Non-invasive screening of bladder cancer using digital microfluidics and FLIM technology combined with deep learning.

Non-invasive screening for bladder cancer is crucial for treatment and postoperative follow-up. This study combines digital microfluidics (DMF) technology with fluorescence lifetime imaging microscopy (FLIM) for urine analysis and introduces a novel non-invasive bladder cancer screening technique. Initially, the DMF was utilized to perform preliminary screening and enrichment of urine exfoliated cells from 54 participants, followed by cell staining and FLIM analysis to assess the viscosity of the intracellular microenvironment. Subsequently, a deep learning residual convolutional neural network was employed to automatically classify FLIM images, achieving a three-class prediction of high-risk (malignant), low-risk (benign), and minimal risk (normal) categories. The results demonstrated a high consistency with pathological diagnosis, with an accuracy of 91% and a precision of 93%. Notably, the method is sensitive for both high-grade and low-grade bladder cancer cases. This highly accurate non-invasive screening method presents a promising approach for bladder cancer screening with significant clinical application potential.

Evaluating Differentiation Status of Mesenchymal Stem Cells by Label-Free Microscopy System and Machine Learning.

Mesenchymal stem cells (MSCs) play a crucial role in tissue engineering, as their differentiation status directly affects the quality of the final cultured tissue, which is critical to the success of transplantation therapy. Furthermore, the precise control of MSC differentiation is essential for stem cell therapy in clinical settings, as low-purity stem cells can lead to tumorigenic problems. Therefore, to address the heterogeneity of MSCs during their differentiation into adipogenic or osteogenic lineages, numerous label-free microscopic images were acquired using fluorescence lifetime imaging microscopy (FLIM) and stimulated Raman scattering (SRS), and an automated evaluation model for the differentiation status of MSCs was built based on the K-means machine learning algorithm. The model is capable of highly sensitive analysis of individual cell differentiation status, so it has great potential for stem cell differentiation research.

Fast and Reproducible ELISA Laser Platform for Ultrasensitive Protein Quantification.

Optofluidic lasers are currently of high interest for sensitive intracavity biochemical analysis. In comparison with conventional methods such as fluorescence and colorimetric detection, optofluidic lasers provide a method for amplifying small concentration differences in the gain medium, thus achieving high sensitivity. Here, we report the development of an on-chip ELISA (enzyme-linked immunosorbent assay) laser platform that is able to complete an assay in a short amount of time with small sample/reagent volumes, large dynamic range, and high sensitivity. The arrayed microscale reaction wells in the ELISA lasers can be microfabricated directly on dielectric mirrors, thus significantly improving the quality of the reaction wells and detection reproducibility. The details of the fabrication and characterization of those reaction wells on the mirror are described and the ELISA laser assay protocols are developed. Finally, we applied the ELISA laser to detecting IL-6, showing that a detection limit of about 0.1 pg/mL can be achieved in 1.5 h with 15 µL of sample/reagents per well. This work pushes the ELISA laser a step closer to solving problems in real-world biochemical analysis.

Chromatin laser imaging reveals abnormal nuclear changes for early cancer detection.

We developed and applied rapid scanning laser-emission microscopy (LEM) to detect abnormal changes in cell nuclei for early diagnosis of cancer and cancer precursors. Regulation of chromatins is essential for genetic development and normal cell functions, while abnormal nuclear changes may lead to many diseases, in particular, cancer. The capability to detect abnormal changes in "apparently normal" tissues at a stage earlier than tumor development is critical for cancer prevention. Here we report using LEM to analyze colonic tissues from mice at-risk for colon cancer (induced by a high-fat diet) by detecting pre-polyp nuclear abnormality. By imaging the lasing emissions from chromatins, we discovered that, despite the absence of observable lesions, polyps, or tumors under stereoscope, high-fat mice exhibited significantly lower lasing thresholds than low-fat mice. The low lasing threshold is, in fact, very similar to that of adenomas and is caused by abnormal cell proliferation and chromatin deregulation that can potentially lead to cancer. Our findings suggest that conventional detection methods, such as colonoscopy followed by histopathology, by itself, may be insufficient to reveal hidden or early tumors under development. We envision that this innovative work will provide new insights into LEM and support existing tools for early tumor detection in clinical diagnosis, and fundamental biological and biomedical research of chromatin changes at the biomolecular level of cancer development.

Turbidimetric inhibition immunoassay revisited to enhance its sensitivity via an optofluidic laser.

Turbidimetric inhibition immunoassay (TIIA) is a classic immunodiagnostic method that has been extensively used for biomarker detection. However, the low sensitivity of this technique hinders its applications in the early diagnosis of diseases. Here, a new concept, optofluidic laser TIIA (OFL-TIIA), is proposed and demonstrated for sensitive protein detection. In contrast to the immunoreaction in traditional TIIA, in which the single-pass laser loss is detected, the immunoreaction in the OFL-TIIA method takes place in a laser cavity, which considerably increases the loss induced by antigen-antibody complexes (AACs) via the amplification effect of the laser. A commercial IgG TIIA kit was selected as a demonstrative model to characterize the performance of OFL-TIIA. A wide dynamic range of five orders of magnitude with an exceptional limit of detection (LOD) (1.8 × 10-10 g/L) was achieved. OFL-TIIA is a fast, sensitive, and low-cost immunoassay with a simple homogeneous and wash-free process and low-volume sample consumption, thus providing a new detection platform for disease diagnostics.

A robust tissue laser platform for analysis of formalin-fixed paraffin-embedded biopsies.

Laser emission-based detection and imaging technology has attracted significant interest in biomedical research due to its high sensitivity, narrow linewidth, and superior spectral and spatial resolution. Recent advances have further revealed the potential to use laser emission to investigate chromatin dynamics, as well as to diagnose cancer tissues based on nuclear biomarkers. To move the laser emission based detection technology a step further towards practical use, in this work, we developed a highly robust tissue laser platform by microfabricating an SU8 spacer with a fixed height on the top mirror of the Fabry-Pérot (FP) cavity, which allows generation of reproducible and stable lasing results regardless of tissue thickness. Then we applied this platform to achieve lasing emission from formalin-fixed, paraffin-embedded (FFPE) lung tissues, which account for an overwhelming fraction of tissues collected for research and clinical use worldwide. We further showed that the cancer and normal FFPE lung tissues can be distinguished by their respective lasing thresholds. Two different tissue thicknesses (10 µm and 5 µm) commonly used in pathological labs were explored. Finally, we tested three additional types of tissues (colon, stomach, and breast) that were prepared independently by lab technicians in a pathology lab in China and shipped to the US in order to validate the general applicability and practicality of the laser emission-based technology as well as the corresponding sample preparation protocol and the tissue laser platform. Our work will not only vastly broaden the applications of laser emission-based detection/imaging technology but also help translate it from the laboratory to an automated system for clinical practice that may eventually benefit biomedicine and biological research.

Nanowire lasers as intracellular probes.

We investigate a cadmium sulfide (CdS) nanowire (NW) laser that is spontaneously internalized into a single cell to serve as a stand-alone intracellular probe. By pumping with nano-joule light pulses, green laser emission (500-520 nm) can be observed inside cells with a peak linewidth as narrow as 0.5 nm. Due to the sub-micron diameter (∼200 nm), the NW has an appreciable fraction of the evanescent field outside, facilitating a sensitive detection of cellular environmental changes. By monitoring the lasing peak wavelength shift in response to the intracellular refractive index change, our NW laser probe shows a sensitivity of 55 nm per RIU (refractive index units) and a figure of merit of approximately 98.

Distributed fibre optofluidic laser for chip-scale arrayed biochemical sensing.

Optofluidic lasers (OFLs) are an emerging technological platform for biochemical sensing, and their good performance especially high sensitivity has been demonstrated. However, high-throughput detection with an OFL remains a major challenge due to the lack of reproducible optical microcavities. Here, we introduce the concept of a distributed fibre optofluidic laser (DFOFL) and demonstrate its potential for high-throughput sensing applications. Due to the precise fibre geometry control via fibre drawing, a series of identical optical microcavities uniformly distributed along a hollow optical fibre (HOF) can be achieved to obtain a one-dimensional (1D) DFOFL. An enzymatic reaction catalysed by horseradish peroxidase (HRP) can be monitored over time, and the HRP concentration is detected by DFOFL-based arrayed colorimetric detection. Experimentally, five-channel detection in parallel with imaging has been demonstrated. Theoretically, spatial multiplexing of hundreds of channels is achievable with DFOFL-based detection. The DFOFL wavelength is tuned over hundreds of nanometers by optimizing the dye concentration or reconfiguring the liquid gain materials. Extending this concept to a two-dimensional (2D) chip through wavelength multiplexing can further enhance its multi-functionality, including multi-sample detection and spectral analysis. This work opens the door to high-throughput biochemical sensing.

Versatile tissue lasers based on high-Q Fabry-Pérot microcavities.

Biolasers are an emerging technology for next generation biochemical detection and clinical applications. Progress has recently been made to achieve lasing from biomolecules and single living cells. Tissues, which consist of cells embedded in an extracellular matrix, mimic more closely the actual complex biological environment in a living body and therefore are of more practical significance. Here, we developed a highly versatile tissue laser platform, in which tissues stained with fluorophores are sandwiched in a high-Q Fabry-Pérot microcavity. Distinct lasing emissions from muscle and adipose tissues stained respectively with fluorescein isothiocyanate (FITC) and boron-dipyrromethene (BODIPY), and hybrid muscle/adipose tissue with dual staining were achieved with a threshold of only ∼10 µJ mm-2. Additionally, we investigated how the tissue structure/geometry, tissue thickness, and staining dye concentration affect the tissue laser. Lasing emission from FITC conjugates (FITC-phalloidin) that specifically target F-actin in muscle tissues was also realized. It is further found that, despite the large fluorescence spectral overlap between FITC and BODIPY in tissues, their lasing emissions could be clearly distinguished and controlled due to their narrow lasing bands and different lasing thresholds, thus enabling highly multiplexed detection. Our tissue laser platform can be broadly applicable to various types of tissues/diseases. It provides a new tool for a wide range of biological and biomedical applications, such as diagnostics/screening of tissues and identification/monitoring of biological transformations in tissue engineering.

Reproducible fiber optofluidic laser for disposable and array applications.

Disposable sensors are widely used in biomedical detection due to their inherent safety, ease of use and low cost. An optofluidic laser is a sensitive bioassay platform; however, demonstrating its fabrication cheaply and reproducibly enough for disposable use has been challenging. Here, we report a low-cost, reproducible fiber optofluidic laser (FOFL) using a microstructured optical fiber (MOF). The MOF not only supports the whispering gallery modes for lasing but also serves as a microfluidic channel for sampling the liquid gain medium via capillary force. Because of the precise control of its geometry (δ < 0.4%) during the fiber-drawing process, good reproducibility in laser intensity (δ = 6.5%) was demonstrated by changing 10 sections of the MOF. The strong coupling between the in-fiber resonator and gain medium enables a low threshold of 3.2 µJ mm-2. The angular dependence of the laser emission was observed experimentally and analyzed with numerical simulations. An array of the FOFLs was also demonstrated. This technology has great potential for low-cost bioassay applications.

Laser-emission imaging of nuclear biomarkers for high-contrast cancer screening and immunodiagnosis.

Detection of nuclear biomarkers such as nucleic acids and nuclear proteins is critical for early-stage cancer diagnosis and prognosis. Conventional methods relying on morphological assessment of cell nuclei in histopathology slides may be subjective, whereas colorimetric immunohistochemical and fluorescence-based imaging are limited by strong light absorption, broad-emission bands and low contrast. Here, we describe the development and use of a scanning laser-emission-based microscope that maps lasing emissions from nuclear biomarkers in human tissues. 41 tissue samples from 35 patients labelled with site-specific and biomarker-specific antibody-conjugated dyes were sandwiched in a Fabry-Pérot microcavity while an excitation laser beam built a laser-emission image. We observed multiple sub-cellular lasing emissions from cancer cell nuclei, with a threshold of tens of µJ/mm2, sub-micron resolution (<700 nm), and a lasing band in the few-nanometre range. Different lasing thresholds of nuclei in cancer and normal tissues enabled the identification and multiplexed detection of nuclear proteomic biomarkers, with a high sensitivity for early-stage cancer diagnosis. Laser-emission-based cancer screening and immunodiagnosis might find use in precision medicine and facilitate research in cell biology.

An integrated microwell array platform for cell lasing analysis.

Biological cell lasers are emerging as a novel technology in biological studies and biomedical engineering. The heterogeneity of cells, however, can result in various lasing behaviors from cell to cell. Thus, the capability to track individual cells during laser investigation is highly desired. In this work, a microwell array was integrated with high-quality Fabry-Pérot cavities for addressable and automated cell laser studies. Cells were captured in the microwells and the corresponding cell lasing was achieved and analyzed using SYTO9-stained Sf9 cells as a model system. It is found that the presence of the microwells does not affect the lasing performance, but the cell lasers exhibit strong heterogeneity due to different cell sizes, cycle stages and polyploidy. Time series laser measurements were also performed automatically with the integrated microarray, which not only enables the tracking and multiplexed detection of individual cells, but also helps identify "abnormal" cells that deviate from a large normal cell population in their lasing performance. The microarrayed cell laser platform developed here could provide a powerful tool in single cell analysis using lasing emission that complements conventional fluorescence-based cell analysis.

Optofluidic FRET lasers using aqueous quantum dots as donors.

An optofluidic FRET (fluorescence resonance energy transfer) laser is formed by putting FRET pairs inside a microcavity acting as a gain medium. This integration of an optofluidic laser and the FRET mechanism provides novel research frontiers, including sensitive biochemical analysis and novel photonic devices, such as on-chip coherent light sources and bio-tunable lasers. Here, we investigated an optofluidic FRET laser using quantum dots (QDs) as FRET donors. We achieved lasing from Cy5 as the acceptor in a QD-Cy5 pair upon excitation at 450 nm, where Cy5 has negligible absorption by itself. The threshold was approximately 14 µJ mm(-2). The demonstrated capability of QDs as donors in the FRET laser greatly improves the versatility of optofluidic laser operation due to the broad and large absorption cross section of the QDs in the blue and UV spectral regions. The excitation efficiency of the acceptor molecules through a FRET channel was also analyzed, showing that the energy transfer rate and the non-radiative Auger recombination rate of QDs play a significant role in FRET laser performance.

Digital DNA detection based on a compact optofluidic laser with ultra-low sample consumption.

DNA lasers self-amplify optical signals from a DNA analyte as well as thermodynamic differences between sequences, allowing quasi-digital DNA detection. However, these systems have drawbacks, such as relatively large sample consumption and complicated dye labelling. Moreover, although the lasing signal can detect the target DNA, it is superimposed on an unintended fluorescence background, which persists for non-target DNA samples as well. From an optical point of view, it is thus not truly digital detection and requires spectral analysis to identify the target. In this work, we propose and demonstrate an optofluidic laser that has a single layer of DNA molecules as the gain material. A target DNA produces intensive laser emission comparable to existing DNA lasers, while any unnecessary fluorescence background is successfully suppressed. As a result, the target DNA can be detected with a single laser pulse, in a truly digital manner. Since the DNA molecules cover only a single layer on the surface of the laser microcavity, the DNA sample consumption is a few orders of magnitude lower than that of existing DNA lasers. Furthermore, the DNA molecules are stained by simply immersing the microcavity in the intercalating dye solution, and thus the proposed DNA laser is free of any complex dye-labelling process prior to analysis.

Optofluidic chlorophyll lasers.

Chlorophylls are essential for photosynthesis and also one of the most abundant pigments on earth. Using an optofluidic ring resonator of extremely high Q-factors (>10(7)), we investigated the unique characteristics and underlying mechanism of chlorophyll lasers. Chlorophyll lasers with dual lasing bands at 680 nm and 730 nm were observed for the first time in isolated chlorophyll a (Chla). Particularly, a laser at the 730 nm band was realized in 0.1 mM Chla with a lasing threshold of only 8 µJ mm(-2). Additionally, we observed lasing competition between the two lasing bands. The presence of laser emission at the 680 nm band can lead to quenching or significant reduction of laser emission at the 730 nm band, effectively increasing the lasing threshold for the 730 nm band. Further concentration-dependent studies, along with theoretical analysis, elucidated the mechanism that determines when and why the laser emission band appears at one of the two bands, or concomitantly at both bands. Finally, Chla was exploited as the donor in fluorescence resonance energy transfer to extend the laser emission to the near infrared regime with an unprecedented wavelength shift as large as 380 nm. Our work will open a door to the development of novel biocompatible and biodegradable chlorophyll-based lasers for various applications such as miniaturized tunable coherent light sources and in vitro/in vivo biosensing. It will also provide important insight into the chlorophyll fluorescence and photosynthesis processes inside plants.

Lasing in blood.

Indocyanine green (ICG) is the only near-infrared dye approved by the U.S. Food and Drug Administration for clinical use. When injected in blood, ICG binds primarily to plasma proteins and lipoproteins, resulting in enhanced fluorescence. Recently, the optofluidic laser has emerged as a novel tool in bio-analysis. Laser emission has advantages over fluorescence in signal amplification, narrow linewidth, and strong intensity, leading to orders of magnitude increase in detection sensitivity and imaging contrast. Here we successfully demonstrate, to the best of our knowledge, the first ICG lasing in human serum and whole blood with the clinical ICG concentrations and the pump intensity far below the clinically permissible level. Furthermore, we systematically study ICG laser emission within each major serological component (albumins, globulins, and lipoproteins) and reveal the critical elements and conditions responsible for lasing. Our work marks a critical step toward eventual clinical and biomedical applications of optofluidic lasers using FDA approved fluorophores, which may complement or even supersede conventional fluorescence-based sensing and imaging.

Monolithic optofluidic ring resonator lasers created by femtosecond laser nanofabrication.

We designed, fabricated, and characterized a monolithically integrated optofluidic ring resonator laser that is mechanically, thermally, and chemically robust. The entire device, including the ring resonator channel and sample delivery microfluidics, was created in a block of fused-silica glass using a 3-dimensional femtosecond laser writing process. The gain medium, composed of Rhodamine 6G (R6G) dissolved in quinoline, was flowed through the ring resonator. Lasing was achieved at a pump threshold of approximately 15 µJ mm(-2). Detailed analysis shows that the Q-factor of the optofluidic ring resonator is 3.3 × 10(4), which is limited by both solvent absorption and scattering loss. In particular, a Q-factor resulting from the scattering loss can be as high as 4.2 × 10(4), suggesting the feasibility of using a femtosecond laser to create high quality optical cavities.

Optofluidic lasers with a single molecular layer of gain.

We achieve optofluidic lasers with a single molecular layer of gain, in which green fluorescent protein, dye-labeled bovine serum albumin, and dye-labeled DNA, are used as the gain medium and attached to the surface of a ring resonator via surface immobilization biochemical methods. It is estimated that the surface density of the gain molecules is on the order of 10(12) cm(-2), sufficient for lasing under pulsed optical excitation. It is further shown that the optofluidic laser can be tuned by energy transfer mechanisms through biomolecular interactions. This work not only opens a door to novel photonic devices that can be controlled at the level of a single molecular layer but also provides a promising sensing platform to analyze biochemical processes at the solid-liquid interface.

Optofluidic laser for dual-mode sensitive biomolecular detection with a large dynamic range.

Enzyme-linked immunosorbent assay (ELISA) is a powerful method for biomolecular analysis. The traditional ELISA employing light intensity as the sensing signal often encounters large background arising from non-specific bindings, material autofluorescence and leakage of excitation light, which deteriorates its detection limit and dynamic range. Here we develop the optofluidic laser-based ELISA, where ELISA occurs inside a laser cavity. The laser onset time is used as the sensing signal, which is inversely proportional to the enzyme concentration and hence the analyte concentration inside the cavity. We first elucidate the principle of the optofluidic laser-based ELISA, and then characterize the optofluidic laser performance. Finally, we present the dual-mode detection of interleukin-6 using commercial ELISA kits, where the sensing signals are simultaneously obtained by the traditional and the optofluidic laser-based ELISA, showing a detection limit of 1 fg ml(-1) (38 aM) and a dynamic range of 6 orders of magnitude.

Highly sensitive fluorescent protein FRET detection using optofluidic lasers.

We achieved optofluidic protein lasing using genetically encoded fluorescent protein FRET pairs linked by length-tunable peptides. Up to 25-fold reduction in the donor laser emission was observed when the donor and the acceptor were brought to close proximity, as compared to only 17% reduction in the donor emission using the conventional FRET detection. Our work opens a door to a broad range of applications in studying protein-protein interactions and protein-drug interactions.