Prothrombin, or factor II, is vital in blood coagulation, transforming into active thrombin. The prothrombin time test (PTT) measures blood clotting time, crucial for heart disease patients on anticoagulants. Our study introduces a novel approach using refractive index to measure prothrombin time, validated through experimental outcomes. This method leverages light refraction principles, offering rapid results, simplicity, and point-of-care potential. Testing with clinical samples showed strong correlation with traditional PTT methods. Developing a point-of-care device for prothrombin time measurement promises to enhance patient care by enabling real-time monitoring of clotting efficiency, allowing timely adjustments to anticoagulant therapy, thus reducing risks of bleeding or thrombosis. The simplicity and accessibility of this method can revolutionize anticoagulant management, especially in resource-limited settings.
Label-free quantification of polymerization is of significant importance in today's research as recent advancements in the polymer field have sparked numerous applications in the biomedical industry. Poly-((Ethylene Glycol) Methyl Ether)- Methacrylate (POEGMA) has been recognized as a replacement for PNIPAM for biomedical applications due to various factors such as biocompatibility and thermal response rate. With a rapid thermal response, POEGMA allows for a variety of biomedical applications including a potential drug delivery mechanism. In this study, a Photonic Crystal-Total Internal Reflection (PC-TIR) biosensor has been utilized to monitor POEGMA-144, which has been synthesized via an Atom Transfer Radical Polymerization (ATRP) grafting-from approach. Monitoring the optical resonance of this biosensor, it allows for quantification of the growth of a layer of an initiator on the sensor surface as well as the growth of the POEGMA on top of it. In addition, the PC-TIR sensor further allows real-time analysis of the polymer’s thermal responsiveness in an aqueous environment. At a well-defined, molecular weight specific, Lower Critical Solution Temperature (LCST) of 42°C, the POEGMA-144 polymer conformationally changes, becoming hydrophobic and collapsing after expelling any water between its PEG side chains. Using the PC-TIR biosensor, a definitive polymer collapse was observed at the LCST; similarly, a re-swelling of the polymer was observed as the ambient temperature of the polymer was allowed to cool back to room temperature.
Label-Free detection of cardiac biomarkers has become an area of great interest with respect to point of care (POC) analysis of acute myocardial infarction and drug cardiotoxicity assays. DNA aptamers have become a potential replacement to traditional antibody detection of antigens in bioassays. In comparison to antibodies, DNA aptamers provide the advantages of lower cost, high flexibility, high batch-to-batch uniformity, stability at 37°C when immobilized on the sensor surface, and reusability with a regeneration solution. However, aptamer usage requires novel binding pathways that must be explored to ensure efficiency and consistency. Herein, a direct approach for Cardiac Troponin I (cTnI) detection was tested utilizing UV immobilization of Amino- and PolyT-modified aptamers on APTES or MPTMS modified and unmodified sensor surfaces composed of SiO2 for a Photonic Crystal-Total Internal Reflection biosensor (PC-TIR). The detection of aptamer functionalization, and ultimately antigen detection, were monitored with a label-free bioassay system enabled by a PC-TIR sensor. Results from this study indicated that the binding pathways with the highest aptamer immobilization were: Amino modified aptamer on an APTES modified surface and PolyT modified aptamer on an MPTMS surface. Detection of the antigen was dependent on both aptamer secondary structure formation and aptamer immobilization following UV exposure.
The detection of cardiac troponin I (cTnI) is clinically used to monitor myocardial infarctions (MI) and other heart diseases. The development of highly sensitive detection assays for cTnI is needed for the efficient diagnosis and monitoring of cTnI levels. Traditionally, enzyme-based immunoassays have been used for the detection of cTnI. However, the use of labelfree sensing techniques have the advantage of potentially higher speed and lower cost for the assays. We previously reported a Photonic Crystal-Total Internal Reflection (PC-TIR) biosensor for label-free quantification of cTnI. To further improve on this, we present a comparative study between an antibody based PC-TIR sensor that relies on recombinant protein G (RPG) for the proper orientation of anti-cTnI antibodies, and an aptamer-based PC-TIR sensor for improved sensitivity and performance. Both assays relied on the use of polyethylene glycol (PEG) linkers to facilitate the modification of the sensor surfaces with biorecognition elements and to provide fluidity of the sensing surface. The aptamer-based PC-TIR sensor was successfully able to detect 0.1 ng/mL of cTnI. For the antibody-based PC-TIR sensor, the combination of the fluidity of the PEG and the increased number of active antibodies allowed for an improvement in assay sensitivity with a low detection limit of 0.01 ng/mL. The developed assays showed good performance and potential to be applied for the detection of cTnI levels in clinical samples upon further development.
During prostate cancer progression, cancerous epithelial cells can undergo epithelial-mesenchymal transition (EMT). EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. It is associated with biochemical changes such as epithelial cell markers Ecadherin and occludins being down-regulated, and mesenchymal markers vimentin and N-cadherin being upregulated. These changes in protein expression, specifically in the cell membrane, may be monitored via biophysical principles, such as changes in the refractive index (RI) of the cell membrane. In our previous research, we demonstrated the feasibility of using cellular RI as a unique contrast parameter to accomplish label-free detection of prostate cancer cells. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free biosensing system, which allows for ultra-sensitive quantification of the changes in cellular RI due to EMT. We induced prostate cancer cells to undergo EMT by exposing these cells to soluble Transforming Growth Factor Beta 1 (TGF-β1). The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced cancer cells. Our study shows promising clinical potential in utilizing the PC-TIR biosensing system not only to detect prostate cancer cells, but also to evaluate changes in prostate cancer cells due to EMT.
Changes in extracellular calcium concentrations ([Ca2+]e) can mediate a variety of biological responses in both excitable and nonexcitable cells. These changes can be seen in both physiological and pathological conditions; however, little is still known about their effects to neuronal excitability. Fluorescent calcium probes are essential tools for studying the fluctuation of calcium ions both in and out of cells. Unfortunately, current techniques utilizing these calcium probes have many limitations that have yet to be addressed, including lack of penetration depth and concurrent multiple site analysis in the whole brain. For example, fluorescence imaging suffers from light diffusion, a fundamental constraint that limits the imaging depth in tissue (< 1 mm). Photoacoustic tomography (PAT) has emerged as a promising imaging modality that overcomes this challenge. In this paper, we utilized a near infrared (NIR) ratiometric calcium fluorescent probe (Ca-NIR) as a unique photoacoustic calcium probe. Ca-NIR is based on fusing a selective calcium ligand BAPTA (1,2-bis-(o-aminophenoxy)ethane-N,N,N0,N0-tetraacetic acid) moiety to a dihydroxanthenehemicyanine fluorophore. We report the use of Ca-NIR as an efficient PA generating agent in various artificial cerebral-spinal fluid (aCSF) solutions with varying Ca2+ concentrations. Our result indicates high sensitivity of Ca-NIR to [Ca2+]e fluctuations in aCSF and great potential of utilizing Ca-NIR in PAT as a method for noninvasive whole brain [Ca2+]e imaging.
The current clinical standard for mass screening of prostate cancer are prostate-specific antigen (PSA) biomarker assays. Unfortunately, the low specificity of PSA’s bioassays to prostate cancer leads to high false-positive rates, as such there is an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we have successfully demonstrated, with the use of our Photonic-Crystal based biosensor in a Total-Internal- Reflection (PC-TIR) configuration, detection of prostate cancer (PC-3) cells against benign prostate hyperplasia (BPH-1) cells. The PC-TIR biosensor achieved detection of individual prostate cancer cells utilizing cellular refractive index (RI) as the only contrast parameter. To further study this methodology in vitro, we report a comprehensive study of the cellular RI’s of various prostate cancer and noncancerous cell lines (i.e. RWPE-1, BPH-1, PC-3, DU-145, and LNCaP) via reflectance spectroscopy and single-cell RI imaging utilizing the PC-TIR biosensor. Our study shows promising clinical potential in utilizing the PC-TIR biosensor system for the detection of prostate cancer against noncancerous prostate epithelial cells.
Bladder cancer is the fourth most common cancer in men and is considered to have the highest rate of recurrence of all cancers at ~70%, and transitional cell carcinoma (TCC) is the most common form of intrabladder malignancy. Current standard-of-care for Stages 2 or higher is radical cystectomy, which involves removal of the urinary bladder and nearby lymph nodes. Alternative, organ-sparing treatments such as chemo- or radiotherapy are relatively ineffective against these cancers. The latter is effective when precisely targeted, but suffers from accuracy issues due to low contrast from computed tomography guidance. These motivate an innovative approach to more precisely visualize and spatially pinpoint TCC. This manuscript presents a novel non-invasive computer vision pipeline that can extract 3D structural information from 2D images obtained during routine flexible cystoscopy. The pipeline utilized camera calibration, adaptive thresholding, Scale Invariant Feature Transform (SIFT), and a Structure from Motion (SFM) implementation to reconstruct 3D point clouds of the inner surface of organ phantoms and an ex vivo porcine bladder. 3D point clouds were processed by Poisson reconstruction to generate a textured, triangle meshed 3D surface. The reconstruction pipeline generated a visually recognizable, qualitative 3D representation of the bladder from 2D video captured via flexible cystoscopy. Once further developed, this approach will enhance the targeting precision of external beam radiotherapy, providing clinicians with better organ-sparing methods to treat TCC.
Advancements in nanotechnology sensors have aided in the detection of subtle, but significant cellular deviations that may mark certain stages of diseases. Gold nanorods (GNRs) are often studied for this purpose due to their tunable optical properties and ease in surface functionalization. The absorption properties of GNRs are governed by the localized surface plasmon resonance (LSPR), which strongly depends on the GNR’s aspect ratio and on interparticle interactions. By controlling the coupling of nearby rods, a sensor can be created to respond to temperature fluctuations in the local environment. Here, we fabricated thermo-responsive gold nanorod assemblies by conjugating GNRs in end-to-end or side-by-side configurations using Poly(N-isopropyl acrylamide) (PNIPAM). End-to-end assemblies were fabricated through mixture of GNRs and PNIPAM in DI water. GNRs and PNIPAM were combined in DI water and dimethylformamide (DMF) under sonication to achieve side-by-side configuration. The optical absorption of the assemblies was measured by UV-Visible spectroscopy at different temperatures. As the temperature increased, the polymer contracted and initiated plasmon coupling between the GNRs. The optical spectrum experienced a blue- or red-shift for side-by-side or end-to-end configurations, respectively. Spectral tunability reversal was observed when cooled. Experimental results were verified by finite-difference time-domain (FDTD) calculations, which demonstrated spectral shifts under similar parameters. We present methods for fabrication of thermo-responsive gold nanorods for use as a local thermal nanosensor.
Prostate-specific antigen (PSA) biomarker assays are the current clinical method for mass screening of prostate cancer. However, high false-positive rates are often reported due to PSA’s low specificity, leading to an urgent need for the development of a more specific detection system independent of PSA levels. In our previous research, we demonstrated the feasibility of using cellular refractive indices (RI) as a unique contrast parameter to accomplish label-free detection of prostate cancer cells via variance testing, but were unable to determine if a specific cell was cancerous or noncancerous. In this paper, we report the use of our Photonic-Crystal biosensor in a Total-Internal-Reflection (PC-TIR) configuration to construct a label-free imaging system, which allows for the detection of individual prostate cancer cells utilizing cellular RI as the only contrast parameter. Noncancerous prostate (BPH-1) cells and prostate cancer (PC-3) cells were mixed at varied ratios and measured concurrently. Additionally, we isolated and induced PC-3 cells to undergo epithelial-mesenchymal transition (EMT) by exposing these cells to soluble factors such as TGF-β1. The biophysical characteristics of the cellular RI were quantified extensively in comparison to non-induced PC-3 cells as well as BPH-1 cells. EMT is a crucial mechanism for the invasion and metastasis of epithelial tumors characterized by the loss of cell-cell adhesion and increased cell mobility. Our study shows promising clinical potential in utilizing the PC-TIR biosensor imaging system to not only detect prostate cancer cells, but also evaluate prostate cancer progression.
Efficient methods for the accurate analysis of drug toxicities are in urgent demand as failures of newly discovered drug candidates due to toxic side effects have resulted in about 30% of clinical attrition. The high failure rate is partly due to current inadequate models to study drug side effects, i.e., common animal models may fail due to its misrepresentation of human physiology. Therefore, much effort has been allocated in the development of organ-on-a-chip models which offer a variety of human organ models mimicking a multitude of human physiological conditions. However, it is extremely challenging to analyze the transient and long-term response of the organ models to drug treatments during drug toxicity tests, as the proteins secreted from the organ-on-a-chip model are minute due to its volumetric size, and current methods for detecting said biomolecules are not suitable for real-time monitoring. As protein biomolecules are being continuously secreted from the human organ model, fluorescence techniques are practically impossible to achieve real-time fluorescence labeling in the dynamically changing environment, thus making a label-free approach highly desirable for the organ-on-achip applications. In this paper, we report the use of a photonic-crystal biosensor integrated with a microfluidic system for sensitive label-free bioassays of secreted protein biomolecules from a heart-on-the-chip model created with cardiomyocytes derived from human induced pluripotent stem cells.
Breast cancer treatment options often include medications that target the overexpression of growth factor receptors, such as the proto-oncogene human epidermal growth factor receptor 2 (HER2/neu) and epidermal growth factor receptor (EGFR) to suppress the abnormal growth of cancerous cells and induce cancer regression. Although effective, certain treatments are toxic to vital organs, and demand assurance that the pursued receptor is present at the tumor before administration of the drug. This requires diagnostic tools to provide tumor molecular signatures, as well as locational information. In this study, we utilized a fiber-optic probe to characterize in vivo HER2 and EGFR overexpressed tumors through the fluorescence of targeted dyes. HER2 and EGFR antibodies were conjugated with ICG-Sulfo-OSu and Alexa Fluor 680, respectively, to tag BT474 (HER2+) and MDA-MB-468 (EGFR+) tumors. The fiber was inserted into the samples via a 30-gauge needle. Different wavelengths of a supercontinuum laser were selected to couple into the fiber and excite the corresponding fluorophores in the samples. The fluorescence from the dyes was collected through the same fiber and quantified by a time-correlated single photon counter. Fluorescence at different antibody-dye concentrations was measured for calibration. Mice with subcutaneous HER2+ and/or EGFR+ tumors received intravenous injections of the conjugates and were later probed at the tumor sites. The measured fluorescence was used to distinguish between tumor types and to calculate the concentration of the antibody-dye conjugates, which were detectable at levels as low as 40 nM. The fiber-optic probe presents a minimally invasive instrument to characterize the molecular signatures of breast cancer in vivo.
Almost since its discovery, Limulus amoebocyte lysate (LAL) testing has been an important part of the pharmaceutical quality control toolkit. It allows for in vitro endotoxin testing, which has replaced tests using animals, such as using rabbits’ thermal response to judge pyrogenicity of test samples, thus leading to a less expensive and faster test of parenteral pharmaceuticals and medical devices that contact blood or cerebrospinal fluid. However, limited by the detection mechanisms of the LAL assays currently used in industry, further improvement in their performance is challenging. To address the growing demand on optimizing LAL assays for increased test sensitivity and reduced assay time, we have developed an LAL assay approach based on a detection mechanism that is different from those being used in industry, namely, gel-clot, turbidimetric, and chromogenic detection. Using a unique open-microcavity photonic-crystal biosensor to monitor the change in the refractive index due to the reaction between LAL regents and endotoxins, we have demonstrated that this approach has improved the LAL assay sensitivity by 200 times compared with the commercial standard methods, reduced the time needed for the assay by more than half, and eliminated the necessity to incubate the test samples. This study opens up the possibility of using the significantly improved LAL assays for a wide range of applications.
Biomarker screening for prostate-specific antigen (PSA) is the current clinical standard for detection of prostate cancer. However this method has shown many limitations, mainly in its specificity, which can lead to a high false positive rate. Thus, there is a growing need in developing a more specific detection system for prostate cancer. Using a Photonic- Crystal-based biosensor in a Total-Internal-Reflection (PC-TIR) configuration, we demonstrate the use of refractive index (RI) to accomplish label-free detection of prostate cancer cells against non-cancerous prostate epithelial cells. The PC-TIR biosensor possesses an open microcavity, which in contrast to traditional closed microcavities, allows for easier access of analyte molecules or cells to interact with its sensing surface. In this study, an imaging system was designed using the PC-TIR biosensor to quantify cell RI as the contrast parameter for prostate cancer detection. Non-cancerous BPH-1 prostate epithelial cells and prostate cancer PC-3 cells were placed on a single biosensor and measured concurrently. Recorded image data was then analyzed through a home-built MatLab program. Results demonstrate that RI is a suitable variable for differentiation between prostate cancer cells and non-cancerous prostate epithelial cells. Our study shows clinical potential in utilizing RI test for the detection of prostate cancer.
During emergency medical situations, where the patient has an obstructed airway or necessitates respiratory support, endotracheal intubation (ETI) is the medical technique of placing a tube into the trachea in order to facilitate adequate ventilation of the lungs. Complications during ETI, such as repeated attempts, failed intubation, or accidental intubation of the esophagus, can lead to severe consequences or ultimately death. Consequently, a need exists for a feedback mechanism to aid providers in performing successful ETI. Our study examined the spectral reflectance properties of the tracheal and esophageal tissue to determine whether a unique spectral profile exists for either tissue for the purpose of detection. The study began by using a hyperspectral camera to image excised pig tissue samples exposed to white and UV light in order to capture the spectral reflectance properties with high fidelity. After identifying a unique spectral characteristic of the trachea that significantly differed from esophageal tissue, a follow-up investigation used a fiber optic probe to confirm the detectability and consistency of the different reflectance characteristics in a pig model. Our results characterize the unique and consistent spectral reflectance characteristic of tracheal tissue, thereby providing foundational support for exploiting spectral properties to detect the trachea during medical procedures.
Clinical attrition is about 30% from failure of drug candidates due to toxic side effects, increasing the drug development costs significantly and slowing down the drug discovery process. This partly originates from the fact that the animal models do not accurately represent human physiology. Hence there is a clear unmet need for developing drug toxicity assays using human-based models that are complementary to traditional animal models before starting expensive clinical trials. Organ-on-a-chip techniques developed in recent years have generated a variety of human organ models mimicking different human physiological conditions. However, it is extremely challenging to monitor the transient and long-term response of the organ models to drug treatments during drug toxicity tests. First, when an organ-on-a-chip model interacts with drugs, a certain amount of protein molecules may be released into the medium due to certain drug effects, but the amount of the protein molecules is limited, since the organ tissue grown inside microfluidic bioreactors have minimum volume. Second, traditional fluorescence techniques cannot be utilized for real-time monitoring of the concentration of the protein molecules, because the protein molecules are continuously secreted from the tissue and it is practically impossible to achieve fluorescence labeling in the dynamically changing environment. Therefore, direct measurements of the secreted protein molecules with a label-free approach is strongly desired for organs-on-a-chip applications. In this paper, we report the development of a photonic crystal-based biosensor for label-free assays of secreted protein molecules from a liver-on-a-chip model. Ultrahigh detection sensitivity and specificity have been demonstrated.
During emergency medical situations where the patient has an obstructed airway or necessitates respiratory support, endotracheal intubation (ETI) is the medical technique of placing a tube into the trachea in order to facilitate adequate ventilation of the lungs. In particular, the anatomical, visual and time-sensitive challenges presented in these scenarios, such as in trauma, require a skilled provider in order to successfully place the tube into the trachea. Complications during ETI such as repeated attempts, failed intubation or accidental intubation of the esophagus can lead to severe consequences or ultimately death. Consequently, a need exists for a feedback mechanism to aid providers in performing successful ETI. To investigate potential characteristics to exploit as a feedback mechanism, our study examined the spectral properties of the trachea tissue to determine whether a unique spectral profile exists. In this work, hyperspectral cameras and fiber optic sensors were used to capture and analyze the reflectance profiles of tracheal and esophageal tissues illuminated with UV and white light. Our results show consistent and specific spectral characteristics of the trachea, providing foundational support for using spectral properties to detect features of the trachea.
We present a simple, non-destructive photoacoustic spectroscopy method utilizing a unique open-microcavity optoacoustic sensor to measure the concentration ratio of Methemoglobin (MetHb) in an optically scattering medium. Elevated levels of MetHb, present for example in the blood disorder Methemeglobinemia, cannot be detected by conventional pulse oximetry, and may result in inaccurate arterial oxygen saturation measurements. Samples with different ratios of Oxygenated Hemoglobin (HbO2), Deoxygenated Hemoglobin (HHb), and MetHb were obtained and mixed with nanoscale latex beads to present an optical scattering effect. Polymer encapsulated hemoglobin (PEH) samples were also studied. A sample chamber containing 20 μL of each sample was positioned directly underneath our patented optoacoustic sensor. Unlike a piezoelectric transducer, our optoacoustic sensor allows an excitation laser beam from an OPO laser to pass through and be absorbed by the sample to produce a photoacoustic signal. The cavity layer of the optoacoustic sensor is exposed directly to the resulting ultrasound signal, which causes an intensity modulation of a HeNe laser that is used to monitor the resonance condition of the sensor. A probe laser beam is total internally reflected off of the sensor and detected with a fiber-coupled APD detector. Three wavelengths are chosen for our excitation laser based on the absorption peaks and isobestic points of HHb, HbO2, and MetHb. Using established values of the molar extinction coefficients of HbO2, HHb, and MetHb a set of three simultaneous equations can be solved to accurately determine the concentration ratio of MetHb.
Bacillus anthracis has posed a threat of becoming biological weapons of mass destruction due to its virulence factors encoded by the plasmid-borne genes, such as lef for lethal factor. We report the development of a fast and sensitive anthrax DNA biosensor based on a photonic crystal structure used in a total-internal-reflection configuration. For the detection of the lef gene, a single-stranded DNA lef probe was biotinylated and immobilized onto the sensor via biotin-streptavidin interactions. A positive control, lef-com, was the complementary strand of the probe, while a negative control was an unrelated single-stranded DNA fragment from the 16S rRNA gene of Acinetobacter baumannii. After addition of the biotinylated lef probe onto the sensor, significant changes in the resonance wavelength of the sensor were observed, resulting from binding of the probe to streptavidin on the sensor. The addition of lef-com led to another significant increase as a result of hybridization between the two DNA strands. The detection sensitivity for the target DNA reached as low as 0.1 nM. In contrast, adding the unrelated DNAs did not cause an obvious shift in the resonant wavelength. These results demonstrate that detection of the anthrax lef by the photonic crystal structure in a total-internal-reflection sensor is highly specific and sensitive.
High-resolution ultrasound imaging requires quality sensors with wide bandwidth and high sensitivity, as shown in a wide range of applications, including intravascular imaging of cardiovascular diseases. However, piezoelectric technology, the current dominant approach for hydrophone fabrication, has encountered many technical limitations in the high-frequency range. Using optical techniques for the detection of high-frequency ultrasound signals has attracted much recent attention. One of the most studied approaches is based on a Fabry-Pérot interferometer, consisting of an optical cavity sandwiched between two mirrors. This technique offers promising sensitivity and bandwidth, and a potential alternative to piezoelectric polyvinylidene fluoride (PVDF) hydrophones. We propose an innovative optical ultrasound sensor using only a single mirror in a total-internal-reflection configuration. Besides retaining the advantages of Fabry-Pérot interferometer-based ultrasound sensors, this unique design provides a bandwidth of at least 160 MHz, a potential decrease in fabrication cost, and an increase in signal fidelity.
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.
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.
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.
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 7×10-8 RIU for bulk solvent refractive index, 6×10-5 nm for molecular layer
thickness and 24 fg/mm2 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.
KEYWORDS: Green fluorescent protein, Blood, Signal detection, Flow cytometry, Capillaries, In vivo imaging, Optical spheres, Tumors, Luminescence, Mirrors
We demonstrate enhanced sensitivity in two-photon flow cytometry with an extended cavity laser excitation source. At low power, the home-built 20-MHz oscillator was able to detect a significantly larger fraction, in either phosphate buffered saline (PBS) or whole blood, of green fluorescent protein (GFP)–expressing MCA-207 cells cross-labeled with the membrane-binding lipophilic dye DiD. A geometrical model is used to explain unique features of the signals resulting from the different spatial distribution of DiD and GFP. These unique features include sub-square law scaling of unsaturated two-photon signal, a sigmoidal sensitivity curve for detection under varying powers for cell detection thresholds as low as a single photon, and uncorrelated signal strengths in two detection channels.
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.
KEYWORDS: Flow cytometry, In vivo imaging, Blood, Luminescence, Signal detection, Near infrared, Fluorescent proteins, Quantum dots, Green fluorescent protein, Breast cancer
We have developed a new two-photon system for in vivo flow cytometry, thereby allowing us to
simultaneously quantify different circulating populations in a single animal. The instrument was able to resolve
minute-by-minute depletion dynamics of injected fluorescent microspheres at finer time scales than conventional
flow cytometry. Also observed were the circulation dynamics of human MCF-7 and MDA-MB-435 breast cancer
cells, which have low and high metastatic potential, respectively. After co-injection of both cell types into mice,
markedly greater numbers of MCF-7 cells were present in the circulation at early time points. While low metastatic
MCF-7 cells were cleared from the vascular system within 24 hours, detectable numbers of metastatic MDA-MB-
435 cells in the circulation remained constant over time. When we replace the commercial (80-MHz) NIR
excitation laser with a reduced-repetition-rate (20-MHz) mode-locked oscillator, the signal is enhanced four-fold,
enabling superior detection in blood of cell lines expressing fluorescent proteins tdTomato and mPlum (crosslabeled
with DiI and DiD). Detection sensitivity versus incident laser power is understood in terms of detected
event photon count distribution, which can be predicted with simple fluorophore distribution assumptions. The
technique of two-color, two-photon flow cytometry greatly enhances the capabilities of ex vivo flow cytometry to
investigate dynamics of circulating cells in cancer and other important diseases.
Metal/dendrimer nanocomposites (DNCs) uniquely combine the properties of metallic clusters and the biofriendly
polymer host in a nanosized hybrid particle. DNCs can biochemically target tissues and locally reduce femtosecond
optical breakdown thresholds, making highly precise and selective photodisruption possible. In this study, we have used
high-frequency acoustic monitoring of bubble production dynamics to investigate how DNC properties, solution
concentration, and optical parameters affect threshold reduction, actual waiting time, and mechanical characteristics of
breakdown. Breakdown is defined here as bubble production with an onset of less than 20 seconds after laser exposure.
DNC properties varied include metal content (silver, gold) and terminal group (amino-NH2, glycidol-OH, and carboxyl-
COOH) which determine pH values. Results indicate that DNC metal content markedly influences solution threshold
reduction, while DNC terminal group (and thus net surface charge) and solution concentration influence the details of
breakdown at these reduced threshold fluences. {Ag(0)} DNCs reduce breakdown threshold fluence 1-2 orders of
magnitude more than {Au(0)} DNCs. Furthermore, concentrated DNC solutions and DNCs carrying a net negative
charge (carboxyl terminal groups) increase bubble production up to four times and shorten waiting time for breakdown
from seconds to milliseconds. Increasing laser fluence for a given DNC solution concentration also shortens breakdown
waiting time. Lastly, utilizing the fluorescence properties of silver nanocomposites, we use confocal microscopy to
examine KB cell uptake of folate targeted silver DNCs. Cells incubated with folate targeted silver DNCs exhibit a
measurable increase of intracellular fluorescence compared to control cells (no DNC incubation). However, while we
observe a threshold reduction in KB cells incubated with 500nM folate-targeted DNC solution, there is no threshold
reduction in cells incubated with 50nM folate-targeted DNC solution. This suggests that a specific minimum DNC
concentration may be required for localized reduced-threshold breakdown to occur.
For diagnostic or therapeutic technologies using femtosecond laser-induced optical breakdown (LIOB) in turbid biological tissues, pulses of sufficient fluence must be delivered to the site of interest. As light attenuates and diffuses rapidly due to wavelength-dependent absorption and scattering, it is important to develop penetration optimization schemes. In this study, we use a high frequency (50MHz) ultrasonic technique to investigate the precision and penetration depth limitations of infrared femtosecond laser-induced photodisruption in excised pig skin. Optical parameters varied include laser fluence (energy density in J/cm2) and focusing numerical aperture. Our ultrasonic method uses sensitive detection of laser-induced bubbles to measure breakdown extent. Using a geometrically focused Nd:Glass laser (1053 nm, 800 fs) source, we show that acoustically detectable bubbles can be produced as deep as 900 um into excised porcine skin. As penetration exceeds several hundred microns, however, multiple bubbles stacked at different depths can be produced with a single laser excitation. Secondary bubble creation is more likely at supra-threshold fluences or with low NA (≤ 0.4) focusing, where optical self-focusing may occur near threshold fluences. However, as the numerical aperture is increased (> 0.4) for deeper focusing, aberrations can severely distort the beam, increasing the perceived LIOB-threshold with maximal penetrations of less than 500um. Using an index matching fluid (i.e. aqueous glycerol solutions) to help reduce scattering, we are able to improve penetration. However, multiple breakdown sites and the corresponding reduction in precision is still likely in skin even with glycerol treatment.
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.
Two-photon excitation fluorescence of complex solvated molecules (Rhodamine590, Fluorescein, and G5-dendrimer conjugated Fluorescein) was successfully controlled using adaptive pulse shaping. We were able to maximize and minimize the ratio of fluorescent yield to average incident power or second-harmonic generation (SHG) in a thin optical crystal. The optimal excitation pulse shape was found experimentally using a genetic learning algorithm and no a priori knowledge. Pulses were shaped with an acousto-optic programmable dispersive filter (Dazzler AOPDF) controlling phase and amplitude of 20 individual frequency components. Convergence occurred over the order of 100 generations of experiments from an original set of 50 random individual pulses. Femtosecond laser pulses (~75 fs, 76 MHz repetition, 800 nm center wavelength, 3nJ without shaping) selected to maximize fluorescence yield / SHG were found to be complementary to those minimizing this ratio when visualized with a SHG-frequency resolved optical gating (SHG-FROG) device. At these powers, linear chirp of the pulse was far less significant in establishing coherent control than the more complex pulse shape. Regeneratively amplified pulses (~150 fs, 20 kHz repetition, 795 nm center wavelength, 2 μJ before shaping) were selected for maximum efficiency of fluorescent yield relative to incident power. The peak intensity, as determined by SHG, did not change significantly for optimal pulses when compared to early generations. This indicates that the improved two-photon fluorescent signal was not the result of simple convergence to a transform limited pulse, and suggests that the dye molecule excited state population is being coherently controlled. We are currently investigating the application of this result to enhancing signal in flow-cytometry and improved discrimination for multi-photon microscopy.
Acoustical monitoring of laser-induced optical breakdown can be used as an important tool for diagnostics and therapeutics in living cells. Laser-induced intracellular microbubbles provide measurable contrast when detected with high-frequency ultrasound, and the bioeffects of these bubbles can be controlled to be within two distinct regimes. In the nondestructive regime, a single, transient, detectable bubble can be generated within a cell, without affecting its viability. In the destructive regime, the induced photodisruption can kill a target cell. To generate and monitor this range of effects in real time, we have developed a system integrating a femtosecond pulsed laser source with optical and acoustical microscopy. Experiments were performed on monolayers of Chinese hamster ovary cells. A Ti:Sapphire laser (793 nm wavelength, 100 fs pulse duration) was pulsed at 3.8 kHz and tightly focused to a 1 μm spot within each cell, and a high-frequency (50 MHz) ultrasonic transducer monitored the generated bubble with continuous pulse-echo recordings. The photodisruption was also observed with bright field optical microscopy, and cell viability was assessed after laser exposure using a colorimetric live/dead stain. By controlling laser pulse fluence, exposure duration, and the intracellular location of the laser focus, either nondestructive or destructive bubbles could be generated.
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 (T3FC) 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 in vivo.
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 in vivo 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 ex vivo and in vivo) 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, in vivo, quantitative fluorescence measurements.
Femtosecond pulsed laser beams can induce precise photodisruption in tissue and tissue-like materials. Both geometrical and biochemical manipulation of laser-induced optical breakdown (LIOB) produces highly localized photodisruption without residual damage to surrounding tissue. Measurable effects associated with LIOB are shock wave emission and microbubble formation. In previous work, we presented techniques for monitoring site-targeted, LIOB microbubbles with high-frequency (>50MHz) ultrasonic imaging. In this study, we used these techniques to study the stability of LIOB-induced bubbles in water-based gelatin. Successive recordings taken before, during, and after laser exposure illustrated bubble creation and stability. Bubbles with a range of lifetimes (20 - 400 ms) and dissolution behaviors were produced by varying either laser fluence (0.7 - 2.1 J/cm2/pulse) or total number of laser pulses delivered (30 - 500 pulses at 18kHz repetition rate). While both increases in pulse fluence and pulse number lengthened bubble lifetime by an order of magnitude and decreased the rate of bubble dissolution, bubble stability was nonlinearly related to total laser exposure. A few pulses at high laser fluence created initially large bubbles with long lifetimes and slow dissolution rates. In contrast, pulses at near-threshold laser fluence created initially smaller, shorter lifetime bubbles that were stabilized with subsequent pulses. Increased stability could be maintained only above a threshold bubble size. Below that critical size, dissolution rate rapidly increased, causing bubble collapse. Ultimately, these results demonstrated an ability to control the size, lifetimes, and stability of laser-induced microbubbles with various optical parameters, increasing their utility as site-activated contrast agents that can be sensitively monitored with high-frequency ultrasound.
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