Superoxide anion is the primary oxygen free radical generated in mitochondria that causes intracellular oxidative stress. The lack of a method to directly monitor superoxide concentration in vivo in real time has severely hindered our understanding on its pathophysiology. We made transgenic zebrafish to specifically express fluorescent proteins, which are recently developed as reversible superoxide-specific indicators, in the liver. A fiber-optic fluorescent probe was used to noninvasively monitor superoxide generation in the liver in real time. The fish were placed in microfluidic channels for manipulation and reagents administration. Several superoxide-inducing and scavenging reagents were administrated onto the fish to investigate their effects on superoxide anion balancing. The biochemical dynamics of superoxide due to the application reagents were revealed in the transient behaviors of fluorescence time courses. With the ability to monitor superoxide dynamics in vivo in real time, this method can be used as an in vivo pharmaceutical screening platform.
We combine pulsed laser, supercontinuum radiation source and fast single-photon counting peripherals to obtain a multifunctional
micro/nano-scope. This provides us with better spatial and temporal resolution to observe fast dynamics.
Performing fluorescence correlation spectroscopy for fast dynamics (<μs) with sub-diffraction limit resolution to observe
the initial state of single-lipid dynamics in supported lipid bilayers and living cells is our goal. Lipid raft serves as a
platform for recruiting signaling components of effective signal transduction. However, the dynamics of sub-200nm
rapidly aggregated lipid rafts are still not elucidated in living cells. We here report our recent progress on the
construction of this multi-functional stage-scanning fluorescence micro/nanoscope for single-lipid dynamics study.
We report an ultracompact Mach-Zehnder index sensor with a device length of 438.6 μm using two micro-abrupt-tapers
in a cladding-reduced strongly-guiding fiber based on a focused CO2 laser beam. The cladding of strongly-guiding fiber
is chemically etched before irradiated by a focused CO2 laser beam. The index sensitivity is 600 nm/refractive index unit
respectively at around 1.37 μm wavelength with a volume of 31.2 picoliter optical liquid trapping at one micro-abrupt-taper.
A 72 picoliter glucose liquid with concentration of 200 mg/dL can lead to a red-shift of 0.8 nm at 1.3 μm.
We report the use of a sensitive double-clad fiber (DCF) probe for in situ cell flow velocity measurements and cell
analysis by means of two-photon excited fluorescence correlation spectroscopy (FCS). We have demonstrated the
feasibility to use this fiber probe for in vivo two-photon flow cytometry previously. However, because of the viscosity of
blood and the non-uniform flow nature in vivo, it is problematic to use the detected cell numbers to estimate the sampled
blood volume. To precisely calibrate the sampled blood volume, it is necessary to conduct real time flow velocity
measurement. We propose to use FCS technique to measure the flow velocity. The ability to measure the flow velocities
of labeled cells in whole blood has been demonstrated. Our two-photon fluorescence fiber probe has the ability to
monitor multiple fluorescent biomarkers simultaneously. We demonstrate that we can distinguish differently labeled
cells by their distinct features on the correlation curves. The ability to conduct in situ cell flow analysis using the fiber
probe may be useful in disease diagnosis or further comprehension of the circulation system.
KEYWORDS: Blood, In vivo imaging, Green fluorescent protein, Luminescence, Flow cytometry, Fiber optics, Photon counting, Signal detection, Tumors, Absorption
Circulating tumor cells in the bloodstream are sensitive indicators for metastasis and disease prognosis. Circulating cells have usually been monitored via extraction from blood, and more recently in vivo using free-space optics; however, long-term intravital monitoring of rare circulating cells remains a major challenge. We demonstrate the application of a two-photon-fluorescence optical fiber probe for the detection of cells in whole blood and in vivo. A double-clad fiber was used to enhance the detection sensitivity. Two-channel detection was employed to enable simultaneous measurement of multiple fluorescent markers. Because the fiber probe circumvents scattering and absorption from whole blood, the detected signal strength from fluorescent cells was found to be similar in phosphate-buffered saline (PBS) and in whole blood. The detection efficiency of cells labeled with the membrane-binding dye 1,1-dioctadecyl-3,3,3,3-tetramethylindoldicarbocyanine, 4-chlorobenzenesulfonate (DiD) was demonstrated to be the same in PBS and in whole blood. A high detection efficiency of green fluorescent protein (GFP)-expressing cells in whole blood was also demonstrated. To characterize in vivo detection, DiD-labeled untransfected and GFP-transfected cells were injected into live mice, and the cell circulation dynamics was monitored in real time. The detection efficiency of GFP-expressing cells in vivo was consistent with that observed ex vivo in whole blood.
A photoacoustic correlation spectroscopy (PACS) technique was proposed for the first time. This technique is inspired
by its optical counterpart-the fluorescence correlation spectroscopy (FCS), which is widely used in the characterization
of the dynamics of fluorescent species. The fluorescence intensity is measured in FCS while the acoustic signals are
detected in PACS. To proof of concept, we demonstrated the flow measurement of light-absorbing beads probed by a
pulsed laser. A PACS system with temporal resolution of 0.8 sec was built. Polymer microring resonators were used to
detect the photoacoustic signals, which were then signal processed and used to obtain the autocorrelation curves. Flow
speeds ranging from 249 to 15.1 μm/s with corresponding flow time from 4.42 to 72.5 sec were measured. The
capability of low-speed flow measurement can potentially be used for detecting blood flow in relatively deep capillaries
in biological tissues. Moreover, similar to FCS, PACS may have many potential applications in studying the dynamics of
photoacoustic beads.
KEYWORDS: Blood, Green fluorescent protein, In vivo imaging, Signal detection, Photon counting, Luminescence, Flow cytometry, Absorption, Scattering, Veins
We have demonstrated the use of a double-clad fiber probe to conduct two-photon excited flow cytometry in vitro and in
vivo. We conducted two-channel detection to measure fluorescence at two distinct wavelengths simultaneously. Because
the scattering and absorption problems from whole blood were circumvented by the fiber probe, the detected signal
strength from the cells were found to be similar in PBS and in whole blood. We achieved the same detection efficiency
of the membrane-binding lipophilic dye DiD labeled cells in PBS and in whole blood. High detection efficiency of green
fluorescent protein (GFP)-expressing cells in whole blood was demonstrated. DiD-labeled untransfected and GFP-transfected
cells were injected into live mice and the circulation dynamics of the externally injected cells were monitored.
The detection efficiency of GFP-expressing cells in vivo was consistent with that observed in whole blood.
Fluorescence quantification in tissues using conventional techniques can be difficult due to the absorption and scattering of light in tissues. Our previous studies have shown that a single-mode optical fiber (SMF)–based, two-photon optical fiber fluorescence (TPOFF) probe could be effective as a minimally invasive, real-time technique for quantifying fluorescence in solid tumors. We report improved results with this technique using a solid, double-clad optical fiber (DCF). The DCF can maintain a high excitation rate by propagating ultrashort laser pulses down an inner single-mode core, while demonstrating improved collection efficiency by using a high–numerical aperture multimode outer core confined with a second clad. We have compared the TPOFF detection efficiency of the DCF versus the SMF with standard solutions of the generation 5 poly(amidoamine) dendrimer (G5) nanoparticles G5-6TAMRA (G5-6T) and G5-6TAMRA-folic acid (G5-6T-FA). The DCF probe showed three- to five-fold increases in the detection efficiency of these conjugates, in comparison to the SMF. We also demonstrate the applicability of the DCF to quantify the targeted uptake of G5-6T-FA in mouse tumors expressing the FA receptor. These results indicate that the TPOFF technique using the DCF probe is an appropriate tool to quantify low nanomolar concentrations of targeted fluorescent probes from deep tissue.
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