Elements advanced research imaging software

The amount of blue light delivered to the cells was normalized throughout all experiments using a power meter to measure the amount of light output at the 20× and 60× objectives of widefield and confocal microscopes, respectively. On the Nikon TE2000E widefield microscope using a 20× with a 0.75 numerical aperture (NA) lens and an FITC HyQ filter from chroma, blue light output was measured at approximately 12.8 microwatts (μW) at the objective. The Nikon A1R confocal microscope at 4% of total output power of the 488 nm laser delivered 12.5 to 13 μW of light through a 60× Plan Apo 1.4 NA objective lens. Nikon Elements Advanced Research imaging software (Nikon instruments) was used for the automated exposure of blue-light pulses to the cells. To administer the blue light stimulation to cells using the widefield microscope, a 30-s pulse was delivered to the entire plate. In live-imaging experiments such as uptake, the 488-nm laser was set to 4% of total power output to deliver a 30-s pulse to the cells before initiating the experiment.

All data are shown as the mean ± SE unless otherwise noted. For colocalization measurements, Nikon Elements Advanced Research imaging software was used to determine Pearson’s correlation coefficient. Data were analyzed using a Student's t test or one-way ANOVA with Tukey’s multiple comparisons test for post hoc analysis. Data are considered statistically relevant when p < 0.05. Statistical analysis was run on GraphPad Prism 9.0 (GraphPad Software). The specific tests used to determine significance for each figure are described in the figure legends.