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Cell enlargement of plant tissue explants oscillates with a temperature-compensated period of ca. 24 min

Rate of plant cell enlargement, measured at intervals of 3 min using a sensitive linear transducer, oscillates with a minimum period of about 24 min that parallels the 24-min periodicity observed with the oxidation of NADH by the external plasma membrane NADH oxidase and of single cells measured previously by video-enhanced light microscopy. Also exhibiting 24-min oscillations is the steady-state rate of cell enlargement induced by the addition of the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) or the natural auxin indole-3-acetic acid (IAA). Immediately following 2,4-D addition, a very complex pattern of oscillations is frequently observed. However, after several hours a dominant 24-min period emerges. The length of the 24-min period is temperature compensated and remains constant at 24 min when measured at 15, 25 or 35 degrees C, despite the fact that the rate of cell enlargement approximately doubles for each 10 degree C rise over this same range of temperatures.

Non-NASA Center

NADH oxidase activity (NOX) and enlargement of HeLa cells oscillate with two different temperature-compensated period lengths of 22 and 24 minutes corresponding to different NOX forms

NOX proteins are cell surface-associated and growth-related hydroquinone (NADH) oxidases with protein disulfide-thiol interchange activity. A defining characteristic of NOX proteins is that the two enzymatic activities alternate to generate a regular period length of about 24 min. HeLa cells exhibit at least two forms of NOX. One is tumor-associated (tNOX) and is inhibited by putative quinone site inhibitors (e.g., capsaicin or the antitumor sulfonylurea, LY181984). Another is constitutive (CNOX) and refractory to inhibition. The periodic alternation of activities and drug sensitivity of the NADH oxidase activity observed with intact HeLa cells was retained in isolated plasma membranes and with the solubilized and partially purified enzyme. At least two activities were present. One had a period length of 24 min and the other had a period length of 22 min. The lengths of both the 22 and the 24 min periods were temperature compensated (approximately the same when measured at 17, 27 or 37 degrees C) whereas the rate of NADH oxidation approximately doubled with each 10 degrees C rise in temperature. The rate of increase in cell area of HeLa cells when measured by video-enhanced light microscopy also exhibited a complex period of oscillations reflective of both 22 and 24 min period lengths. The findings demonstrate the presence of a novel oscillating NOX activity at the surface of cancer cells with a period length of 22 min in addition to the constitutive NOX of non-cancer cells and tissues with a period length of 24 min.

Non-NASA Center

A comparison of imaging methods for use in an array biosensor

An array biosensor has been developed which uses an actively-cooled, charge-coupled device (CCD) imager. In an effort to save money and space, a complementary metal-oxide semiconductor (CMOS) camera and photodiode were tested as replacements for the cooled CCD imager. Different concentrations of CY5 fluorescent dye in glycerol were imaged using the three different detection systems with the same imaging optics. Signal discrimination above noise was compared for each of the three systems.

Evaluation Studies

Novel software for analysis of root gravitropism: comparative response patterns of Arabidopsis wild-type and axr1 seedlings

In an earlier study (Evans, Ishikawa & Estelle 1994, Planta 194, 215-222) we used a video digitizer system to compare the kinetics of auxin action on root elongation in wild-type seedlings and seedlings of auxin response mutants of Arabidopsis thaliana (L.) Heynh. We have since modified the system software to allow determination of elongation on opposite sides of vertical or gravistimulated roots and to allow continuous measurement of the angle of orientation of sequential subsections of the root during the response. We used this technology to compare the patterns of differential growth that generate curvature in roots of the Columbia ecotype and in the mutants axr1-3, axr1-12 and axr2, which show reduced gravitropic responsiveness and reduced sensitivity to inhibition by auxin. The pattern of differential growth during gravitropism differed in roots of wild-type and axr1 seedlings. In wild-type roots, initial curvature resulted from differential inhibition of elongation in the distal elongation zone (DEZ). This was followed by an acceleration of elongation along the top side of the DEZ. In roots of axr1-3, curvature resulted from differential stimulation of elongation whereas in roots of axr1-12 the response was variable. Roots of axr2 did not exhibit gravitropic curvature. The observation that the pattern of differential growth causing curvature is dramatically altered by a change in sensitivity to auxin is consistent with the classical Cholodny-Went theory of gravitropism which maintains that differential growth patterns induced by gravistimulation are mediated primarily by gravi-induced shifts in auxin distribution. The new technology introduced with this report allows automated determination of stimulus response patterns in the small but experimentally popular roots of Arabidopsis.

Non-NASA Center

Fracture behavior of large-scale thin-sheet aluminum alloy

A series of fracture tests on large-scale, pre-cracked, aluminum alloy panels is being carried out to examine and to characterize the process by which cracks propagate and link up in this material. Extended grips and test fixtures were specially designed to enable the panel specimens to be loaded in tension, in a 1780-kN-capacity universal testing machine. Twelve panel specimens, each consisting of a single sheet of bare 2024-T3 aluminum alloy, 3988 mm high, 2286 mm wide, and 1.016 mm thick are being fabricated with simulated through-cracks oriented horizontally at mid-height. Using existing information, a test matrix has been set up that explores regions of failure that are controlled by fracture mechanics, with additional tests near the boundary between plastic collapse and fracture. In addition, a variety of multiple site damage (MSD) configurations have been included to distinguish between various proposed linkage mechanisms. All tests but one use anti-buckling guides. At this writing seven specimens have been tested. Three were fabricated with a single central crack, three others had multiple cracks on each side of the central crack, and one had a single crack but no anti-buckling guides. Each fracture event was recorded on film, video, computer, magnetic tape, and occasionally optical microscopy. The visual showed the crack tip with a load meter in the field of view, using motion picture film for one tip and SVHS video tape for the other. The computer recorded the output of the testing machine load cell, the stroke, and twelve strain gages at 1.5 second intervals. A wideband FM magnetic tape recorder was used to record data from the same sources. The data were analyzed by two different procedures: (1) the plastic zone model based on the residual strength diagram; and (2) the R-curve. The first three tests were used to determine the basic material properties, and these results were then used in the analysis of the two subsequent tests with MSD cracks. There is good agreement between measured values and results obtained from the model.

Dewit, Roland

Electron spectroscopy analysis

The Surface Science Laboratories at the University of Alabama in Huntsville (UAH) are equipped with x-ray photoelectron spectroscopy (XPS or ESCA) and Auger electron spectroscopy (AES) facilities. These techniques provide information from the uppermost atomic layers of a sample, and are thus truly surface sensitive. XPS provides both elemental and chemical state information without restriction on the type of material that can be analyzed. The sample is placed into an ultra high vacuum (UHV) chamber and irradiated with x-rays which cause the ejection of photoelectrons from the sample surface. Since x-rays do not normally cause charging problems or beam damage, XPS is applicable to a wide range of samples including metals, polymers, catalysts, and fibers. AES uses a beam of high energy electrons as a surface probe. Following electronic rearrangements within excited atoms by this probe, Auger electrons characteristic of each element present are emitted from the sample. The main advantage of electron induced AES is that the electron beam can be focused down to a small diameter and localized analysis can be carried out. On the rastering of this beam synchronously with a video display using established scanning electron microscopy techniques, physical images and chemical distribution maps of the surface can be produced. Thus very small features, such as electronic circuit elements or corrosion pits in metals, can be investigated. Facilities are available on both XPS and AES instruments for depth-profiling of materials, using a beam of argon ions to sputter away consecutive layers of material to reveal sub-surface (and even semi-bulk) analyses.

Gregory, John C.

Biological Data for Deep Space Mission Support

Increased biomedical risks and challenges associated with deep space missions (cis-Lunar, Mars transit, Mars surface) require new knowledge discovery and development of novel ecosystem and biomedical support capabilities. This paradigm shift supporting distant and long-duration missions requires biological data to be findable, accessible, interoperable, reusable (FAIR), and maximally open-access (i.e., there is a data governance continuum from closed to mediated to embargoed to open). The NASA “Open Science Data Repositories” (OSDR) aims to meet scientific, technical, and operational spaceflight needs, and offers the ability to upload, download, search, share, analyze, and visualize data across physiological, behavioral, ‘omics, and environmental monitoring telemetry datasets. OSDR includes NASA GeneLab, NASA Ames Life Sciences Data Archive (ALSDA), and NASA Biological Institutional Scientific Collection (NBISC). In the past year, ALSDA has undergone a transformation in its data collection, curation, and architecture methods. Standardizing non-genomic (phenotypic) datasets was, and will continue to be, a challenge because of their diverse nature (e.g., molecular, cellular, tissue, whole organism behavior; micro-computed tomography, intraocular pressure, fluorescence microscopy, western blot, ultrasonography; tabular, images, video). This year ALSDA, alongside GeneLab, introduced the Biological Data Management Environment (BDME) with the purpose to accept submission of data from space relevant experiments including spaceflight, radiation, simulated gravity, gravitropism, isolation and confinement, hostile closed environments and/or distance from Earth. In addition to bringing together omics, phenotypic, physiological, bioimaging, and behavioral data into one repository. By integrating with GeneLab a multi-project submission portal aims to reduce the burden on PIs submitting data and enabling the discovery of both omics and phenotypic data. The purpose of ALSDA is to collect, curate, and make all non-human space-relevant biological data maximally findable, accessible, interoperable, and reusable (FAIR). These scope of ALSDA data collected and submitted by PIs include study design metadata, subject metadata, assay metadata (parameters), raw and processed assay data, assay imagery/video, and subject-experienced mission data telemetry (radiation, temperature, humidity, acoustics, vibrations, etc.). In 2021, a community of researchers rallied to form the ALSDA Analysis Working Group (AWG) and provided scientific consensus on dataset sample and assay metadata. The community and excitement around the ALSDA/OSDR system has already led to several data reuse studies, demonstrating value using machine learning (ML), knowledge graphs, and meta-analysis approaches.

space biology

Biological Data for Deep Space Mission Support

Increased biomedical risks and challenges associated with deep space missions (cis-Lunar, Mars transit, Mars surface) require new knowledge discovery and development of novel ecosystem and biomedical support capabilities. This paradigm shift supporting distant and long-duration missions requires biological data to be findable, accessible, interoperable, reusable (FAIR), and maximally open-access (i.e., there is a data governance continuum from closed to mediated to embargoed to open). The NASA “Open Science Data Repositories” (OSDR) aims to meet scientific, technical, and operational spaceflight needs, and offers the ability to upload, download, search, share, analyze, and visualize data across physiological, behavioral, ‘omics, and environmental monitoring telemetry datasets. OSDR includes NASA GeneLab, NASA Ames Life Sciences Data Archive (ALSDA), and NASA Biological Institutional Scientific Collection (NBISC). In the past year, ALSDA has undergone a transformation in its data collection, curation, and architecture methods. Standardizing non-genomic (phenotypic) datasets was, and will continue to be, a challenge because of their diverse nature (e.g., molecular, cellular, tissue, whole organism, behavior; micro-computed tomography, intraocular pressure, fluorescence microscopy, western blot, ultrasonography; tabular, images, video). This year ALSDA, alongside GeneLab, introduced the Biological Data Management Environment (BDME) with the purpose to accept submission of data from space relevant experiments including spaceflight, radiation, simulated gravity, gravitropism, isolation and confinement, hostile closed environments and/or distance from Earth. In addition to bringing together omics, phenotypic, physiological, bioimaging, and behavioral data into one repository. By integrating with GeneLab a multi-project submission portal aims to reduce the burden on PIs submitting data and enabling the discovery of both omics and phenotypic data. The purpose of ALSDA is to collect, curate, and make all non-human space-relevant biological data maximally findable, accessible, interoperable, and reusable (FAIR). These scope of ALSDA data collected and submitted by PIs include study design metadata, subject metadata, assay metadata (parameters), raw and processed assay data, assay imagery/video, and subject-experienced mission data telemetry (radiation, temperature, humidity, acoustics, vibrations, etc.). In 2021, a community of researchers rallied to form the ALSDA Analysis Working Group (AWG) and provided scientific consensus on dataset sample and assay metadata. The community and excitement around the ALSDA/OSDR system has already led to several data reuse studies, demonstrating value using machine learning (ML), knowledge graphs, and meta-analysis approaches.

space biology

Summary of 2016 Light Microscopy Module (LMM) Physical Science Experiments on ISS. Update of LMM Science Experiments and Facility Capabilities

This presentation will feature a series of short, entertaining, and informative videos that describe the current status and science support for the Light Microscopy Module (LMM) facility on the International Space Station. These interviews will focus on current experiments and provide an overview of future capabilities. The recently completed experiments include nano-particle haloing, 3-D self-assembly with Janus particles and a model system for nano-particle drug delivery. The videos will share perspectives from the scientists, engineers, and managers working with the NASA Light Microscopy program.

Sicker, Ronald J.

Space station microscopy: Beyond the box

Microscopy aboard Space Station Freedom poses many unique challenges for in-flight investigations. Disciplines such as material processing, plant and animal research, human reseach, enviromental monitoring, health care, and biological processing have diverse microscope requirements. The typical microscope not only does not meet the comprehensive needs of these varied users, but also tends to require excessive crew time. To assess user requirements, a comprehensive survey was conducted among investigators with experiments requiring microscopy. The survey examined requirements such as light sources, objectives, stages, focusing systems, eye pieces, video accessories, etc. The results of this survey and the application of an Intelligent Microscope Imaging System (IMIS) may address these demands for efficient microscopy service in space. The proposed IMIS can accommodate multiple users with varied requirements, operate in several modes, reduce crew time needed for experiments, and take maximum advantage of the restrictive data/ instruction transmission environment on Freedom.

Hunter, N. R.

KLASS: Kennedy Launch Academy Simulation System

Software provides access to many sophisticated scientific instrumentation (Scanning Electron Microscope (SEM), a Light Microscope, a Scanning Probe Microscope (covering Scanning Tunneling, Atomic Force, and Magnetic Force microscopy), and an Energy Dispersive Spectrometer for the SEM). Flash animation videos explain how each of the instruments work. Videos on how they are used at NASA and the sample preparation. Measuring and labeling tools provided with each instrument. Hands on experience of controlling the virtual instrument to conduct investigations, much like the real scientists at NASA do. Very open architecture. Open source on SourceForge. Extensive use of XML Target audience is high school and entry-level college students. "Many beginning students never get closer to an electron microscope than the photos in their textbooks. But anyone can get a sense of what the instrument can do by downloading this simulator from NASA's Kennedy Space Center." Science Magazine, April 8th, 2005

Garner, Lesley C.

Small Particle Impact Damage on Different Glass Substrates

Impact experiments using sand particles were performed on four distinct glass substrates. The sand particles were characterized using the X-Ray micro-CT technique; 3-D reconstruction of the particles was followed by further size and shape analyses. High-speed video footage from impact tests was used to calculate the incoming and rebound velocities of the individual sand impact events, as well as particle volume. Further, video analysis was used in conjunction with optical and scanning electron microscopy to relate the incoming velocity and shape of the particles to subsequent fractures, including both radial and lateral cracks. Analysis was performed using peridynamic simulations.

Waxman, R.

IMIS: An intelligence microscope imaging system

Until recently microscope users in space relied on traditional microscopy techniques that required manual operation of the microscope and recording of observations in the form of written notes, drawings, or photographs. This method was time consuming and required the return of film and drawings from space for analysis. No real-time data analysis was possible. Advances in digital and video technologies along with recent developments in article intelligence will allow future space microscopists to have a choice of three additional modes of microscopy: remote coaching, remote control, and automation. Remote coaching requires manual operations of the microscope with instructions given by two-way audio/video transmission during critical phases of the experiment. When using the remote mode of microscopy, the Principal Investigator controls the microscope from the ground. The automated mode employs artificial intelligence to control microscope functions and is the only mode that can be operated in the other three modes as well. The purpose of this presentation is to discuss the advantages and disadvantages of the four modes of of microscopy and how the IMIS, a proposed intelligent microscope imaging system, can be used as a model for developing and testing concepts, operating procedures, and equipment design of specifications required to provide a comprehensive microscopy/imaging capability onboard Space Station Freedom.

Caputo, Michael

Radiation Dosimetry via Automated Fluorescence Microscopy

A developmental instrument for assessment of radiation-induced damage in human lymphocytes includes an automated fluorescence microscope equipped with a one or more chargecoupled- device (CCD) video camera(s) and circuitry to digitize the video output. The microscope is also equipped with a three-axis translation stage that includes a rotation stage, and a rotary tray that holds as many as thirty specimen slides. The figure depicts one version of the instrument. Once the slides have been prepared and loaded into the tray, the instrument can operate unattended. A computer controls the operation of the stage, tray, and microscope, and processes the digital fluorescence-image data to recognize and count chromosomes that have been broken, presumably by radiation. The design and method of operation of the instrument exploit fluorescence in situ hybridization (FISH) of metaphase chromosome spreads, which is a technique that has been found to be valuable for monitoring the radiation dose to circulating lymphocytes. In the specific FISH protocol used to prepare specimens for this instrument, metaphase lymphocyte cultures are chosen for high mitotic index and highly condensed chromosomes, then several of the largest chromosomes are labeled with three of four differently colored whole-chromosome-staining dyes. The three dyes, which are used both individually and in various combinations, are fluorescein isothiocyanate (FITC), Texas Red (or equivalent), and Cy5 (or equivalent); The fourth dye 4',6-diamidino- 2-phenylindole (DAPI) is used as a counterstain. Under control by the computer, the microscope is automatically focused on the cells and each slide is scanned while the computer analyzes the DAPI-fluorescence images to find the metaphases. Each metaphase field is recentered in the field of view and refocused. Then a four-color image (more precisely, a set of images of the same view in the fluorescent colors of the four dyes) is acquired. By use of pattern-recognition software developed specifically for this instrument, the images in the various colors are processed to recognize the metaphases and count the chromosome fragments of each color within the metaphases. The intermediate results are then further processed to estimate the proportion of cells that have suffered genetic damage. The prototype instrument scans at an average areal rate of 4.7 mm2/h in unattended operation, finding about 14 metaphases per hour. The false-alarm rate is typically less than 3 percent, and the metaphase-miss rate has been estimated to be less than 5 percent. The counts of chromosomes and fragments thereof are 50 to 70 percent accurate.

Castleman, Kenneth R.

Three-dimensional visualization and display technologies; Proceedings of the Meeting, Los Angeles, CA, Jan. 18-20, 1989

Special attention was given to problems of stereoscopic display devices, such as CAD for enhancement of the design process in visual arts, stereo-TV improvement of remote manipulator performance, a voice-controlled stereographic video camera system, and head-mounted displays and their low-cost design alternatives. Also discussed was a novel approach to chromostereoscopic microscopy, computer-generated barrier-strip autostereography and lenticular stereograms, and parallax barrier three-dimensional TV. Additional topics include processing and user interface isssues and visualization applications, including automated analysis and fliud flow topology, optical tomographic measusrements of mixing fluids, visualization of complex data, visualization environments, and visualization management systems.

Robbins, Woodrow E.

Advancement of Solidification Processing Technology Through Real Time X-Ray Transmission Microscopy: Sample Preparation

Two types of samples were prepared for the real time X-ray transmission microscopy (XTM) characterization. In the first series directional solidification experiments were carried out to evaluate the critical velocity of engulfment of zirconia particles in the Al and Al-Ni eutectic matrix under ground (l-g) conditions. The particle distribution in the samples was recorded on video before and after the samples were directionally solidified. In the second series samples of the above two type of composites were prepared for directional solidification runs to be carried out on the Advanced Gradient Heating Facility (AGHF) aboard the space shuttle during the LMS mission in June 1996. X-ray microscopy proved to be an invaluable tool for characterizing the particle distribution in the metal matrix samples. This kind of analysis helped in determining accurately the critical velocity of engulfment of ceramic particles by the melt interface in the opaque metal matrix composites. The quality of the cast samples with respect to porosity and instrumented thermocouple sheath breakage or shift could be easily viewed and thus helped in selecting samples for the space shuttle experiments. Summarizing the merits of this technique it can be stated that this technique enabled the use of cast metal matrix composite samples since the particle location was known prior to the experiment.

Stefanescu, D. M.

The Effects of Gravity on the Crystallization Behavior of Heavy Metal Fluoride Glasses

Heavy metal fluoride glasses are used in such applications as fiber lasers and laser amplifiers. ZrF4-BaF2-LaF3-AlF3-NaF (ZBLAN) is one of the more commonly used heavy metal fluoride glasses. ZBLAN is an infrared transmitter and has a theoretical attenuation coefficient of 0.002 db/km. However, due to impurities and small crystallites this attenuation coefficient has not been achieved to date. ZBLAN is a fragile glass which can lead to rapid crystallization, if the glass is not cooled rapidly to below the glass transition temperature or if the glass is reheated near the crystallization temperature for any period of time. Studies carried on at Marshall Space Flight Center and the University of Alabama in Huntsville since 1993 have shown that heating ZBLAN glass at the crystallization temperature in reduced gravity results in a suppression of crystallization when compared to ZBLAN processed in unit gravity. These studies utilized NASA's KC-135 aircraft and the Conquest sounding rocket. In the first series of experiments, short lengths of ZBLAN fiber were heated to the crystallization temperature in reduced gravity on board the KC- 135 and the Conquest sounding rocket and compared with fibers heated in unit gravity. The fibers processed in reduced gravity showed no evidence of crystallization when studied with x-ray diffraction and scanning electron microscopy. However, the fibers processed in unit gravity were completely crystallized. Subsequent experiments included heating small pieces of ZBLAN glass at the crystallization temperature while viewing with a video camera to follow the crystallization phenomenon. In this experiment crystallization was observed in reduced gravity, however, it was suppressed when compared to heating in unit gravity. In the most recent experiment on board the KC-135, rapid thermal analysis of ZBLAN was performed. A mechanism to explain the observations has been proposed. This mechanism is based on shear thinning whereby, the glass undergoes a reduction in viscosity in the 10(exp 5) - 10(exp 7) poise range, allowing more rapid diffusion and thus crystallization. It is proposed that this mechanism is suppressed in reduced gravity. An experiment is presently being conducted to test this theory. With increased knowledge of ZBLAN behavior in reduced gravity, three low earth orbit tiber drawing facilities have been designed. One would be suitable for use on the International Space Station, another while aboard the Space Shuttle and the third system is a fully automated facility which would operate independently of the ISS or Shuttle in a free float mode. The primary benefits of free floating a facility in LEO includes a higher quality of microgravity and reduced safety concerns since it is not in a manned environment.

Tucker, Dennis S.

Super-Resolved Single-Molecule Tracking Studies of Rhodamine B Accumulation on Fresh and Aged Polyethylene Terephthalate

It is well-known that toxic organic micropollutants (OMs) accumulate on the surfaces of microplastics. However, much remains to be learned about the exact molecular level mechanisms of OM accumulation and how these evolve as the plastics age. In this work, super-resolved single-molecule tracking (SMT) is used for the first time to investigate the accumulation of Rhodamine B (RhB) dye on fresh and artificially aged polyethylene terephthalate (PET) surfaces. PET thin films serve as models for microplastics, while RhB serves as a proxy for the OMs they accumulate. Artificial aging of the films is accomplished by exposing them in a UV-ozone chamber. Water contact angle, spectroscopic ellipsometry, and carbonyl index measurements reveal a gradual decrease in film hydrophobicity, thickness, and carbonyl content with age. Atomic force microscopy (AFM) data reveal an increase in surface roughness and confirm that the films remain largely intact and continuous across the aging times explored. In SMT experiments, wide-field fluorescence videos acquired from the water/PET interface under 7.5 pM RhB reveal both mobile and immobile dye molecules. Measurements of the frame-to-frame displacements of the dye show that diffusion occurs by a desorption-mediated mechanism and that the diffusion rate varies with PET film age. The surface density of mobile dye molecules decreases with increasing PET age, while the population of immobile molecules becomes relatively larger, suggesting an age-dependent transformation of the mechanism(s) by which the dye is accumulated. SMT data reveal that both mobile and immobile molecules repeatedly adsorb over the same surface sites, consistent with the emergence of nanoscale PET surface heterogeneity also revealed by AFM. Estimates of the adsorption coefficients are obtained using a nearest-neighbor analysis, giving values from 9.9 × 10 5 to 2.1 × 10 6 M –1 for immobile molecules and from 1.8 × 10 5 to 2.5 × 10 5 M –1 for mobile molecules on fresh and 5 min aged PET, respectively. Here, anomalous age-dependent variations in the velocity of molecular motion on the PET surface and in the population of immobile molecules are shown to correlate with changes in the strength of RhB adsorption.

Adsorption