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.

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.

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.