Acl2520u

The experiments (Figs 1–5 and 7) were conducted using wide field imaging on an ASI-XYZ stage fitted with a modular infinity microscope. The objective imaged onto the detector chip of a Hamamatsu ORCA-Flash 4.0 scientific-CMOS camera. The images were collected using either MATLAB scripts that controlled the camera, ASI stage, SH1-Thorlabs shutter, and ThorLabs DC4100 Four Channel-LED Driver, or by using μManager [46 (link)]. A custom illumination system was arranged. Briefly, a dichroic mirror was positioned at the entrance of the microscope to combine blue light LED illumination with 561 laser illumination. An SH1-Thorlabs shutter was used to create brief illumination from a blue ThorLABS LED (488nm) for rapid stimulation of bPAC. At 100% the blue light illumination was 70mW/cm². The yellow illumination was provided with a Sapphire laser (561nm, 50mW, Coherent). The laser beam was steered with two mirrors (arranged in a periscope) into an entrance aperture of the ASI microscope. Before entering the microscope, the laser beam passed through a 50⁰ diffuser (ED1 C50 MD, Thorlabs) placed in the focus of an f = 20 mm aspheric lens (ACL2520U, Thorlabs) that collimated the beam for further traveling into the microscope.

A rapid-scan spectrophotometer was constructed by arranging an actinic blue light projection LED module (Luxenon Rebel Royal Blue 447.5 nm, full width at half maximum of 20 nm [Philips Lumileds Lighting Company] equipped with an aspheric condenser lens, Φ of 25 mm, F of 20 mm [ACL2520U; Thorlabs Inc.]), a tungsten projection lamp for absorption spectroscopy (262154; Olympus Corp., Tokyo, Japan), a Photonic Multichannel Analyzer (C7473-36; Hamamatsu Photonics K.K.), and an electro-magnetic shutter module (C-79; Chuo Precision Ind. Co. Ltd., Tokyo, Japan) in the optical path. This rapid-scan spectrophotometer was driven by a multiple array of electric relays (R4 ichan; Data Six Co., Ltd., Kobe, Japan). OaPAC solution in a quartz cuvette (diameter of 1 cm) was placed in the optical path and irradiated by actinic blue light (447.5 nm) for 20 s. Immediately after this, the absorption spectrum between 350 nm and 550 nm was recorded using the rapid-scan spectrophotometer, at every 0.4 s, until OaPAC was fully relaxed. Absorption differences and time constants of conversion from light-adapted states to dark-adapted states at 492 nm were analyzed using one-way ANOVA, followed by the Tukey–Kramer test.