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Fig. 5 | Plant Methods

Fig. 5

From: Design of a comprehensive microfluidic and microscopic toolbox for the ultra-wide spatio-temporal study of plant protoplasts development and physiology

Fig. 5

a Schematic representation of the in-house microscope built for long-term studies. The white illumination is provided by a white LED controlled with an Arduino micro-controller. The LED is turned on intermittently for a short amount of time to allow the picture recording by the CMOS camera. The camera is controlled by a Matlab script and the Micro-Manager software. The microscopic setup is encased in a box equipped with a LED tile on the ceiling, used to set the experimental photoperiod (night: 8 h, day: 16 h). The chamber temperature is recorded all along the experiment using a thermocouple sensing module. b Picture of the experimental setup during an experiment. c Flowchart of the movie recording and automatic frame tagging routine. The LED tile is used to impose the photoperiodic illumination of the sample. Within each day/night cycle, τ represents the time increment between two pictures, and is usually equal to 10 min. tacq corresponds to exposure time of the camera and hence the time during which the Arduino-controlled LED is turned on to allow imaging. To automatically tag the pictures with their relevant photoperiodic step (day or night), a picture of the sample is recorded prior to each acquisition with the Arduino-controlled LED turned off, to measure the light intensity in the chamber. After averaging, the intensity of each picture is compared to a reference value to discriminate if the picture of the microchip has beed acquired by night or by day, and the information is stored in a metadata file for further analysis. d Time-lapse microscopic recording of the growth of P. patens in the microdevice achieved with the custom-built setup, from day 6 after immobilization. Scalebar = 100 µm

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