On-Chip Photoacoustics-Activated Cell Sorting (PA-ACS) for Label-Free and High-Throughput Detection and Screening of Microalgal Cells.

Microalgae play a crucial role in global carbon cycling as they convert carbon dioxide into various valuable macromolecules. Among them, Haematococcus pluvialis (H. pluvialis) is the richest natural source of astaxanthin (AXT), which is a valuable antioxidant, anti-inflammatory, and antiapoptosis agent. These benefits make AXT highly commercially valuable in pharmaceuticals, cosmetics, and nutritional industries. However, intrinsic genetic characteristics and extrinsic cultivation conditions influence biomass gains, leading to low productivity and extraction as the main techno-economic bottlenecks in this industry. Thus, detecting AXT in H. pluvialis is essential to determine the influence of multiple parameters on biocompound accumulation, enabling optimization of cultivation and enrichment of AXT-rich H. pluvialis cells. This work developed an opto-acousto-fluidic microplatform for detection, analysis, and sorting of microalgae. Via label-free monitoring and extraction of sample-induced ultrasonic signals, a photoacoustic microscopic system was proposed to provide a full-field visualization of AXT's content and distribution inside H. pluvialis cells. When employed as on-chip image-based flow cytometry, our microplatform can also offer high-throughput measurements of intracellular AXT in real time, which demonstrates similar results to conventional spectrophotometry methods and further reveals the heterogeneity of AXT content at the single-cell level. In addition, a solenoid valve-pump dual-mode cell sorter was integrated for effective sorting of cells with a maximum working frequency of 0.77 Hz, reducing the fluid response time by 50% in rising and 40-fold in recovery. The H. pluvialis cells which have more AXT accumulation (>30 µm in diameter) were 4.38-fold enriched with almost no dead empty and small green cells. According to the results, automated and reliable photoacoustics-activated cell sorting (PA-ACS) can screen AXT-rich cells and remove impurities at the terminal stage of cultivation, thereby increasing the effectiveness and purity of AXT extraction. The proposed system can be further adopted to enrich strains and mutants for the production of biofuels or other rare organic substances such as ß-carotene and lutein.

Growth Characteristic Analysis of Haematococcus pluvialis in a Microfluidic Chip Using Digital in-Line Holographic Flow Cytometry.

In order to obtain high yield of astaxanthin, a high-value compound with ultrastrong antioxidant capacity, it is necessary to identify the growth characteristics (biomass, morphology, and size) of Haematococcus pluvialis. The current detection methods have the disadvantages of labor-consuming operation or complicated measurement system. It is an urgent need to explore a simple and cost-effective method for the detection of H. pluvialis with large size distribution during its growth period. In this work, a digital in-line holographic flow cytometry using a linear array sensor is proposed to measure the growth characteristics of H. pluvialis in a two-dimensional (2-D) hydrodynamic focusing microfluidic chip. Based on the modified angular spectrum method, the distorting holograms caused by the asynchrony of sample flow velocity and acquisition speed of the linear array sensor were rectified and reconstructed. In addition, the depth-of-focus of the imaging system were digitally extended to cover the entire depth of the microfluidic channel for optimized imaging quality. We have utilized the proposed method to statistically investigate the biomass, morphology and size of H. pluvialis under different culture conditions and growth durations.

Acoustic Standing Wave Aided Multiparametric Photoacoustic Imaging Flow Cytometry.

Photoacoustic imaging reveals great potential for the study of individual cells due to the rich imaging contrast for both label-free and labeled cells. However, previously reported photoacoustic imaging flow cytometry configuration suffers from inadequate imaging quality and challenge to distinguish multiple cells. In order to solve such issues, we propose a novel acoustic standing wave aided multiparametric photoacoustic imaging flow cytometry (MPAFC) system. The acoustic standing wave is introduced to improve the imaging quality and speed. Multispectral illumination along with cell geometry, photoacoustic amplitude, and acoustic frequency spectrum enables the proposed system to precisely identify multiple types of cells with one scanning. On the basis of the identification, elimination of melanoma cells, and targeted labeled glioma cells have been performed with an elimination efficiency of >95%. Additionally, the MPAFC system is able to image and capture melanoma cells at a lowest concentration of 100 cells mL-1 in pure blood. Current results suggest that the proposed MPAFC may provide a precise and efficient tool for cell detection, manipulation, and elimination in both fundamental and clinical studies.

On-Chip Multicolor Photoacoustic Imaging Flow Cytometry.

On-chip imaging flow cytometry has been widely used in cancer biology, immunology, microbiology, and drug discovery. Pure optical imaging combined with flow cytometry to derive chemical, structural, and morphological features of cells provides systematic insights into biological processes. However, due to the high concentration and strong optical attenuation of red blood cells, preprocessing is necessary for optical flow cytometry while dealing with whole blood. In this study, we develop an on-chip photoacoustic imaging flow cytometry (PAIFC), which combines multicolor high-speed photoacoustic microscopy and microfluidics for cell imaging. The device employs a micro-optical scanner to achieve a miniaturized outer size of 30 × 17 × 24 mm3 and ultrafast cross-sectional imaging at a frame rate of 1758 Hz and provides lateral and axial resolutions of 2.2 and 33 µm, respectively. Using a multicolor strategy, PAIFC is able to differentiate cells labeled by external contrast agents, detect melanoma cells with an endogenous contrast in whole blood, and image melanoma cells in blood samples from tumor-bearing mice. The results suggest that PAIFC has sufficient sensitivity and specificity for future cell-on-chip applications.

Study on the dual-Fano resonance generation and its potential for self-calibrated sensing.

Sensors based on Fano resonance (FR) have become a promising platform for various biological and chemical applications. However, most investigations on FR are limited to the generation of individual resonance. In this paper, based on the coupling between surface plasmon polariton (SPP) and two photonic waveguide modes, a dual-FR system is designed and analyzed. To explain the coupling mechanism, an extended temporal coupled-mode model is established to provide the physical insight. The spectral response obtained from the model matches well with the numerical one. Due to the decoupled nature of the FRs, a self-calibrated or dual-parameter sensing scheme for refractive index and temperature is proposed. The refractive index sensitivity up to 765 nm/RIU and temperature sensitivity up to 0.087 nm/°C are obtained by wavelength interrogation with figure-of-merit (FOM) up to 33260.9 RIU-1 and 3.78 °C-1 respectively. The proposed sensor provides great potential in fields of the multi-parameter sensing.

Cloaking object on an optofluidic chip: its theory and demonstration.

Recently, the design of metamaterial guided by transformation optics (TO) has emerged as an effective method to hide objects from optical detection, based on arranging a bended light beam to detour. However, this TO-based solution involves fabrication of material with complicated distribution of permittivity and permeability, and the device falls short of tunability after fabrication. In this work, we propose an optofluidic model employing the method of streamline tracing-based transformation optofluidics (STTOF) to hydrodynamically reconfigure light propagation in a given flow field for object-cloaking purposes. The proof-of-concept is demonstrated and tested on an optofluidic chip to validate our proposed theory. Experimental results show that our proposed STTOF method can be used to successfully detour the light path from the object under cloaking in a mathematically pre-defined manner.

Reconfigurable multiwavelength fiber laser based on multimode interference in highly germanium-doped fiber.

A reconfigurable multiwavelength erbium-doped fiber laser based on an all-fiber multimode interferometer (MMI) is proposed and experimentally demonstrated. The interferometer is constructed by sandwiching a section of highly germanium-doped fiber (HGDF) between two sections of single-mode fiber. The insertion loss of the interferometer is as low as 2 dB. Due to the polarization-dependent spectral filtering effect formed by the MMI, by rotating the intracavity polarization controller, the laser output can be switched among single-, dual-, and triple-wavelength lasing states with optical signal-to-noise ratio up to 50 dB. In particular, the obtained dual-wavelength state shows high stability with wavelength shift within $ \pm {0.04}\;{\rm nm}$±0.04nm, wavelength spacing variation within $ \pm {0.03}\;{\rm nm}$±0.03nm, and power fluctuation within $ \pm {0.04}\;{\rm dB}$±0.04dB by monitoring the output spectra over 8 h at room temperature. By changing the length of the HGDF, the wavelength spacing can also be flexibly manipulated. Taking the advantages of reconfiguration, low cost, and easy fabrication, this fiber laser may have great potential in various optical applications.

Optofluidic refractive index sensor based on asymmetric diffraction.

A novel optofluidic refracrtive index (RI) sensor was proposed based on asymmetric Fraunhofer diffraction. In-plane optofluidic lens, light source, slit, diffraction pattern visualization zone and optical path were integrated into the microfluidic networks to avoid the manual alignment of the optical components as well as to reduce the cost of external bulky components. Unlike the conventional RI sensor, this device visualizes the bulk refractive index change of the liquid through a diffraction image, which is readily read-out for clinical diagnosis right at the point-of-care or on-site security check. In the experiment, the device can measure a RI change of as low as ~10-5 RIU. A low noise-equivalent detection limit (NEDL) of ~10-6 refractive index unit (RIU) and high sensitivity of ~1.1 × 104/RIU were achieved. The new device is practical and suitable to be extended for high throughput applications by simultaneously reading multiple chips with an 2D-array image sensor.

An opto-acousto-fluidic microscopic system with a high spatiotemporal resolution for microfluidic applications.

In this work, we develop a new opto-acouto-fludic microsopic system, which employs a high-speed one-dimensional galvanometer scanner and an ultrafast pulse laser (600 kHz). The new system has achieved a high two-dimensional frame rate of up to 2500 Hz with a lateral resolution of 1.7 µm and an axial resolution of 36 µm at the imaging plane. To demonstrate the improved performance of the new system compared to our previous one, we carried out experiments to image the flowing droplets generated with T-junction and flow focusing configurations. We also successfully imaged dynamic migration of magneto particles subjected to non-uniform magnetic field in the microchannel. The results suggest that our new system has sufficient spatiotemporal resolutions to carry out studies for high throughput microfluidic applications.

Photoacoustic endomicroscopy based on a MEMS scanning mirror.

In this Letter, we present a high-resolution photoacoustic endomicroscopy probe based on a microelectromechanical systems (MEMS) scanning mirror. The built-in optical assembly consists of a 0.7 mm graded-index (GRIN) lens for light focusing and a ϕ1 mm MEMS mirror to reflect and scan the beam. A miniaturized unfocused ultrasound transducer with a center frequency of 10 MHz is used for photoacoustic detection. Sharp blades, carbon fibers, and black tapes were utilized to evaluate the performance of the system. In vivo mouse ears and resected rectums were imaged to further demonstrate the feasibility of this probe for potential biological and clinical applications.

Confocal photoacoustic microscopy using a single multifunctional lens.

Photoacoustic microscopy (PAM) has remained one of the fastest developing biomedical imaging modalities in the past decade. The confocal strategy of optical illumination and acoustic detection is a way to boost the sensitivity of PAM. To achieve confocal PAM, current PAM systems utilize separate acoustic and optical converging devices, making the systems bulky and complicated. In this Letter, we demonstrate the use of a single-liquid lens to successfully achieve acoustic and optical confocal configuration for optical-resolution PAM (ORPAM). Using the lens with a numerical aperture of 0.43, we show that the resolution of the ORPAM system is 4.8 µm with a significantly improved sensitivity of acoustic detection. We also apply this compact ORPAM system to in vivo imaging of the vasculature of a rat ear.

Liquid acoustic lens for photoacoustic tomography.

Recently, intensive research has been conducted to accelerate the development of photoacoustic (PA) imaging modality for biomedical applications. The use of acoustic lenses to collect ultrasound signals is of great interest. This Letter presents the design and fabrication of a liquid acoustic diverging lens, which can enlarge the acceptance angle of an ultrasound transducer. This lens possesses an inherent advantage of low acoustic impedance and the convenience to be attached to or detached from a commercial flat transducer. Phantom experiments have been carried out to demonstrate the improvement of using such a liquid lens over using a bare transducer for PA tomography.

Dispersive phase microscopy incorporated with droplet-based microfluidics for biofactory-on-a-chip.

Biomolecular imaging of intracellular structures of a single cell and subsequent screening of the cells are of high demand in metabolic engineering to develop strains with the desired phenotype. However, the capability of current methods is limited to population-scale identification of cell phenotyping. To address this challenge, we propose to utilize dispersive phase microscopy incorporated with a droplet-based microfluidic system that combines droplet volume-on-demand generation, biomolecular imaging, and droplet-on-demand sorting, to achieve high-throughput screening of cells with an identified phenotype. Particularly, cells are encapsulated in homogeneous environments with microfluidic droplet formation, and the biomolecule-induced dispersive phase can be investigated to indicate the biomass of a specific metabolite in a single cell. The retrieved biomass information consequently guides the on-chip droplet sorting unit to screen cells with the desired phenotype. To demonstrate the proof of concept, we showcase the method by promoting the evolution of the Haematococcus lacustris strain toward a high production of natural antioxidant astaxanthin. The validation of the proposed system with on-chip single-cell imaging and droplet manipulation functionalities reveals the high-throughput single-cell phenotyping and selection potential that applies to many other biofactory scenarios, such as biofuel production, critical quality attribute control in cell therapy, etc.

Tunable optofluidic aperture configured by a liquid-core/liquid-cladding structure.

Miniaturized and tunable optical components, such as the waveguide, lens, and prism, have been of great interest for lab-on-chip systems. This Letter reports an optofluidic aperture stop formed by the liquid-core/liquid-cladding flow. The aperture size can be tuned accordingly by adjusting the flow rates. Manipulation of the aperture size allows control of the amount of light passing through the corresponding optical system as well as the angular aperture on the image side. This optofluidic aperture enables lab-on-chip optical systems to have a greater flexibility and more functionalities.

Disposable flow cytometer with high efficiency in particle counting and sizing using an optofluidic lens.

Flow cytometers are widely applied to environmental monitoring, industrial testing, and biochemical studies. Integrating a flow cytometer into microfluidic networks helps to miniaturize the system and make it portable for field use. The integration of optical components, such as lenses, further improves the compactness and thus has been intensively studied recently. However, the current designs suffer from severe light scattering due to the roughness of the solid-based lens interface. In this Letter, we propose a flow cytometer using an optofluidic lens to focus the light beam. Benefiting from the smooth liquid-liquid lens interface and the refractive-index matching liquid as cladding streams, a light beam can be well focused without scattering. The variations of the signal peak values are reduced, owing to the small beam width at the beam waist. The device presents an efficient and accurate performance on both the counting and sizing of particles.

The Fusion of Microfluidics and Optics for On-Chip Detection and Characterization of Microalgae.

It has been demonstrated that microalgae play an important role in the food, agriculture and medicine industries. Additionally, the identification and counting of the microalgae are also a critical step in evaluating water quality, and some lipid-rich microalgae species even have the potential to be an alternative to fossil fuels. However, current technologies for the detection and analysis of microalgae are costly, labor-intensive, time-consuming and throughput limited. In the past few years, microfluidic chips integrating optical components have emerged as powerful tools that can be used for the analysis of microalgae with high specificity, sensitivity and throughput. In this paper, we review recent optofluidic lab-on-chip systems and techniques used for microalgal detection and characterization. We introduce three optofluidic technologies that are based on fluorescence, Raman spectroscopy and imaging-based flow cytometry, each of which can achieve the determination of cell viability, lipid content, metabolic heterogeneity and counting. We analyze and summarize the merits and drawbacks of these micro-systems and conclude the direction of the future development of the optofluidic platforms applied in microalgal research.

Three-dimensional visualization and analysis of flowing droplets in microchannels using real-time quantitative phase microscopy.

Recent years have witnessed the development of droplet-based microfluidics as a useful and effective tool for high-throughput analysis in biological, chemical and environmental sciences. Despite the flourishing development of droplet manipulation techniques, only a few methods allow for label-free and quantitative inspection of flowing droplets in microchannels in real-time and in three dimensions (3-D). In this work, we propose and demonstrate the application of a real-time quantitative phase microscopy (RT-QPM) technique for 3-D visualization of droplets, and also for full-field and label-free measurement of analyte concentration distribution in the droplets. The phase imaging system consists of a linear-CCD-based holographic microscopy configuration and an optofluidic phase-shifting element, which can be used for retrieving quantitative phase maps of flowing objects in the microchannels with a temporal resolution only limited to the frame rate of the CCD camera. To demonstrate the capabilities of the proposed imaging technique, we have experimentally validated the 3-D image reconstruction of the droplets generated in squeezing and dripping regimes and quantitatively investigated the volumetric and morphological variation of droplets as well as droplet parameters related to the depth direction under different flow conditions. We also demonstrated the feasibility of using this technique, as a refractive index sensor, for in-line quantitative measurement of carbamide analyte concentration within the flowing droplets.

Tunable micro-optofluidic prism based on liquid-core liquid-cladding configuration.

The integration of optical components into microfluidic systems has the potential to reduce the amount of bulky external devices and thus reduce the cost. However, one of the challenges of this concept is the accurate alignment of the optical path among multiple optical components inside a chip. We propose a tunable micro-optofluidic prism based on the liquid-core liquid-cladding structure formed in a sector-shape chamber. The optical interface of the prism is maintained in a straight line shape by distributing a row of pressure barriers in the chamber. By adjusting the flow rate ratio between core and cladding streams, the apex angle of the prism can be tuned accordingly. As a consequence, the deviation angle of the light beam refracted by the prism can be changed continuously. This tunability of our optofluidic prism can be utilized for the alignment of the optical path inside a chip or for the development of optical switches.

Modelling and optimization of micro optofluidic lenses.

This paper reports the modelling and experimental results of a liquid-core liquid-cladding optofluidic lens. The lens is based on three laminar streams in a circular chamber. The stream lines and the curvature of the interface can be predicted accurately using the theory of two-dimensional dipole flow in a circularly bounded domain. The model establishes basic relations between the flow rate ratio of the core/cladding streams and the radius of curvature and consequently the focal length of the lens. Compared to a rectangular chamber, this new circular design allows the formation of a liquid-core liquid-cladding lens with perfect curvatures. The circular design allows tuning a perfect curvature ranging from the chamber radius itself to infinity. The test device with a circular lens chamber with 1 mm diameter and 50 microm height was fabricated in PDMS. The lens shape as well as the stream lines were characterized using fluorescent dye and tracing particles. Experimental results agree well with the analytical results predicted by the model.

Biconcave micro-optofluidic lens with low-refractive-index liquids.

One of the current problems of micro-optofluidics is the choice of a suitable liquid with a high refractive index (RI). We report the use of a low-RI liquid in a biconcave liquid-core liquid-cladding lens for focusing light. For the characterization of the lens, a telescope system was constructed from polydimethylsiloxane lenses to collimate and expand a light beam emitted from an optical fiber. The tunable optofluidic biconcave lens focuses the parallel beam. Fluorescent dye diluted in an index-matching liquid was used for the visualization of the light rays in a beam-tracing chamber. The focused beam is tuned by adjusting the flow rate ratio between core and cladding streams.