W objective

Cytoplasmic [Ca²⁺] imaging was performed using the customized fluorescence microscope with a 40×/0.8 W objective (Olympus) for time-lapse imaging and an Optoscan monochromator (Cairn Research Ltd.) as a light source. Cells were stained with 5 μM Fura2-AM in Tyrode’s solution for 30 min at 37 °C. Then, Fura2-AM was washed out using Tyrode’s solution. The pseudocolor of the 380-nm channel is red (excited by 380-nm light with 10-nm bandwidth and collected with 505–535-nm bandpass filter), and the 340-nm channel is green (sequentially excited by 340-nm light with 10-nm bandwidth and collected with 505–535-nm bandpass filter). Ratiometric values of the 340-nm channel to the 380-nm channel were calculated pixel by pixel to represent the relative [Ca²⁺] of the sample. The other conditions of imaging, data acquisition and analysis for the cytoplasmic [Ca²⁺] study were the same as the conditions in the redox study.

Redox (FAD/FAD+NADH) imaging was performed using a customized fluorescence microscope with a 40×/0.8 W objective (Olympus) for time-lapse imaging. The endogenous autofluorescence images^{16 (link)} were excited with an Optoscan monochromator (Cairn Research Ltd., Kent, UK). The pseudocolor of the FAD channel is red (excited by 430-nm light with 20-nm bandwidth and collected with 525–575-nm bandpass filter), whereas the NADH+FAD channel is green (sequentially excited by 340-nm light with 20-nm bandwidth and collected with 420-nm long pass filter). In addition, the fluorescence images were acquired with an Evolve 512 EMCCD (Photometrics Ltd., Tucson, UK). Time-lapse imaging of BA was performed in 4 ml of Tyrode’s solution at 33 °C. Sixty frames were recorded in a time-lapse imaging at 30-s intervals. NE (0.1 μM) was injected as early as the 11th frame of time-lapse imaging for the redox studies in BA. The other conditions of imaging, data acquisition and analysis for the redox study were the same as the conditions in the thermogenic study.

Redox (FAD/FAD + NADH) imaging was performed using a customized fluorescence microscope with a 40/0.8 W objective (Olympus) for time-lapse imaging. The endogenous autofluorescence images were excited with Optoscan monochromator (Cairn Research Ltd., UK). FAD channel is excited by 430 nm light with 20 nm bandwidth and collected with 525–575 nm band pass filter whilst NADH + FAD channel sequentially excited by 340 nm light with 20 nm bandwidth and collected with 420 nm long pass filter. The fluorescence images were acquired with Evolve 512 EMCCD (Photometrics Ltd., UK). Time-lapse images of the cells were performed in 4 mL Tyrode’s solution at 33 °C. MATLAB (MathWorks Inc. USA) and ImageJ (NIH, USA) were applied to analyze images.