Autonomous technologies are needed which are capable of sensing real time changes in biophysical transport
across cell membranes/organelles. These technologies must not only be highly sensitive/selective, but must also be
minimally invasive/intrusive, causing no significant physical/chemical effects on cell behavior. Challenges with
mainstream technologies (e.g., assays, fluorescent dyes, microsensors) include signal noise/drift, low temporal
resolution, requirement of large sample sizes, cytoxicity, organelle sequestration, and intracellular buffering. Recent
advancements in fiber optics have greatly enhanced the performance of microsensors (e.g., increased
sensitivity/selectivity, response time), but used in concentration mode near cells/tissues these sensors suffer from poor
signal to noise ratio. Work over the last few decades has advanced microsensor utility through sensing modalities that
extend and enhance the data recorded by sensors. This technique, known as self-referencing, converts static
micro/nanosensors with otherwise low signal-to-noise ratios into dynamic flux sensors capable of filtering out signals
not associated with active transport by acquisition and amplification of differential signals. Here, we demonstrate the use
of a self-referencing referencing frequency domain fiber optic microsensor containing a quenched dye (platinum
tetrakis-pentafluorophenyl porphyrin) for quantifying cell/tissue flux in biomedical, agricultural, and environmental
applications.
Image subtraction has been an extremely useful tool for capturing subtle changes in pixel intensity with extremely high
temporal resolution, and has been used for decades in the astronomy and metal corrosion fields. However, to date, image
subtraction has not been used as a mainstream technique for investigating morphological changes in cells, tissues, or
whole organisms. We introduce a user-friendly differential imaging technique for monitoring real time (~msec) changes
in morphology within the micrometer to millimeter spatial scale. The technique is demonstrated by measuring
morphological changes morphology for biomedical (bone stress), agricultural (crop root elongation), and environmental
(zooplankton ecotoxicology) applications. Subtle changes in growth that would typically only be observed by highly
skilled experts are easily resolved via image subtraction and the use of convolution kernels. When coupled with
techniques characterizing real time biochemical transport (e.g., respiration, ion/substrate transport), physiology can be
directly quantified with a high temporal and spatial resolution. Because of the ease of use, this technique can be readily
applied to any field of science concerned with bridging the gap between form and function.
Traditionally micro-well plate based platforms used in biology utilize fluorescence intensity based methods to measure
processes of biological relevance. However, fluorescence intensity measurements suffer from calibration drift due to a
variety of factors. Photobleaching and self-quenching of the fluorescent dyes cause the intensity signal to drop over the
lifetime of sensor immobilized inside the well. Variation in turbidity of the sample during the course of the measurement
affects the measured fluorescence intensity. In comparison, fluorescence lifetime measurements are not significantly
affected by these factors because fluorescence lifetime is a physico-chemical property of the fluorescent dye. Reliable
and inexpensive frequency domain fluorescence lifetime instrumentation platforms are possible because the greater
tolerance for optical alignment, and because they can be performed using inexpensive light sources such as LEDs. In this
paper we report the development of a frequency domain fluorescence lifetime well-plate platform utilizing an oxygen
sensitive transition-metal ligand complex fluorophore with a lifetime in the microsecond range. The fluorescence
lifetime dye is incorporated in a polymer matrix and immobilized on the base of micro-well of a 60 well micro-well
plate. Respiration measurements are performed in both aqueous and non-aqueous environment. Respirometry
measurements were recorded from single Daphnia magna egg in hard water. Daphnia is an aquatic organism, important
in environmental toxicology as a standard bioassay and early warning indicator for water quality monitoring. Also
respirometry measurements were recorded from Tribolium castaneum eggs, which are common pests in the processed
flour industry. These eggs were subjected to mitochondrial electron transport chain inhibitor such as potassium cyanide
(KCN) and its effects on egg respiration were measured in real-time.
The detection of harmful chemicals and biological agents in real time is a critical need for protecting water quality. We
studied the real-time effects of five environmental contaminants with differing modes of action (atrazine,
pentachlorophenol, cadmium chloride, malathion, and potassium cyanide) on respiratory oxygen consumption in 2-day
post-fertilization fathead minnow (Pimephales promelas) eggs. Our objective was to assess the sensitivity of fathead
minnow eggs using the self-referencing micro-optrode technique to detect instantaneous changes in oxygen consumption
after brief exposures to low concentrations of contaminants. Oxygen consumption data indicated that the technique is
indeed sensitive enough to reliably detect physiological alterations induced by all contaminants. After 2 h of exposure,
we identified significant increases in oxygen consumption upon exposure to pentachlorophenol (100 and 1000 μg/L),
cadmium chloride (0.0002 and 0.002 μg/L), and atrazine (150 μg/L). In contrast, we observed a significant decrease in
oxygen flux after exposures to potassium cyanide (5.2, 22, and 44 μg/L) and atrazine (1500 μg/L). No effects were
detected after exposures to malathion (200 and 340 μg/L). We have also tested the sensitivity of Daphnia magna
embryos as another animal model for real-time environmental biomonitoring. Our results are so far encouraging and
support further development of this technology as a physiologically coupled biomonitoring tool for the detection of
environmental toxicants.
The reference optical path is essential for optical systems which function on the basis of light interference. In the case of frequency domain (FD) fluorescence life-time optrodes, a reference LED is used as a standard for calculating the phase angle. The reference LED is configured so that radiation travels the same length to the detector as that of the fluorescence signal being analyzed. The phase shift, which provides details of fluorescence lifetime, is measured between these two signals - the fluorescence signal and reference LED signal, using a photodetector. We have designed, developed and implemented a FD optrode system without a reference LED. The key requirement of such a system is that phase shifts due to optics at wavelength of fluorescence and electronics have to be calibrated. In the reference-free system, the reference signal comes from the lock-in-amplifier which also drives the excitation LED. The lock-in-amplifier measures the phase shift between the excitation signal and the fluorescence emission signal from the photodetector and is locked at the frequency of modulation of the excitation signal. This insures higher signal to noise ratio and low-noise measurements. The reference-free optrode system removes some constraints on the coupling optics, which help improve the overall performance of the system. After development of electronics, and optimization of coupling optics, the system was calibrated in different oxygen concentration solutions to measure fluorescence intensity and lifetime of the oxygen sensitive dye platinum tetrakis (pentafluorophenyl) porphine (PtTFPP).
Fiber optic based sensor technologies have many significant advantages over electrochemical sensors, and as a result
have broad application for sensing in biology, agriculture and medicine. An important component of fiber optic
biosensor is the sensing element. Usually, a polymer matrix containing the analyte specific fluorescent dye is
immobilized on one end of the fiber optic probe. The polymer matrix provides mechanical stability to the immobilized
membrane and entraps the fluorescent dye molecules. The target analyte diffuses into the membrane polymer and
quenches the fluorescent dye. This optical interaction between the analyte and fluorescent dye dynamically changes the
fluorescence lifetime of the dye. These changing fluorescent lifetimes reveal information about the target analyte
concentration. Although the fluorescent lifetime of the dye is of primary interest, high signal-to-noise ratio (SNR) is also
very desirable. Conventionally, complex electronics is implemented to achieve high SNR. Various signal processing
methods such as signal amplification and filtering are implemented to achieve high SNR. In this study, we report optical
signal amplification by modification of the dye-polymer matrix by addition of titanium oxide particles. The addition of
titanium oxide particles enhanced the optical signal intensity. Optical performances of different sizes and concentrations
of titanium oxide particles are compared. We believe that this increased optical intensity is due to increased optical
scattering in the dye-polymer matrix. We also compare the performance of titanium oxide particles to gold and other
material particles to experimentally probe the exact nature of light scattering in the dye-polymer matrix.
Trans-cellular calcium currents play a central role in the establishment of polarity in differentiating cells. Typically these
currents are measured and studied experimentally using ion selective glass microelectrodes. We have recently developed
an in silico cell electrophysiology lab-on-a-chip device with the specific science objectives of measuring these
transcellular calcium currents in an advanced throughput format. The device consists of 16 pyramidal pores on a silicon
substrate with four Ag/AgCl electrodes leading into each pore on the four poles. An SU-8 layer is used as the structural
and insulating layer and a calcium ion selective membrane is used to impart ion selectivity to the Ag/AgCl electrodes. In
this paper we demonstrate the utility of the cell electrophysiology biochip in measuring these transcellular calcium
currents from single cells using the model biological system Ceratopteris richardii. We monitored these fern spores
during germination and pharmacologically inhibited biophysical calcium transport. These results demonstrate the utility
and versatility of the in silico cell electrophysiology biochip. While this version of the biochip was engineered to fulfill
the specific science objectives of measuring trans-cellular calcium currents from Ceratopteris fern spores, the chip can
easily be modified for a variety of biomedical and pharmacological applications. Future
Optically transduced sensors (optrodes, or optodes) offer significant advantages over polarographic techniques for
measuring oxygen. In biology and medicine, how we make measurements is very important, and this is especially true in
terms of physiological exchange. Cellular and tissue oxygenation is a function of background concentration and
respiratory demand, and in pure physical terms this is best expressed in terms of molecular flux based on Fick's law.
Measuring dynamic flux from biological systems requires sensing technology that can measure activity in multiple
dimensions. Here we report the development of a self-referencing oxygen optrode (SRO) for reliably making noninvasive
measurements of oxygen flux from a variety of biological systems. The self-referencing microsensor technique
was adapted to operate optrodic oxygen sensors through the integration of optical sensing instrumentation with software-controlled
data acquisition and micro-stepping motion control. This allows the sensor to scan biologically active
gradients of oxygen flux directly, as it relates to cellular and tissue respiratory activity. The technique was validated first
using artificially generated oxygen gradients, which are theoretically modelled and compare with measured signals.
Subsequently, the SRO was applied in basic research applications to non-invasively measure molecular oxygen flux
from a variety of animal and plant systems.
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