was designed to capture chemiluminescent images and to digitize the images automatically. Thus, new capabilities added to gel imaging systems make them capable of detecting and displaying multiple signals more conveniently.     

Quantitation and mapping of integrated human papillomavirus on human metaphase chromosomes using a fluorescence microscope imaging system.

Integrated human papillomavirus type 16 (HPV-16) DNA was directly visualized on metaphase chromosomes in the two human cervical carcinoma cell lines SiHa and CaSki by fluorescence in situ hybridization with a biotinylated DNA probe (7.9 kb). The fluorescence intensities of hybridization signals from single copies and dispersed clusters of integrated HPV-16 DNA were quantified using a microscope equipped with a cooled-CCD camera that was interfaced to an image processor and host computer. Hybridization signals were localized on chromosomes using separate, registered images of 4',6-diamidino-2-phenylindole (DAPI) or propidium iodide stained metaphase chromosome spreads. In both SiHa and CaSki spreads, a single fluorescein signal was observed on one or both chromatids of chromosome 13, which was identified by simultaneous hybridization with a biotinylated centromere probe specific for chromosomes 13 and 21. Ratios of the distance from 13pter to the HPV-16 signals to the entire chromosome length were approximately 0.63 +/- 0.05 in both SiHa and CaSki cells, indicating the possibility of a common integration domain on chromosome 13. In SiHa cells, no additional signals were observed on other chromosomes. This observation, taken together with literature reports that SiHa cells contain 1 to 2 copies of the HPV-16 genome in this region of chromosome 13, suggests that each fluorescein signal on chromosome 13 represents one equivalent of the HPV-16 genome. The total integrated fluorescence intensity in isolated CaSki metaphase chromosome spreads was approximately two orders of magnitude greater than that of a single copy of HPV-16 DNA in SiHa cells, indicating an increase in HPV-16 copy number.(ABSTRACT TRUNCATED AT 250 WORDS)     

Portable telepathology: methods and tools

Telepathology is becoming easier to implement in most pathology departments. In fact e-mail image transmit can be done from almost any pathologist as a simplistic telepathology system. We tried to develop a way to improve capabilities of communication among pathologists with the idea that the system should be affordable for everybody. We took the premise that any pathology department would have microscopes and computers with Internet connection, and selected a few elements to convert them into a telepathology station. Needs were reduced to a camera to collect images, a universal microscope adapter for the camera, a device to connect the camera to the computer, and a software for the remote image transmit. We found out a microscope adapter (MaxView Plus) that allowed us connect almost any domestic digital camera to any microscope. The video out signal from the camera was sent to the computer through an Aver Media USB connector. At last, we selected a group of portable applications that were assembled into a USB memory device. Portable applications are computer programs that can be carried generally on USB flash drives, but also in any other portable device, and used on any (Windows) computer without installation. Besides, when unplugging the device, none of personal data is left behind. We selected open-source applications, and based the pathology image transmission to VLC Media Player due to its functionality as streaming server, portability and ease of use and configuration. Audio transmission was usually done through normal phone lines. We also employed alternative videoconferencing software, SightSpeed for bi-directional image transmission from microscopes, and conventional cameras allowing visual communication and also image transmit from gross pathology specimens. All these elements allowed us to install and use a telepathology system in a few minutes, fully prepared for real time image broadcast.     

DNA staining in agarose and polyacrylamide gels by methyl green

ABSTRACT

Methyl green (MG) is an inexpensive, nonproprietary, traditional histological stain for cell nuclei. When bound to DNA and upon excitation with orange-red light, it fluoresces brightly in the far red region. We compared MG with ethidium bromide (EtBr), the conventional stain for DNA in gels, and Serva DNA stain G? (SDsG), a proprietary stain marketed as a safer alternative to EtBr for staining of electrophoresed DNA bands in agarose and polyacrylamide gels. DNA-MG fluorescence was recorded and 2.4 μg/ml MG produced crisp images of electrophoresed DNA after incubation for 10 min. Stain solutions were stable and detection limits for faint bands as well as relative densitometric quantitation were equivalent to EtBr. MG, EtBr and SDsG cost 0.0192, 0.024 and 157.5 US cents/test, respectively. MG is an effective stain for visualizing DNA in agarose and polyacrylamide gels. Its major advantages including low cost, comparable quality of staining, storage at room temperature, photo-resistance and low mutagenic profile outweigh its disadvantages such as staining of tracking dye and requirement for a gel documentation system with a red filter.     

Measurement of marine picoplankton cell size by using a cooled, charge-coupled device camera with image-analyzed fluorescence microscopy.

Accurate measurement of the biomass and size distribution of picoplankton cells (0.2 to 2.0 microns) is paramount in characterizing their contribution to the oceanic food web and global biogeochemical cycling. Image-analyzed fluorescence microscopy, usually based on video camera technology, allows detailed measurements of individual cells to be taken. The application of an imaging system employing a cooled, slow-scan charge-coupled device (CCD) camera to automated counting and sizing of individual picoplankton cells from natural marine samples is described. A slow-scan CCD-based camera was compared to a video camera and was superior for detecting and sizing very small, dim particles such as fluorochrome-stained bacteria. Several edge detection methods for accurately measuring picoplankton cells were evaluated. Standard fluorescent microspheres and a Sargasso Sea surface water picoplankton population were used in the evaluation. Global thresholding was inappropriate for these samples. Methods used previously in image analysis of nanoplankton cells (2 to 20 microns) also did not work well with the smaller picoplankton cells. A method combining an edge detector and an adaptive edge strength operator worked best for rapidly generating accurate cell sizes. A complete sample analysis of more than 1,000 cells averages about 50 min and yields size, shape, and fluorescence data for each cell. With this system, the entire size range of picoplankton can be counted and measured.