KEYWORDS: Surface plasmons, Life sciences, Fluorescent markers, Cameras, Digital imaging, Biological research, Tissues, Current controlled current source
Distinguishing between intact cells, dead but still whole cells, and cell debris is an important but difficult task in life sciences. The most common way to identify dead cells is using a cell-impermeant DNA binding dye, such as propidium iodide. A healthy living cell has an intact cell membrane and will act as a barrier to the dye so that it cannot enter the cell. A dead cell has a compromised cell membrane, and it will allow the dye into the cell to bind to the DNA and become fluorescent. The dead cells therefore will be positive and the live cells will be negative. The dead cells later deteriorate quickly into debris. Different pieces of debris from a single cell can be incorrectly identified as separate dead cells. Although a flow cytometer can quickly perform numerous quantitative, sensitive measurements on each individual cell to determine the viability of cells within a large, heterogeneous population, it is bulky, expensive, and only large hospitals and laboratories can afford them. In this work, we show that the distance-dependent coupling of fluorophore light to surface plasmon coupled emission (SPCE) from fluorescently-labeled cells can be used to distinguish whole cells from cell debris. Once the fluorescent labels are excited by a laser, the fluorescently-labeled whole cells create two distinct intensity rings in the far-field, in contrast to fluorescently-labeled cell debris, which only creates one ring. The distinct far-field patterns can be captured by camera and used to distinguish between whole cells and cell debris.
In biotechnology, the ability to instantly detect contaminants is key to running a reliable bioprocess. Bioprocesses are prone to be contaminated by cells that are abundant in our environment; detection and quantification of these cells would aid in the preservation of the bioprocess product. This paper discusses the design and development of a portable kinetics fluorometer which acts as a single-excitation, single-emission photometer that continuously measures fluorescence intensity of an indicator dye, and plots it. Resazurin is used as an indicator dye since the viable contaminant cells reduce Resazurin toResorufin, the latter being strongly fluorescent. A photodiode detects fluorescence change by generating current proportional to the intensity of the light that reached it, and a trans-impedance differential op-amp ensures amplification of the photodiodes’ signal. A microfluidic chip was designed specifically for the device. It acts as a fully enclosed cuvette, which enhances the Resazurin reduction rate. E. coli in LB media, along with Resazurin were injected into the microfluidic chip. The optical sensor detected the presence of E. coli in the media based on the fluorescence change that occurred in the indicator dye in concentrations as low as 10 CFU/ml. A method was devised to detect and determine an approximate amount of contamination with this device. This paper discusses application of this method to detect and estimate sample contamination. This device provides fast, accurate, and inexpensive means to optically detect the presence of viable cells.
Fluorescent proteins are often used as reporters of protein concentration in biology and biomedicine applications. They can be detected using a fluorimeter equipped with fiber optics for ease of access. However, small changes in the path length due to change in the position, or immersion depth of the optical fiber results in large changes in readings. To alleviate the situation, the fiber is equipped with a fixed-length-extension that provides constant path length. The operation of the fiber equipped fluorimeter is theoretically modelled and practically verified in this paper.
The main bioprocess variables that are continuously measured are pH, dissolved oxygen (DO), and dissolved car- bon dioxide (DCO2). Less common variables are redox, concentrations of substrate and product concentrations, product activity, etc. Recently, pH and DO have been measured using optical chemical sensors due to their small form factor and convenience in use. These sensors are typically interrogated using a lab grade spectrometer, or with the help of a low-cost, tailor-made optoelectronic transducer that is designed around the optical sensor. Recently, we proposed a new class of optoelectronic transducers that are capable of monitoring several different optical sensors without the need to switch the optics or hardware when changing the type of sensor. This allows flexibility closer to the lab-grade devices at a price point of a dedicated sensor.
In this work, we have demonstrated a universal optical platform capable of monitoring pH or DO sensors. It uses the principle of ratio-metric fluorescence measurements for pH and fluorescence lifetime measurements for DO. The platform is capable of seamlessly switching between these two modes. It is also capable of auto recognition of the sensor type. The platform can operate both with patch-type or fiber optic type of sensors. The platform has measurement accuracy of about 0.08 pH units and approximately 5 % air saturation with oxygen. Additionally, an approach to obtain identical calibrations between several devices is presented.
The described platform has been tested in actual bioprocesses and has been found adequate for continuous bioprocess monitoring.
Publisher’s Note: This paper, originally published on 5/13/2015, was replaced with a corrected/revised version on 7/1/2015. If you downloaded the original PDF but are unable to access the revision, please contact SPIE Digital Library Customer Service for assistance.
Plants exhibit complex responses to changes in environmental conditions such as radiant heat flux, water quality, airborne pollutants, soil contents. We seek to utilize the natural chemical and electrophysiological response of plants to develop novel plant-based sensor networks. Our present work focuses on plant responses to high-energy radiation – with the goal of monitoring natural plant responses for use as benchmarks for detection and dosimetry. For our study, we selected a plants cactus, Arabidopsis, Dwarf mango (pine), Euymus and Azela. We demonstrated that the ratio of Chlorophyll a to Chlorophyll b of the leaves has changed due to the exposure gradually come back to the normal stage after the radiation die.
We used blue laser-induced blue fluorescence-emission spectra to characterize the pigment status of the trees. Upon blue laser excitation (400 nm) leaves show a fluorescence emission in the red spectral region between 650 and 800nm (chlorophyll fluorescence with maxima near 690nm and 735 nm). Sample tree subjects were placed at a distance of 1m from NIST-certified 241AmBe neutron source (30 mCi), capable of producing a neutron field of about 13 mrem/h. This corresponds to an actual absorbed dose of ~ 1 mrad/h.
Our results shows that all plants are sensitive to nuclear radiation and some take longer time to recover and take less. We can use their characteristics to do differential detection and extract nuclear activity information out of measurement results avoid false alarms produced environmental changes. Certainly the ultimate verification can be obtained from genetic information, which only need to be done when we have seen noticeable changes on plant optical spectra, mechanical strength and electrical characteristics.
Plants have mechanisms to perceive and transmit information between its organs and tissues. These signals had long been considered as hormonal or hydraulic in nature, but recent studies have shown that electrical signals are also produced causing physiological responses. In this work we show that Venus Flytrap, Dionaea muscipula, can respond to both electrical and optical signals beside mechanical stimulations. While the Venus Flytrap does not have any neurons, it does contain transport cells with very similar characteristics to neurotransmitters and uses ionic mechanisms, as human neurons do, to generate action potentials. In our electrical stimulation study, electrodes made out of soft cloth were soaked in salt water before being placed to the midrib (+) and lobe (-). The flytrap's surface resistance was determined by subtracting out the average electrode resistance from the measured electrode to plant surface resistance, yielding an average contact resistance of around 0.98MΩ. A logarithmic amplifier was used to monitor mechanically generated electrical signals. Two electrical pulses were generated by mechanically touching the trigger hairs in the lobe twice within 20 seconds. By discharging around 600μC charge stored in a capacitor we demonstrated electrically closing of the flytrap. For optical excitation we found in our FTIR study it's tissue contains very similar protein absorption peaks to that of insects. A 7.35μm laser with ~50mw power was then used for the stimulation study. Electrical action potential was generated twice by mid-infrared photons before closure of the flytrap.
Neuronal optical excitation can provide non-contacting tools to explore brain circuitry and a durable stimulation
interface for cardiac pacing and visual as well as auditory sensory neuronal stimulation. To obtain accurate absorption
spectra, we scan the transmission of neurons in cell culture medium, and normalize it by subtracting out the absorption
spectrum of the medium alone. The resulting spectra show that the main neuronal absorption peaks are in the 3000-
6000nm band, although there is a smaller peak near 1450nm. By coupling the output of a 3μm interband cascade laser
(ICL) into a mid-IR fluorozirconate fiber, we can effectively deliver more than 1J/cm2 photon intensity to the excitation
site for neuronal stimulation.
We present a biosensing platform that uses spatial electroluminescent (EL) illumination combined with charge-coupled
device (CCD)-based detection for fluorescence measurements. The resulting EL-CCD detector platform was used to
monitor different protease activities with substrates labeled for fluorescence resonance energy transfer (FRET)-based
assays. The first uses a commercial FITC/DABCYL-SNAP-25 peptide substrate to monitor the activity of the light
chain derivative (LcA) of botulinum neurotoxin A, achieving a limit of detection (LOD) of 1.25 nM (62 ng/ml). The
second protease activity assay measured trypsin proteolysis using peptide substrates immobilized onto semiconductor
quantum dot (QD) nanoparticles with a LOD of 6.2 nM trypsin (140 ng/ml). The specific ovomucoid inhibition of
trypsin activity was also monitored. The highlighted studies clearly demonstrate the utility of the EL-CCD detector
platform for monitoring fluorescent-based protease activity assays with potential healthcare applications, including
point-of-care diagnostics.
A baculovirus expression system was used to produce DsRed fusion protein in insect larvae. As the baculovirus/insect larvae system requires precise harvest timing to achieve high yield of protein, a low-cost miniature all-solid state optical probe was used for detection of the protein concentrations in the frozen larvae. Three batches of infected larvae were monitored at different post-infection times. The calibration curve of the probe was obtained by simultaneous measurements both in laboratory fluorimeter and using gel electrophoresis analysis. The results show good correlation between the optical measurements and the standard laboratory technique.
Glucose is the major source of carbon, and glutamine is the major source of nitrogen in cell culture media. Thus, glucose and glutamine monitoring are important in maintaining optimal conditions in industrial bioprocesses. Here we report reagentless glucose and glutamine sensors using the E. coli glucose binding protein (GBP) and the glutamine binding protein (GlnBP). Both of these proteins are derived from the permease system of the gram-negative bacteria. The Q26C variant of GBP was labeled at the 26-position with anilino-naphthalene sulfonate (ANS), while the S179C variant of GlnBP was labeled at the 179-position with acrylodan. The ANS and acrylodan emissions are quenched in the presence of glucose and glutamine, respectively. The acrylodan-labeled GlnBP was labeled at the N-terminal with ruthenium bis-(2,2’-bipyridyl)-1,10-phenanthroline-9-isothiocyanate. The ruthenium acts as a non-responsive long-lived reference. The apparent binding constant, Kd’, of 8.0 μM glucose was obtained from the decrease in intensity of ANS in GBP. The reliability of the method in monitoring glucose during yeast fermentation was determined by comparison with the YSI Biochemistry Analyzer. The apparent binding constant, Kd’, of 0.72 μM glutamine was calculated from the ratio of emission intensities of acrylodan and ruthenium (I515/I610) in GlnBP. The presence of the long-lived ruthenium allowed for modulation sensing at lower frequencies (1-10 MHz) approaching an accuracy of ± 0.02 μM. The conversion of the GBP into a similar ratiometric sensor was described.
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