Novel detection schemes and automated image analysis algorithms for planar chromatography and gel electrophoresis

After a detailed introduction to Charge Coupled Devices (CCDs) and a discussion of personal computer capabilities, examples of their applications involving novel analytical techniques are presented;A new laser-based indirect fluorometric detection method for thin-layer chromatography is demonstrated with digitally controlled two-dimensional scanning and computer-aided image acquisition and vision. Utilizing this mode, universal detection is possible without derivatization, with a detection limit of 6 pg; 100 times lower than with human vision;A detection scheme for DNA in agarose slab gel electrophoresis based on ultraviolet absorption allows for convenient in situ monitoring of conventional gels via a CCD camera system, which greatly improves detectability compared to previous applications of this detection mode. With the use of the CCD system, low light level images can be acquired rapidly with minimal noise. Further on-line processing of the image allows one to correct for background and lighting variations and other non-random noise. The detection limit is about 5 ng of double-stranded DNA per band, which is at least 25 times lower than those obtained previously for UV absorption;The UV imaging method, along with a native fluorescence scheme, is demonstrated for detection of proteins in agarose slab gels. Detectability is comparable to traditional visualization schemes based on coomassie blue staining;A computerized method for correcting systematic signal distortions of images from slab gel electrophoresis is presented. The method is useful for interpretation of one-dimensional slab gel electrophoresis images where the information is manifested in rectangular shaped bands and the distortions can be described by low order polynomial functions. The purpose is to eliminate human judgement and interaction from the process, which is necessary for automated analysis of electrophoretic gels;A computerized expert system is introduced for the reduction of data acquisition in random access two-dimensional scanners or imaging devices where multiple scans are necessary to identify all features. The various areas of the image are probed to different extents based on the accumulated signal to noise ratio (SNR) of each area, resulting in a constant SNR throughout the final image. The method is applied to sequencing gel interpretation

Indirect fluorometric detection techniques on thin layer chromatography and effect of ultrasound on gel electrophoresis

Thin-layer chromatography (TLC) is a broadly applicable separation technique. It offers many advantages over high performance liquid chromatography (HPLC), such as easily adapted for two-dimensional separation, for whole-column detection and for handling multiple samples, etc. However, due to its draggy development of detection techniques comparing with HPLC, TLC has not received the attention it deserves. Therefore, exploring new detection techniques is very important to the development of TLC. It is the principal purpose of this dissertation to present a new detection method for TLC--indirect fluorometric detection method. This detection technique is universal, sensitive, nondestructive, and simple. This will be described in detail from Sections I through Section V;Section I and III describe the indirect fluorometric detection of anions and nonelectrolytes in TLC. They are based on the displacement of fluorophore by the sample molecule, which cause a decrease of fluorescence. The negative signals can be monitored, i.e., analytes can be detected indirectly;In Section II, a detection method for cations based on fluorescence quenching of ethidium bromide is presented. Cations such as Cu[superscript]2+, Cr[superscript]3+ and Hg[superscript]+ can be separated on silica gel TLC plate and detected by quenching the red fluorescence of ethidium bromide;In Section IV, a simple and interesting TLC experiment is designed, three different fluorescence detection principles are used for the determination of caffeine, saccharin and sodium benzoate in beverages;A laser-based indirect fluorometric detection technique in TLC is developed in Section V. The new technique can be easily used for quantitative measurements because of its two-dimension scanning capability. With this technique a detection limit of 6 pg can be achieved;Section VI is totally different from Sections I through V. An ultrasonic effect on the separation of DNA fragments in agarose gel electrophoresis is investigated. It is found that the ultrasound improves the separation of DNA fragments without degrading them, a phenomenon that is totally different from that of ultrasonic scission

Quantitation of thin-layer chromatograms with an Apple II computer-based videodensitometer

An inexpensive, high-speed densitometer made from an Apple II computer, a black-and-white video camera, and an image digitizing board is described. By supplementing the computer with a very fast coprocessor, one can obtain a measurement of a typical thin-layer spot in 30-40 s. Only minimal electronic expertise is required for assembling the system from the commercially available components. Coefficients of variation for multiple readings of a single 500-ng spot of charred lipid were about 0.5%. For lipid spots weighing 250 to 2000 ng, a second-order relationship between weight and density reading was obtained with a correlation coefficient of 0.998.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25516/1/0000057.pd

Viability, Functionality, and Genetic Assessment of Cells After Laser Guidance

We systematically vary the controllable laser parameters, namely, wavelength, intensity, and exposure time of the laser on single cells to clearly establish laser parameters that allow negligible cell damage with significant cellular position control. To accomplish this goal, embryonic day 7 (E7) chick forebrains neurons were cultured with control and test cells selected one hour after seeding to allow attachment, at which time test cells were subjected to the laser. Cells were imaged at 4, 12, 24, and 36 hours after laser exposure to evaluate viability and functionality by using the Live/Dead Viability Cytotoxicity Kit (Molecular Probes L3224) and measuring neurite outgrowth, respectively. We have modified the COMET (single-cell gel electrophoresis) assay to quantify DNA damage on individual patterned cells and, thus, more extensively explore possible cell damage. For all parameters tested, our results show that there is no significant effect of laser exposure on cell viability or neurite outgrowth

Index to 1984 NASA Tech Briefs, volume 9, numbers 1-4

Short announcements of new technology derived from the R&D activities of NASA are presented. These briefs emphasize information considered likely to be transferrable across industrial, regional, or disciplinary lines and are issued to encourage commercial application. This index for 1984 Tech B Briefs contains abstracts and four indexes: subject, personal author, originating center, and Tech Brief Number. The following areas are covered: electronic components and circuits, electronic systems, physical sciences, materials, life sciences, mechanics, machinery, fabrication technology, and mathematics and information sciences

Application of polymeric microfluidic devices for separation of single-stranded DNA

Microsystems targeted for DNA sequencing, especially those focused on electrophoretic separations, are rapidly proving their viability to genomic research, mimicking the progress made when capillary electrophoresis developed from miniaturizing slab gel electrophoresis techniques. Being the more recent electrophoretic separation platform, the commercial availability of microchip electrophoresis devices remains relatively limited. To this extent, high-aspect ratio microstructures formed in thermo plastics have been developed using rapid fabrication methods from molding tools designed for mass replication of high-aspect ratio microfeatures. In this work, the choice and compatibility of poly(methylmethacrylate) (PMMA) – the primary substrate for DNA separations in this work – was investigated for use with our fluorescence lifetime detection instrument. The accuracy and precision of the fluorescence lifetime values of dye-labeled primers used for construction of single-stranded DNA (ssDNA) sequencing tracts was determined to discern the influence of PMMA as a substrate material to the discrimination method. The separation performance of ssDNA was evaluated for potential use of the polymer-based microchip electrophoresis devices as a platform for rapid, high-throughput DNA sequencing. To enhance these separations, a scheme to modify the surface of PMMA employing chemical and photochemical methods was developed. Once optimized, a linear polyacrylamide-modified PMMA surface demonstrated an electroosmotic flow, which varied from chip to chip, lowered by two orders of magnitude and demonstrated increased efficiencies for the separation of ssDNA fragments. As part of a modular system for the analysis of DNA material being developed in our labs, a purification device fabricated in polycarbonate was used to reversibly immobilize DNA sequencing fragments. The purified ssDNA was collected and analyzed by capillary electrophoresis to evaluate the device’s efficiency in removal of contaminants from fragments constructed with dye-labeled primers. One significant result showed the necessity for a down-stream concentration method. Thus, we have investigated the use of a thermally responsive polymer, poly-N-isopropylacrylamide (pNIPAAm) grafted onto the surface of PMMA to serve as a concentration medium for the purified fragments. Results suggest pNIPAAm will be effective in concentrating and releasing fragments when changing the temperature from above its critical temperature (32°), where it exhibits a hydrophobic nature, to below it where it becomes hydrophilic

Transient absorption imaging of hemeprotein in fresh muscle fibers

2022 Summer.Includes bibliographical references.Mitochondrial diseases affect 1 in 4000 individuals in the U.S. among adults and children of all races and genders. Nevertheless, these diseases are hard to diagnose because they affect each person differently. Meanwhile the gold standard diagnosis methods are usually invasive and time- consuming. Therefore, a non-invasive and in-vivo diagnosis method is highly demanded in this area. Our goal is to develop a non-invasive diagnosis method based on the endogenous nonlinear optical effect of the live tissues. Mitochondrial disease is frequently the result of a defective electron transport chain (ETC). Our goal is to develop a non-invasive way to measure redox within the ETC, specifically, of cytochromes. Cytochromes are iron porphyrins that are essential to the ETC. Their redox states can indicate cellular oxygen consumption and mitochondrial ATP production. So being able to differentiate the redox states of cytochromes will offer us a method to characterize mitochondrial function. Meanwhile, Chergui's group found out that the two redox states of cytochrome c have different pump-probe spectroscopic responses, meaning that the transient absorption (TA) decay lifetime can be a potential molecular contrast for cytochrome redox state discrimination. Their research leads us to utilize the pump-probe spectroscopic idea to develop a time-resolved optical microscopic method to differentiate not only cytochromes from other chemical compounds but also reduced cytochromes from oxidized ones. This dissertation describes groundbreaking experiments where transient absorption is used to reveal excited-state lifetime differences between healthy controls and an animal model of mitochondrial disease, in addition to differences between reduced and oxidized ETC in isolated mitochondria and fresh preparations of muscle fibers. For our initial experiments, we built a pump-probe microscopic system with a fiber laser source, producing 530nm pump and 490nm probe using a 3.5kHz laser scanning rate. The pulse durations of pump and probe are both 800fs. For the preliminary results, we have successfully achieved TA decay contrast between reduced and oxidized cytochromes in solution form. Then we have achieved SNR enhanced pump-probe image of BGO crystal particles with the help of the software- based adaptive filter noise canceling method. We also have installed a FPGA-based adaptive filter to enhance the pump-probe signals of the electrophoresis gels that contain different mitochondrial respiratory chain supercomplexes. However, because the noise floor was still 30 dB higher than shot noise limit, cytochrome imaging in live tissues was still problematic. We then built another pump-probe microscope with a solid- state ultrafast laser source. In that way, we do not need to worry about laser relative intensity noise (RIN) anymore, since the noise floor of the solid-state laser source can reach the shot noise limit at MHz region. One other advantage of the new laser source is that it can provide one tunable laser output that can be directly converted to the probe pulse with tunable center wavelength. Its tunability can cover the entire visible spectrum. We realized a pump-probe microscopy with a 520nm pump pulse and a tunable probe pulse. The tunability on the probe arm allows us to explore better pump-probe contrast between two redox states. What's more, I will introduce my preliminary results of utilizing supercontinuum generation in a photonic crystal fiber (PCF) to realize tunability on pump wavelength. In that way, more possibilities will be unlocked. And the hyperspectral pump-probe microscope will be able to distinguish more molecules