Jeffrey Sipior

Single quantum well light emitting diodes demonstrated as excitation sources for nanosecond phase‐modulation fluorescence lifetime measurements

We have characterized the output of inexpensive, commercially available single quantum well (SQW) blue and green light emitting diodes (LEDs). The SQW LEDs were amplitude modulated with the output from a frequency generator while biased through a bias tee with 5 mA of current. The blue SQW LED produced 800 μW of light centered at 466 nm, with a −3 dB bandwidth of 58 MHz. The green SQW LED produced 543 μW of light centered at 522 nm, with a −3 dB bandwidth of 26 MHz. Modulated light was available to approximately 100 MHz, allowing the measurement of ns fluorescence lifetimes. The fluorescence lifetime of a standard fluorophore (fluorescein) was measured in the frequency domain using the phase‐modulation technique, and gave results similar to those obtained with a 488 nm argon ion laser modulated with a Pockels cell. To demonstrate the usefulness of the SQW LED source, we also performed measurements with the fluorescent pH indicator SNAFL‐2. Again, these results compared favorably with those obtained with t...

On‐line green fluorescent protein sensor with LED excitation

We present an intensity based sensor designed for on-line monitoring of green fluorescent protein, a revolutionary marker of protein expression. The device consisted of a blue light emitting diode as the excitation source. A band pass excitation filter cut off light longer than 490 nm. The light was directed into a bifurcated optical fiber bundle with the common end inserted into a stainless steel housing equipped with a quartz window. The fiber bundle and stainless steel housing are steam sterilizable. The emission radiation was collected through a long wave pass filter to reject the excitation light shorter than 505 nm and was detected by a photomultiplier tube. The signal was amplified and sent to a computer for recording time course data. The sensor was tested in an Escherichia coli fermentation of JM105 transformed with pBAD-GFP. The on-line signal was compared to off-line fluorescence spectrophotometer measurements. The on-line profile closely followed the off-line. Western blot data showed that with a time shift, the sensor was able to both continuously and quantitatively monitor expression of green fluorescent protein on-line in real time.

Blue light-emitting diode demonstrated as an ultraviolet excitation source for nanosecond phase-modulation fluorescence lifetime measurements

We have produced amplitude-modulated near-ultraviolet light, centered at 390 nm, using an inexpensive, commercially available blue light-emitting diode (LED). The LED was amplitude modulated with the +13 dBm ac output from a frequency generator while biased through a bias tee with 60 mA of dc current. The LED produced 45 to 54 μW of UV light over the modulation bandwidth of 0.01 to 200 MHz, when measured after optical filters to remove the residual blue output. Since the filter attenuated the UV output about 3 dB, more than 100 μW of UV light was initially produced. Modulated UV light was available to approximately 200 MHz, with a −3 dB point of 31 MHz, allowing the measurement of ns fluorescence lifetimes. The fluorescence lifetimes of standard fluorophores (9-cyanoanthracene and green fluorescent protein) were measured in the frequency domain using the phase-modulation technique, producing lifetimes that closely agree with those reported in the literature, confirming that the UV-emitting blue LED is pra...

Low cost phase-modulation measurements of nanosecond fluorescence lifetimes using a lock-in amplifier

The use of a 200 MHz lock-in amplifier was demonstrated as a low cost instrument for frequency domain measurements of nanosecond fluorescence lifetimes. The lock-in directly provided both the dc bias and the ac signal used to modulate the intensity of a blue light emitting diode excitation source. The emission was measured by a photomultiplier tube and the resulting signal was sent back through a dc block to the lock-in with no external signal processing or heterodyning required. The system was highly accurate at measuring phase and modulation up to 80 MHz and moderately so up to 100 MHz. The fluorescence lifetimes of several standard fluorophores (Fluorescein, Rhodamine B, and [Ru(bpy)3]2+) were measured by the lock-in, and the results agreed closely with those made on a research grade fluorometer. The entire lock-in based system costs less than US

Phase Fluorometric Optical Carbon Dioxide Gas Sensor for Fermentation Off-Gas Monitoring

10,000 to build and can be controlled by any standard computer through a GPIB or serial connection. The system is also portable, consumes little power, and c...

Lifetime-based optical sensing of pH using resonance energy transfer in sol-gel films

We demonstrated an optical carbon dioxide gas sensor suitable for replacement of gas chromatographs and mass spectrometers for the measurement of carbon dioxide in the off‐gas of a bioreactor for fermentation and cell culture applications. The sensor is based upon the change in lifetime of a donor fluorophore, sulforhodamine 101 (SR101), induced by fluorescence resonance energy transfer to a pH‐sensitive, nonfluorescent acceptor, m‐cresol purple (MCP). Carbon dioxide diffusing into the sensor produces carbonic acid, changing the absorbance spectrum of the MCP, and thus its spectral overlap with the SR101, changing its lifetime. This lifetime change was measured in the frequency, rather than the time domain, as a change in the phase angle of the fluorescence relative to the modulated excitation light. The sensor was calibrated by correlating the phase response to carbon dioxide concentrations. The calibration remained valid over the life of the sensor, which has been shown to be greater than 2 weeks. The sensor was most sensitive at low CO2 concentrations and responded to concentration changes in seconds. The sensor film is very inexpensive to produce and the light source is an inexpensive light‐emitting diode. Furthermore, lower cost detection electronics can be developed since only one modulation frequency is required. In addition, this sensor can potentially be used in vivo, with a fiber optic both delivering the excitation light and collecting the emission.

Directly modulated diode laser frequency doubled in a KTP waveguide as an excitation source for CO2 and O2 phase fluorometric sensors

Abstract We describe the fabrication and testing of an optical pH sensor based on fluorescence lifetime measurements and sol-gel technology. These sensors are potentially suitable for use with simple laser-diode light sources. The sensor is based on the phenomenon of fluorescence resonance energy transfer (FRET) from a pH-insentive donor to a pH-sensitive acceptor. The pH-dependent increase in the bromothymol blue (BTB) acceptor absorbance results in increased energy transfer, reducing the lifetime of the Texas Red Hydrazide (TRH) donor. The lifetimes are measured by the phase and modulation of the emission, relative to the modulated incident light, and are found to be insentive to the total signal level and fluctuations in light intensity. However, the present sensors are sensitive to salt concentration and/or ionic strength. Importantly, this sol-gel sensor is not fragile: it provides stable readings for days and can be repeatedly autoclaved without loss of sensitivity to pH. The use of FRET as the pH transduction mechanism can be reliably extended to longer wavelenghts, and allows the future use of laser-diode excitation sources. Such simple and robust sensors have numerous potential applications in analytical and clinical chemistry.

Lifetime-based optical sensing of pH using resonance energy transfer in sol-gel sensors

A directly modulated infrared diode laser was frequency doubled in an intracavity KTP quasi-phase matched waveguide, producing modulated laser light at 425 nm, which was subsequently employed for phase fluorometric detection of CO2 and O2 gases. The frequency-doubled source was coupled to a single optical fiber to deliver the excitation light to the sensor mounted at the fiber tip. Fluorescence from the sensor returned through the same fiber to a photodetector. This compact, low-power source provided continuously tunable modulation frequencies up to 200 MHz. In the detection of CO2 , the doubled infrared diode laser produced 425-nm light that was modulated to a depth of 27% at a modulation frequency of 95 MHz.

Fluorescence-lifetime-based sensors: oxygen sensing and other biomedical applications

We describe the fabrication and testing of an optical pH sensor based on fluorescence lifetime measurements and sol-gel technology. The sensor is based on the phenomenon of fluorescence resonance energy transfer (FRET), from a pH-insensitive donor to a pH-sensitive acceptor. The pH-dependent increase in the bromothymol blue acceptor absorbance results in increased energy transfer, reducing the lifetime of the Texas red hydrazide donor. The lifetimes were measured by the phase and modulation of the emission, relative to the modulated incident light, and were found to be insensitive to the total signal level and fluctuations in light intensity. However, the present sensors are sensitive to salt concentration and/or ionic strength. Importantly, this sol-gel sensor is not fragile, providing stable readings for days and can be repeatedly autoclaved without loss of sensitivity to pH. The use of FRET as the pH transduction mechanism can be reliably extended to longer wavelengths, and allows the future use of laser diode excitation sources.

Long-lived highly luminescent rhenium (I) metal-ligand complex as a probe of biomolecules

Murine hybridomas were cultivated in tissue culture flasks. Dissolved oxygen tensions in the gas and liquid phases during cell growth were measured non-invasively by an optical oxygen sensor. Readings were made with caps both cracked open and completely closed. During cell growth, gas phase oxygen concentrations remained near atmospheric levels, while the oxygen tension at the bottom of the flasks eventually reached zero. These results suggest that the widespread practice of cracking open tissue culture flask caps during cell growth with a view to supplying adequate oxygen to cells is ineffective and unnecessary. The mass transfer characteristics of the tissue culture flask indicate the dominant resistance to oxygen mass transfer to the cells was the liquid media. The mass transfer rates through the liquid layer under standard laboratory conditions were found to be greater than those predicted by diffusion alone, suggesting microscale mixing. Volumetric and specific oxygen consumption rates were calculated from the sensor data, and were comparable to published values. A recently developed single fiber optic oxygen sensor is described. This new sensor will provide oxygen concentrations at various levels in the tissue culture flasks, allowing more accurate modeling of oxygen diffusion.