Fig. 5: Comparing bioluminescence and fluorescence sensitivity and observing PINK1 subcellular dynamics.

a–d, The fluorescence signal from MEFs expressing a Gamillus–NLuc fusion protein imaged at different dox levels. Exposure time, 0.3 s. The same image contrast settings were applied to each image. e–h, The bioluminescence signal from the same cells imaged in a–d. Exposure time, 0.3 s. Substrate, Nano-Glo Live Cell Substrate. The same image contrast settings were applied to each image. i, The change in bioluminescence intensity of a representative cluster of PINK1–HiBiT/LgBiT cells following CCCP injection. The black arrow indicates the time point of CCCP addition. Substrate, Nano-Glo Vivazine Substrate. j,k, Denoised images of HEK293T cells expressing PINK1–HiBiT/LgBiT without (j) and with (k) 10 μM CCCP treatment (5 h) measured on the EMCCD/LV200. Inset shows a zoomed-in image of one cell. Image contrast settings were adjusted to optimally show bioluminescence localization. Substrate, Nano-Glo Live Cell Substrate. l, The intensity profile of the dotted line in k is plotted. m,n, Denoised images of PINK1–HiBiT/LgBiT cells without (m) and with (n) 10 μM CCCP treatment (5 h) measured on the QIScope/6.5×. Inset shows a zoomed-in image of one cell with the same dimensions as the inset in k. Image contrast settings were adjusted to optimally show bioluminescence localization. Substrate, Nano-Glo Live Cell Substrate. o, Intensity profile of the dotted line in n is plotted. Effective pixel size of a–h, 423 nm. Effective pixel size of LV200/EMCCD, 800 nm. Effective pixel size of QIScope/6.5×, 169.2 nm. Representative results are shown from two to five independent experiments.