Near-Infrared in vivo Fluorescence Sensor with Integrated Dielectric Emission Filter
ABSTRACT We present a monolithically integrated near-infrared fluorescence sensor incorporating a dielectric emission filter for in vivo applications. We successfully integrated a dielectric emission filter (OD3) onto a low-noise detector and sensed 50nM fluorescent dye concentration.
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ABSTRACT: This paper reviews authors' laboratory's work on the development of nitride-based blue-green and ultraviolet microscale LED devices with particular classes of imaging and spectroscopic applications in cellular level biology. Starting from neuroscience, we illustrate the utility of blue-green micro-LEDs for voltage-sensitive dye imaging of individual neural cells, as well as their ultraviolet counterparts for photostimulation of neurons. Arrays of micro-LEDs are also shown to be useful in projecting spatiotemporal patterns of photoexcitation to study the visual system development in living animals. As another illustration of the utility of the emerging nitride microdevice technology, we demonstrate the application of UV micro-LED arrays in bio-sensing technology as the core of a real-time fluorescence spectroscopy biowarning system.Journal of Physics D Applied Physics 01/2008; 41(9). · 2.53 Impact Factor
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ABSTRACT: This article presents a monolithically-integrated semiconductor sensor for fluorescence detection on a microfluidic platform. Vertical-cavity surface-emitting lasers (VCSELs) for 773 nm excitation, PIN photodetectors and optical emission filters have been integrated on one GaAs substrate. These optoelectronic components are optically coupled to a glass microfluidic channel (100 μm width and 45 μm depth) through the use of a discrete micro-lens to form a complete sensor. The experimental limit of detection was 250 nM of IRDye 800 Phosphoramidite. Based on an S/N = 3, the theoretical limit of detection was determined to be 40 nM. Laser background levels currently limit the sensor sensitivity. Large gains in sensitivity are possible through the systematic reduction of laser background by increasing spectral and spatial filtration. The low-cost, compact and parallel architecture makes this sensor a candidate for fluorescence-based sensing applications.Sensors and Actuators B: Chemical. 01/2005;
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ABSTRACT: 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) has extensively been used for clinical diagnosis, staging, and therapy monitoring of cancer and other diseases. Nonradioactive glucose analogues enabling the screening of the glucose metabolic rate of tumors are of particular interest for anticancer drug development. A nonradioactive fluorescent deoxyglucose analogue may have many applications for both imaging of tumors and monitoring therapeutic efficacy of drugs in living animals and may eventually translate to clinical applications. We found that a fluorescent 2-deoxyglucose analogue, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose (2-NBDG), can be delivered in several tumor cells via the glucose transporters (GLUTs). We therefore conjugated D-glucosamine with a near-infrared (NIR) fluorphor Cy5.5 and tested the feasibility of the Cy5.5-D-glucosamine (Cy5.5-2DG) conjugate for NIR fluorescence imaging of tumors in a preclinical xenograft animal model. Cy5.5-2DG was prepared by conjugating Cy5.5 monofunctional N-hydroxysuccinimide ester (Cy5.5-NHS) and D-glucosamine followed by high-performance liquid chromatography purification. The accumulation of Cy5.5-2DG and Cy5.5-NHS in different tumor cell lines at 37 and 4 degrees C were imaged using a fluorescence microscope. Tumor targeting and retention of Cy5.5-2DG and Cy5.5-NHS in a subcutaneous U87MG glioma and A375M melanoma tumor model were evaluated and quantified by a Xenogen IVIS 200 optical cooled charged-coupled device system. Fluorescence microscopy imaging shows that Cy5.5-2DG and Cy5.5-NHS are taken up and trapped by a variety of tumor cell lines at 37 degrees C incubation, while they exhibit marginal uptake at 4 degrees C. The tumor cell uptake of Cy5.5-2DG cannot be blocked by the 50 mM D-glucose, suggesting that Cy5.5-2DG may not be delivered in tumor cells by GLUTs. U87MG and A375M tumor localization was clearly visualized in living mice with both NIR fluorescent probes. Tumor/muscle contrast was clearly visible as early as 30 min postinjection (pi), and the highest U87MG tumor/muscle ratios of 2.81 +/- 0.10 and 3.34 +/- 0.23 were achieved 24 h pi for Cy5.5-2DG and Cy5.5-NHS, respectively. While as a comparison, the micropositron emission tomography imaging study shows that [18F]FDG preferentially localizes to the U87MG tumor, with resulting tumor/muscle ratios ranging from 3.89 to 4.08 after 30 min to 2 h postadministration of the probe. In conclusion, the NIR fluorescent glucose analogues, Cy5.5-2DG and Cy5.5-NHS, both demonstrate tumor-targeting abilities in cell culture and living mice. More studies are warranted to further explore their application for optical tumor imaging. To develop NIR glucose analogues with the ability to target GLUTs/hexokinase, it is highly important to select NIR dyes with a reasonable molecular size.Bioconjugate Chemistry 17(3):662-9. · 4.58 Impact Factor
Near-infrared in vivo fluorescence sensor with integrated
dielectric emission filter
Thomas D. O’Sullivan1, Elizabeth Munro2, Christopher Conca3, Natesh Parashurama4, Adam de la Zerda4,
Sanjiv S. Gambhir4, James S. Harris1, and Ofer Levi2
1Solid State and Photonics Laboratory, Stanford University, Stanford, CA 94305
2Inst.of Biomaterials and Biomedical Engineering, Dept. of Electrical and ComputerEngineering, University of Toronto, Toronto, ON, Canada
3Chroma Technology Corp., 10 Imtec Lane, PO Box 489, Rockingham, VT 05101
4Molecular Imaging Program at Stanford, Departments of Radiology and Bioengineering, Stanford University, Stanford, CA 94305
Abstract: We present a monolithically integrated near-infrared fluorescence sensor incorporating
a dielectric emission filter for in vivo applications. We successfully integrated a dielectric emission
filter (OD3) onto a low-noise detector and sensed 50nM fluorescent dye concentration.
©2009 Optical Society of America
OCIS codes: (170.3890) Medical optics instrumentation; (230.5160) Photodetectors
Optical semiconductor devices are ubiquitous in modern technologies because of their benefits in efficiency, cost,
performance, and integrative ability for complex systems. These devices have revolutionized telecommunications
and are now apparent for applications in high-speed computing, cryptography, and power generation. Optical
semiconductor devices are also an ideal technology for bio-sensing, and have been used in lab-on-a-chip [1,2],
microscopy, and spectroscopy applications. A pioneering application of these devices is to monitor living systems
for biomedical research. Potential future clinical applications exist, including medical diagnostics, monitoring
disease progression, and evaluating treatment efficacy.
We present a miniaturized optical bio-sensor, based on semiconductor technologies, designed for continuous in
vivo fluorescence detection. Fluorescence sensing is a powerful tool, providing the ability to image biological
systems at the molecular level. The primary components of a fluorescence sensing system are an excitation light
source, fluorescence emission filter, and photodetector. Systems currently used for in vivo fluorescence studies are
bulky, employing broad-area excitation sources and cooled-CCD detectors which limit studies to anesthetized,
immobilized animal subjects . We developed a miniaturized optical bio-sensor designed for implantable, long-
term, continuous studies by monolithically integrating the components of a fluorescence system. We discuss this
novel device and design constraints associated with in vivo optical sensing.
Integrating an optical sensor is difficult and there are many design factors to consider based on its application. Our
integrated fluorescence sensor (fig. 1) consists of a 670nm GaAs-based vertical cavity surface emitting laser
(VCSEL) excitation source, a large-area GaAs PIN photodiode, and a dielectric emission filter coating designed for
sensing Cy5.5 fluorescent dye. The PIN structure is epitaxially grown above the GaInP/AlGaInP VCSEL. The
emission filter is coated only on the detector, using a bi-layer resist lift-off technique and optical lithography.
Fig. 1 Integrated fluorescence sensor design
(dielectric coating is removed for clarity)
Fig. 2 Integrated fluorescence sensor response overlaid
with Cy5.5 excitation and emission
Integrated Fluorescence Sensor Response
and Cy5.5 Emission
© 2009 OSA/CLEO/IQEC 2009
For in vivo sensing, the greatest challenge is to detect a weak fluorescence signal in the presence of tissue back-
scattered excitation light. The problem is exacerbated by direct cross-talk between lasers and detectors on-chip. We
chose to use a VCSEL source rather than light-emitting diodes LEDs  because the directionality of the source
greatly reduces on-chip cross-talk. VCSELs also provide high optical power for deeper tissue penetration and they
can be arrayed for directed illumination. However, VCSEL fabrication is difficult at wavelengths below 650nm,
presently making the design unavailable for exciting fluorescent proteins. Fortunately, the optical properties of live
tissue are desirable at near infrared (IR) wavelengths (less scattering and absorption), allowing deeper imaging with
injected dyes. We have achieved lasing at 670nm in oxide-confined, etched mesa/air VCSEL devices emitting up to
2.6mW optical power at room temperature, but reducing to 0.15mW at 40ºC. By optimizing VCSEL fabrication for
thermal management, 2mW power can be expected at mammalian body temperatures (37-40ºC) . VCSELs also
exhibit low-noise , and both single- and multi-mode operation is viable for in vivo sensing.
To achieve high sensitivity, the photodetector must exhibit low-noise performance at in vivo temperatures.
GaAs-based detectors are advantageous due to their low dark current, and thus noise performance compared to
silicon , as well as their integration with optical sources. We have measured dark currents as low as 3pA / mm2
(100mV bias, room temperature), limited by surface generation at the mesa sidewalls. Growing the detector above
the VCSEL does not increase dark current or reduce performance. To reduce noise, the detector mesa must be
etched last to avoid subjecting the sidewalls to damaging fabrication steps associated with the VCSEL (high
temperature oxidation), followed by detector sidewall cleaning and passiviating techniques.
The final component of the sensor is a high-quality emission filter above the detector to prevent excitation light
from interfering with the detection of fluorescence signals. The filter is critical for implantable applications because
of the large fraction of light backscattered from tissue. We estimate that, to realize the full sensitivity performance of
the detector, one must extract a fluorescence signal amidst a tissue-backscattered background that is 7 orders of
magnitude higher. We elected to combine a high performance dielectric emission filter (fig. 2) and opaque sidewall
blocking layers designed to reduce detected excitation light by 6 orders of magnitude (OD6). The dielectric filter is
advantageous over other methods because of its (patterning via lift-off) and performance. The dielectric filter was
deposited by Chroma Technology Corp. on both fused silica (FS) and GaAs detector substrates. We overcame
several difficulties to integrate the dielectric filter with the GaAs detector including a photoresist lift-off design that
minimizes stress in the film, and matching the transmission spectrum to Cy5.5 emission. We have realized greater
than OD6 blocking at 670nm on FS, while GaAs-coated filter is limited to OD3. A drawback of dielectric stack
filters is the limited angular acceptance range outside which OD decreases (0-30 degrees for this sensor). We
attribute reduced blocking on the GaAs to be partly due to this effect and are investigating methods to improve filter
We have fabricated and verified the performance of separate sensor components from the same epitaxial structure. A
prototype sensor, with a VCSEL adjacent to the integrated emission detector, detected concentrations of Cy5.5 dye
as low as 50nM (50µL volume), matching the sensitivity of a previous integrated fluorescence sensor employing a
semiconductor distributed-Bragg reflector filter . Sensor performance is limited by excitation light leaking
through the emission filter. The dielectric approach results in more desirable detection spectra (fig. 2), holds
promise for increased performance and thus device sensitivity and, since it is patterned via lift-off, enables multiple
emission detectors to be incorporated a single chip for multiplexing. We plan to improve filter performance and
fabricate monolithically integrated sensors to demonstrate continuous in vivo fluorescence monitoring.
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microfluidic electrophoresis devices," Applied Physics Letters, vol. 89, pp. 114101-1-3, 2006.
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© 2009 OSA/CLEO/IQEC 2009