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.
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.
We have demonstrated real-time, label-free detection of small molecule binding using a novel optical biosensor. This
sensor is a recently developed sensing platform incorporating a one-dimensional photonic crystal (PC) structure in a
total-internal-reflection (TIR) geometry (PC-TIR). This simple configuration functions as an open Fabry-Perot resonator
which provides a narrow optical resonance to enable label-free, highly sensitive detection of analyte molecules on the
sensing surface in the enhanced evanescent field. Moreover, when the differential intensity modulation during binding is
measured, a very high detection sensitivity can be obtained, and real-time binding observed. The well-studied biotinstreptavidin
system was chosen to calibrate the detection limit for small molecule detection. Effective surface
functionalization methods for streptavidin immobilization on the silica sensing surface were investigated, and analyte
biotin molecules specifically binding to the sensing surface were monitored in real time. The binding of the smallest
molecule D-Biotin, with a molecular weight of 244 Da, was easily experimentally observed with a high signal to noise
ratio, which shows that the PC-TIR sensor has great potential to be a high-sensitivity and high-throughput sensing
technology for small molecule binding analysis.
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.
A novel optical biosensor using a one-dimensional photonic crystal structure in a total-internal-reflection geometry (PCTIR)
is presented and investigated for label-free biosensing applications. This simple configuration forms a micro Fabry-
Perot resonator in the top layer which provides a narrow optical resonance to enable label-free, highly sensitive
measurements for the presence of analytes on the sensing surface or the refractive index change of the surrounding
medium in the enhanced evanescent field; and at the same time it employs an open sensing surface for real-time
biomolecular binding detection. The high sensitivity of the sensor was experimentally demonstrated by bulk solvent
refractive index changes, ultrathin molecular films adsorbed on the sensing surface, and real-time analytes binding,
measuring both the spectral shift of the photonic crystal resonance and the change of the intensity ratio in a differential
reflectance measurement. Detection limits of 7×10<sup>-8</sup> RIU for bulk solvent refractive index, 6×10<sup>-5</sup> nm for molecular layer
thickness and 24 fg/mm<sup>2</sup> for mass density were obtained, which represent a significant improvement relative to state-ofthe-
art surface-plasmon-resonance (SPR)-based systems. The PC-TIR sensor is thus seen to be a promising technology
platform for high sensitivity and accurate biomolecular detection.
Flow cytometry is a powerful technique for quantitative characterization of fluorescence in cells. Quantitation is achieved by ensuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow. Two-photon excitation has the advantages that it enables simultaneous excitation of multiple dyes and achieves a very high SNR through simplified filtering and fluorescence background reduction. We demonstrate that two-photon excitation in conjunction with a targeted multidye labeling strategy enables quantitative flow cytometry even under conditions of nonuniform flow, such as may be encountered in simple capillary flow or in vivo. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling cells with targeted nanoparticles containing multiple fluorophores enables normalization of the fluorescence signal and thus quantitative measurements under nonuniform excitation. Flow cytometry using two-photon excitation is demonstrated for detection and differentiation of particles and cells both in vitro in a glass capillary and in vivo in the blood stream of live mice. The technique also enables us to monitor the fluorescent dye labeling dynamics in vivo. In addition, we present a unique two-beam scanning method to conduct cell size measurement in nonuniform flow.
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.
Real-time fluorescence measurement in deep tumors in live animals (or humans) by conventional methods has significant challenges. We have developed a two-photon optical fiber fluorescence (TPOFF) probe as a minimally invasive technique for quantifying fluorescence in solid tumors in live mice. Here we demonstrate TPOFF for real-time measurements of targeted drug delivery dynamics to tumors in live mice. 50-femtosecond laser pulses at 800 nm were coupled into a single mode optical fiber and delivered into the tumor through a 27-gauge needle. Fluorescence was collected back through the same fiber, filtered, and detected with photon counting. Biocompatible dendrimer-based nanoparticles were used for targeted delivery of fluorescent materials into tumors. Dendrimers with targeting agent folic acid and fluorescent reporter 6-TAMRA (G5-6T-FA) were synthesized. KB cell tumors expressing high levels of FA receptors were developed in SCID mice. We initially demonstrated the specific uptake of the targeted conjugates into tumor, kidney and liver, using the TPOFF probe. The tumor fluorescence was then taken in live mice at 30 min, 2 h and 24 h with the TPOFF probe. G5-6T-FA accumulated in the tumor with maximum mean levels reaching 673 ± 67 nM at the 2 h time point. In contrast, the levels of a control, non-targeted conjugate (G5-6T) at 2 h reached a level of only 136 ± 28 nM in tumors, and decrease quickly. This indicates that the TPOFF probe can be used as a minimally invasive detection system for quantifying the specific targeting of a fluorescent nanodevice on a real-time basis.
Flow cytometry is a powerful technique for obtaining quantitative information from fluorescence in cells. Quantization is achieved by assuring a high degree of uniformity in the optical excitation and detection, generally by using a highly controlled flow such as is obtained via hydrodynamic focusing. In this work, we demonstrate a two-beam, two-channel detection and two-photon excitation flow cytometry (T<sup>3</sup>FC) system that enables multi-dye analysis to be performed very simply, with greatly relaxed requirements on the fluid flow. Two-photon excitation using a femtosecond near-infrared (NIR) laser has the advantages that it enables simultaneous excitation of multiple dyes and achieves very high signal-to-noise ratio through simplified filtering and fluorescence background reduction. By matching the excitation volume to the size of a cell, single-cell detection is ensured. Labeling of cells by targeted nanoparticles with multiple fluorophores enables normalization of the fluorescence signal and thus ratiometric measurements under nonuniform excitation. Quantitative size measurements can also be done even under conditions of nonuniform flow via a two-beam layout. This innovative detection scheme not only considerably simplifies the fluid flow system and the excitation and collection optics, it opens the way to quantitative cytometry in simple and compact microfluidics systems, or <i>in vivo</i>.
Despite the fact that laser scanning confocal microscopy (LSCM) has become an important tool in modern biological laboratories, it is bulky, inflexible and has limited field of view, thus limiting its applications. To overcome these drawbacks, we report the development of a compact dual-clad photonic-crystal-fiber (DCPCF) based multiphoton scanning microscope. In this novel microscope, beam-scanning is achieved by directly scanning an optical fiber, in contrast to conventional beam scanning achieved by varying the incident angle of a laser beam at an objective entrance pupil. The fiber delivers femtosecond laser pulses for two-photon excitation and collects fluorescence back through the same fiber. Conventional fibers, either single-mode fiber (SMF) or multimode fiber (MMF), are not suitable for this detection configuration because of the low collection efficiency for a SMF and low excitation rate for a MMF. Our newly invented DCPCF allows one to optimize collection and excitation efficiency at the same time. In addition, when a gradient-index (GRIN) lens is used to focus the fiber output to a tight spot, the fluorescence signal collected back through the GRIN lens forms a large spot at the fiber tip because of the chromatic aberrations of the GRIN lens. This problem prevents a standard fiber from being applicable, but is completely overcome by the DCPCF. We demonstrate that this next generation scanning confocal microscope has an extremely simple structure and a number of unique features owing to its fundamentally different scanning mechanism: high flexibility, arbitrarily large scan range, aberration-free imaging, and low cost.
Fluorescence is a powerful tool for biosensing, but conventional fluorescence measurements are limited because solid tumors are highly scattering media. To obtain quantitative <i>in vivo </i>fluorescence information from tumors, we have developed a two-photon optical fiber fluorescence (TPOFF) probe where excitation light is delivered and the two-photon fluorescence (TPF) excited at the tip of the fiber is collected back through the same fiber. In order to determine whether this system can provide quantitative information, we measured the fluorescence from a variety of systems including mouse tumors (both <i>ex vivo </i>and <i>in vivo</i>) which were transfected with the gene to express varying amounts of green fluorescence protein (GFP), and tumors which were labeled with targeted dendrimer-based drug delivery agents. The TPOFF technique showed results quantitatively in agreement with those from flow cytometry and confocal microscopy. In order to improve the sensitivity of our fiber probe, we developed a dual-clad photonic-crystal fiber which allowed single-mode excitation and multimode (high numerical aperture) collection of TPF. These experiments indicate that the TPOFF technique is highly promising for real-time, <i>in vivo</i>, quantitative fluorescence measurements.