Fig 1.
Life cycle of the unicellular microalgae Haematococcus pluvialis.
Cultivation stage 1: Bipolar flagellated and motile zoospores with high amounts of chlorophyll. Reproduction takes place in this stage by asexual cell proliferation. Induction: Astaxanthin biosynthesis is triggered in the cells by application of high intensity light stress and nitrate starvation. Target cells in the late stage 1 start producing astaxanthin and partially lose their flagella. Cultivation stage 2: Continuous application of inductive stimuli lead to the transformation of green reproductive and motile zoospores to non-motile red aplanospores with high intracellular amounts of astaxanthin and a thick multilayered cell wall.
Fig 2.
Spectral properties of HP cell lysates from different micro algae sub types.
A) RGB image (left) and monochrome image at spectral band 538–548 nm (right) of three relevant micro algae sub types. B) Spectral properties of cell lysates (diluted in Dichloromethane:Methanol (25:75 v/v) green, green/red and red algae as well as pure Astaxanthin as reference. The blue marked band between 538–548 nm is used for imaging. All other bands are blocked by the bandpass filter.
Fig 3.
Microfluidic chip concept and functional units.
A) Organigram of the implemented functional units of the sorter chip. B) Schematic representations of the three-step flow focusing process. First step the randomly distributed cells are compressed into a vertical lamella at the FFU1. Second step the flow rotation unit (FRU) rotate the vertical lamella for 90 degree into horizontal lamella. In the last step the cells are compressed at FFU2 to single particle file. C) Microscope image of the IFCsort2-Chip.
Fig 4.
Microfluidic sorting principle and actuator bridge integration.
A) Single particle series while passing through the sorting area—sorted (left) and unsorted (right). All particles with the previously defined properties deflected into the sorting channel (upper channel) by the fluid pulse (left). All particles, which are not displaced by a cross flow fluid pulse leave the system through the default outlet (lower channel) (right). The blue rectangles are highlighting the region of interest (ROI) used for in-line image acquisition, digital image analysis and decision making for sorting. The decision point for sorting is precise matched within the time window from leaving the ROI to arriving at the sorting area. B) Cross flow fluid pulses are generated by a pump chamber, covered with a 400 μm thick PDMS membrane. The stroke is created by piezo actors and transferred to the pump chamber membrane via a stamp using a flexure hinge. C) microfluidic chip device built-in a microscopy slide compatible plate covered with 3D printed actor bridge and integrated piezo actors (left). Top view of the optical accessibility for the illumination and imaging system (right).
Fig 5.
System and control diagram for the sorter infrastructure.
Fluid management is provided by the FMU. Sorted particles are collected at the sorter outlet. Two CMOS cameras are implemented into the system. Sorter camera, which images the ROI for sorting and delivers them to the image-based sorting process chain. Validation images of the sorter chip are continuously recorded for independent monitoring and evaluation of the sorting process. Blue connectors indicate tubing’s for fluid transport, black connectors indicate electrical connections for date transfer and power lines.
Fig 6.
Composition of cells in the different fractions of the sorting process.
A) Percentage distribution of the relevant cell type fractions I) Initial population II) Sorted population III) Waste population B) Microscopy images of the three fractions. Sorted population with the given criteria for sorted cells in terms of size, an intact alginate shell, a visible flagellum and the presence of initially formed carotenoids in the center of the cells.