Engineering topics

Fernandez, Salvador M.

Publications and source records attributed to Fernandez, Salvador M..

7 records

NASA NTRS2011

Cytometer on a Chip

A cytometer now under development exploits spatial sorting of sampled cells on a microarray chip followed by use of grating-coupled surface-plasmon-resonance imaging (GCSPRI) to detect the sorted cells. This cytometer on a chip is a prototype of contemplated future miniature cytometers that would be suitable for rapidly identifying pathogens and other cells of interest in both field and laboratory applications and that would be attractive as alternatives to conventional flow cytometers. The basic principle of operation of a conventional flow cytometer requires fluorescent labeling of sampled cells, stringent optical alignment of a laser beam with a narrow orifice, and flow of the cells through the orifice, which is subject to clogging. In contrast, the principle of operation of the present cytometer on a chip does not require fluorescent labeling of cells, stringent optical alignment, or flow through a narrow orifice. The basic principle of operation of the cytometer on a chip also reduces the complexity, mass, and power of the associated laser and detection systems, relative to those needed in conventional flow cytometry. Instead of making cells flow in single file through a narrow flow orifice for sequential interrogation as in conventional flow cytometry, a liquid containing suspended sampled cells is made to flow over the front surface of a microarray chip on which there are many capture spots. Each capture spot is coated with a thin (approximately 50-nm) layer of gold that is, in turn, coated with antibodies that bind to cell-surface molecules characteristic of one the cell species of interest. The multiplicity of capture spots makes it possible to perform rapid, massively parallel analysis of a large cell population. The binding of cells to each capture spot gives rise to a minute change in the index of refraction at the surface of the chip. This change in the index of refraction is what is sensed in GCSPRI, as described briefly below. The identities of the various species in a sample of cells is spatially encoded in the chip by the pattern of capture spots. The number of cells of a particular species is determined from the magnitude of the GCSPRI signal from that spot. GCSPRI as used here can be summarized as follows: The cytometer chip is fabricated with a diffraction grating on its front surface. The chip is illuminated with a light emitting diode (LED) from the front. By proper choice of grating parameters and of the wavelength and the angle of incidence of a laser beam, laser light can be made to be coupled into an electromagnetic mode that resonates with surface plasmons and thus couples light into surface plasmons. Coupling of light into a surface plasmon at a given location reduces the amount of incident light reflected from that location. A change in the index of refraction at the surface of a capture spot gives rise to a change in the resonance condition. Depending on the specific design, the change in the index of refraction could manifest itself as a brightening or darkening, a change in the wavelength needed to excite the plasmon at a given angle of incidence, or a change in the angle of incidence needed to excite the plasmon at a given wavelength. Whereas a multiwavelength laser system with multichannel detection would be needed to detect multiple species in conventional flow cytometry, it suffices to use an LED and a single detector channel in the GCSPRI approach: this contributes significantly to reductions in cost, complexity, size, mass, and power. GCSPRI cytometer chips could be made of plastic and could be mass-produced cheaply by use of molding and other methods adopted from the manufacture of digital video disks. These methods are amenable to a high degree of miniaturization: such additional features as fluidic channels, reaction chambers, and fluid-coupling ports could readily be incorporated into the chips, without incurring substantial additional costs.

Fernandez, Salvador M.↗

NASA NTRS2011

Cytometer on a Chip

A cytometer now under development exploits spatial sorting of sampled cells on a microarray chip followed by use of grating-coupled surface-plasmon-resonance imaging (GCSPRI) to detect the sorted cells. This cytometer on a chip is a prototype of contemplated future miniature cytometers that would be suitable for rapidly identifying pathogens and other cells of interest in both field and laboratory applications and that would be attractive as alternatives to conventional flow cytometers. The basic principle of operation of a conventional flow cytometer requires fluorescent labeling of sampled cells, stringent optical alignment of a laser beam with a narrow orifice, and flow of the cells through the orifice, which is subject to clogging. In contrast, the principle of operation of the present cytometer on a chip does not require fluorescent labeling of cells, stringent optical alignment, or flow through a narrow orifice. The basic principle of operation of the cytometer on a chip also reduces the complexity, mass, and power of the associated laser and detection systems, relative to those needed in conventional flow cytometry. Instead of making cells flow in single file through a narrow flow orifice for sequential interrogation as in conventional flow cytometry, a liquid containing suspended sampled cells is made to flow over the front surface of a microarray chip on which there are many capture spots. Each capture spot is coated with a thin (.50-nm) layer of gold that is, in turn, coated with antibodies that bind to cell-surface molecules characteristic of the cell species of interest. The multiplicity of capture spots makes it possible to perform rapid, massively parallel analysis of a large cell population. The binding of cells to each capture spot gives rise to a minute change in the index of refraction at the surface of the chip. This change in the index of refraction is what is sensed in GCSPRI, as described briefly below. The identities of the various species in a sample of cells is spatially encoded in the chip by the pattern of capture spots. The number of cells of a particular species is determined from the magnitude of the GCSPRI signal from that spot. GCSPRI as used here can be summarized as follows: The cytometer chip is fabricated with a diffraction grating on its front surface. The chip is illuminated with a light emitting diode (LED) from the front. By proper choice of grating parameters and of the wavelength and the angle of incidence of a laser beam, laser light can be made to be coupled into an electromagnetic mode that resonates with surface plasmons and thus couples light into surface plasmons. Coupling of light into a surface plasmon at a given location reduces the amount of incident light reflected from that location. A change in the index of refraction at the surface of a capture spot gives rise to a change in the resonance condition. Depending on the specific design, the change in the index of refraction could manifest itself as a brightening or darkening, a change in the wavelength needed to excite the plasmon at a given angle of incidence, or a change in the angle of incidence needed to excite the plasmon at a given wavelength. Whereas a multiwavelength laser system with multichannel detection would be needed to detect multiple species in conventional flow cytometry, it suffices to use an LED and a single detector channel in the GCSPRI approach: this contributes significantly to reductions in cost, complexity, size, mass, and power. GCSPRI cytometer chips could be made of plastic and could be mass-produced cheaply by use of molding and other methods adopted from the manufacture of digital video disks. These methods are amenable to a high degree of miniaturization: such additional features as fluidic channels, reaction chambers, and fluid-coupling ports could readily be incorporated into the chips, without incurring substantial additional costs.

Fernandez, Salvador M.↗

NASA NTRS2010

Cytometer on a chip

An assay technique for label-free, highly parallel, qualitative and quantitative detection of specific cell populations in a sample and for assessing cell functional status, cell-cell interactions and cellular responses to drugs, environmental toxins, bacteria, viruses and other factors that may affect cell function. The technique includes a) creating a first array of binding regions in a predetermined spatial pattern on a sensor surface capable of specifically binding the cells to be assayed; b) creating a second set of binding regions in specific spatial patterns relative to the first set designed to efficiently capture potential secreted or released products from cells captured on the first set of binding regions; c) contacting the sensor surface with the sample, and d) simultaneously monitoring the optical properties of all the binding regions of the sensor surface to determine the presence and concentration of specific cell populations in the sample and their functional status by detecting released or secreted bioproducts.

Lynes, Michael A.↗

NASA NTRS2004

Phytoplankton-Fluorescence-Lifetime Vertical Profiler

A battery-operated optoelectronic instrument is designed to be lowered into the ocean to measure the intensity and lifetime of fluorescence of chlorophyll A in marine phytoplankton as a function of depth from 0 to 300 m. Fluorescence lifetimes are especially useful as robust measures of photosynthetic productivity of phytoplankton and of physical and chemical mechanisms that affect photosynthesis. The knowledge of photosynthesis in phytoplankton gained by use of this and related instruments is expected to contribute to understanding of global processes that control the time-varying fluxes of carbon and associated biogenic elements in the ocean. The concentration of chlorophyll in the ocean presents a major detection challenge because in order to obtain accurate values of photosynthetic parameters, the intensity of light used to excite fluorescence must be kept very low so as not to disturb the photosynthetic system. Several innovations in fluorometric instrumentation were made in order to make it possible to reach the required low detection limit. These innovations include a highly efficient optical assembly with an integrated flow-through sample interface, and a high-gain, low-noise electronic detection subsystem. The instrument also incorporates means for self-calibration during operation, and electronic hardware and software for control, acquisition and analysis of data, and communications. The electronic circuitry is highly miniaturized and designed to minimize power demand. The instrument is housed in a package that can withstand the water pressure at the maximum depth of 300 m. A light-emitting diode excites fluorescence in the sample flow cell, which is placed at one focal point of an ellipsoidal reflector. A photomultiplier tube is placed at the other focal point. This optical arrangement enables highly efficient collection of fluorescence emitted over all polar directions. Fluorescence lifetime is measured indirectly, by use of a technique based on the same principle as the one described in "Fluorometer for Analysis of Photosynthesis in Phytoplankton" (SSC-00110), NASA Tech Briefs, Vol. 24, No. 1 (November 2000), page 79. The excitation is modulated at a frequency of 70 MHz, and the phase shift between the excitation light and the emitted fluorescence is measured by a detection method in which the 70 MHz signal is down-converted to a 400 Hz signal. The fluorescence lifetime can be computed from the known relationship among the fluorescence lifetime, phase shift, and modulation frequency

Fernandez, Salvador M.↗

NASA NTRS2002

The Use of Chlorophyll Fluorescence Lifetime to Assess Phytoplankton Physiology within a River-Dominated Environment

Chlorophyll a fluorescence lifetime was measured for phytoplankton populations inhabiting the three physical zones surrounding the Mississippi River's terminus in the Gulf of Mexico. Observations of river discharge volume, nitrate + nitrite, silicate, phosphate, PAR (Photosynthetically Active Radiation) diffuse attenuation within the water column, salinity, temperature, SPM, and chl a concentration were used to characterize the distribution of chl fluorescence lifetime within a given region within restricted periods of time. 33 stations extending from the Mississippi River plume to the shelf break of the Louisiana coast were surveyed for analysis of chlorophyll fluorescence lifetime during two cruises conducted March 31 - April 6, 2000, and October 24 - November 1, 2000. At each station, two to three depths were chosen for fluorescence lifetime measurement to represent the vertical characteristics of the water column. Where possible, samples were taken from just below the surface and from just above and below the pycnocline. All samples collected were within the 1% light level of the water column (the euphotic zone). Upon collection, samples were transferred to amber Nalgene bottles and left in the dark for at least 15 minutes to reduce the effects of non-photochemical quenching and to insure that photosynthetic reaction centers were open. Before measurements within the phase fluorometer were begun, the instrument was allowed to warm up for no less than one hour.

Hall, Callie M.↗

NASA NTRS2000

Relationship between the Fluorescence Lifetime of Chlorophyll 'a' and Primary Productivity within the Mississippi River Plume and Adjacent Shelf Region

In situ measurements of chlorophyll fluorescence intensity have been widely used to estimate phytoplankton biomass. However, because the fluorescence quantum yield of chlorophyll a in vivo can be highly variable, measurements of chlorophyll fluorescence intensity cannot be directly correlated with phytoplankton biomass and do not provide information on the physiological state of the phytoplankton under study. Conversely, lifetime-based measurements of chlorophyll fluorescence provide a framework in which photosynthetic rates of phytoplankton can be analyzed according to phytoplankton physiology. Along with the measurement of primary production and ambient nutrient concentrations within the Mississippi River plume in the northern Gulf of Mexico, phytoplankton fluorescence lifetimes were measured using a Fluorescence Lifetime Phytoplankton Analyzer (developed under a NASA Small Business Innovative Research contract to Ciencia, Inc.). Variability of fluorescence lifetimes within the plume can be used as a background from which to interpret variations in the maximum quantum yield of photochemistry. The extent to which nutrient and effluent loading in this dynamic coastal area affect the photosynthetic performance of phytoplankton will be presented as a function of phytoplankton fluorescence lifetimes.

Hall, Callie↗

NASA NTRS1995

Compact, Automated, Frequency-Agile Microspectrofluorimeter

Compact, reliable, rugged, automated cell-culture and frequency-agile microspectrofluorimetric apparatus developed to perform experiments involving photometric imaging observations of single live cells. In original application, apparatus operates mostly unattended aboard spacecraft; potential terrestrial applications include automated or semiautomated diagnosis of pathological tissues in clinical laboratories, biomedical instrumentation, monitoring of biological process streams, and portable instrumentation for testing biological conditions in various environments. Offers obvious advantages over present laboratory instrumentation.

Fernandez, Salvador M.↗