Fgs900s

Imaging was done using bright-field on a home-built microscope coupled to a laser source. A commercial light-emitting diode (λ = 505 nm THORLABS) with a diffuser (ground glass N-BK7 600 grit, THORLABS), a condenser and an iris, to achieve Köhler illumination. The scattered light was picked up by the microscope objective (HCX PL APO ×40 NA = 0.85, LEICA), and a tube lens (B&H), and detected by a digital camera (DCC1545M, IMAGING SOURCE) and acquired using commercial video recording software (IC CAPTURE, IMAGING SOURCE). A heating beam was introduced on a separate optical path (see Supplementary Information). A 1064 nm laser beam (YLR-10-1064-LP, IPG Photonics) was passed through a zero-order half-plate (WPH05M-1064 Thorlabs) and contracted using a custom Galilean telescope to achieve a ~300 μm beam (see Supplementary Information). The laser beam was introduced into the sample using a polarizing beam splitter (PBS CM1-PBS253 THORLABS) and its intensity at the sample was controlled by a combination of the electronic laser head controller and by adjusting the half-plate, and was measured using an optical power meter (PM100D power meter, with S175C sensor, THORLABS). In order to eliminate laser intensity before the camera, stained glasses (FGS900S, THORLABS) were stacked after the objective.

Optical access through the thermal shields is necessary to determine periods during which the flies are at rest, so that the basal heat production can be extracted. However, opening holes in the thermal shields also permits undesirable thermal coupling between the ambient environment and the calorimeter via radiation exchange. To reduce thermal drift in the heat production signal, we use glass bandpass filters (Thorlabs, Inc. FGS900S) as optical windows. These filters transmit in the wavelength range 315–710 nm but absorb longer-wavelength infrared radiation (which primarily contributes to thermal exchange) before it reaches the sensitive thermometry in the calorimeter. The windows were diced and epoxied (3 M Scotch-Weld Epoxy Adhesive 2216 B/A) into narrow viewports to limit the view factor from the calorimeter to the environment.
To perform our imaging, we use low intensity 632 nm LED illumination. Two CCD cameras are positioned above the outer thermal shield to collect images of the illuminated flies during the experiment. Each camera is responsible for collecting light from five calorimeter tubes simultaneously, and the images are logged at two frames per second using LabView software. The resulting images are post-processed using a custom-developed algorithm that estimates the fly center of mass for each individual frame so that activity level can be quantified.

Imaging is performed on a custom-built, bright-field microscope coupled to a laser source. A commercial light emitting diode (λ = 505 nm Thorlabs) with a diffuser (ground glass N-BK7 600 grit, Thorlabs), condenser, and an iris are used to achieve Köhler illumination. The scattered light is picked up by the microscope objective (HCX PL APO 40x NA = 0.85, Leica) and a tube lens (B&H), detected by a digital camera (DCC1545M, Imaging Source), and acquired using commercial video recording software (IC Capture, Imaging Source). A laser beam was introduced on a separate optical path (see Supplementary Fig. 1a). A λ = 1064 nm laser beam (YLR-10-1064-LP, I.P.G. Photonics) passes through a zero-order half-wave plate (WPH05M-1064 Thorlabs) and is contracted using a customized Galilean telescope to achieve a wide beam (Supplementary Fig. 1b). The laser beam is introduced into the sample using a polarizing beam splitter (PBS CM1-PBS253 Thorlabs). Its intensity at the sample is controlled by a combination of the electronic laser head controller and adjustment of the half-plate. The intensity is measured using an optical power meter (PM100D power meter, with S175C sensor, Thorlabs). In order to eliminate laser intensity before the camera, stained glasses (FGS900S, Thorlabs) are stacked after the objective.