W1 spinning disk

HADHA Mut and WT CMs were plated following lactate enrichment at 20,000 cells per Matrigel-coated well in a 24 well, glass bottom plate (Cellvis) and treated with Glc + FA medium for 12 days. Cells were stained using 4.5 mM Rhod-2 (Thermofisher R1244) in DMSO and 2 nM Mitotracker green (Thermofisher M7514) in DMSO for 30 min^{93 (link)}. Cells were rinsed with PBS and returned to culture medium for imaging on the heated, 5%CO2 stage of an inverted Nikon eclipse Ti equipped a Yokogawa W1 spinning disk. Colocalization analysis was performed as previously described^{92 (link)}.

A single section containing both the dorsal and ventral (Nucleus Accumbens, NAc) striatum was used to examine DA terminals unilaterally. The position chosen corresponded to approximate bregma 0.74 vmm in an adult mouse brain. A site 1 mm below the olfactory tubercule within the NAc shell was chosen as this represents a clear projection target of DA neurons within the VTA with no overlap from projections from DA neurons within the SN. A site 1 mm medial from the most lateral point of the striatum was selected as the region within the dorsal striatum to be examined as this represents projections of DA neurons from the SN with little overlap from DA neuron projections from the VTA. The mean of five non-overlapping images (128 × 128 µm) (centre, top, bottom, left and right) of each of the two regions was scanned using a Zeiss Axio Observer Z1 equipped with a W1 spinning disk (Yokogawa) under a ×100 objective lens (1.4 NA) (refer to Supplementary Fig. 4). Huygen Professional software (Scientific Volume Imaging, Netherlands) was used to deconvolve images. Deconvolved TIFF images were then analysed using Imaris software (Bitplane, version 9.4). High-probability axonal DA release sites were identified as Bassoon+ spots within TH varicosities^{19 (link),20 (link)}. One NAc sample was damaged during tissue processing and so could not be assessed.

This assay was performed similarly to the method described in [71 (link)]. In brief, the S. aureus biofilms formed on day 5 at the bottom of a flat-bottom 12-well microplate (Corning, Incorporated, Kennebunk, ME, USA) were washed twice with PBS and stained with 3.3 µM SYTO 9 (Invitrogen, Life Technologies, Carlsbad, CA, USA) and 2 µg/mL propidium iodide (PI; Sigma, St. Louis, MO, USA) and kept in the dark at RT for 20 min. Following the incubation, the stained biofilms were washed with PBS and fixed in 4% paraformaldehyde for 40 min. After fixation, the biofilms were washed again with PBS and mounted with 1 mL of 50% glycerol in DDW. The stained biofilms were visualized under a Nikon Spinning Disk confocal microscope (SDCM) (Nikon Corporation, Tokyo, Japan) connected to Yokogawa W1 Spinning Disk (Yokogawa Electric Corporation, Tokyo, Japan). Imaris software (Imaris version 9.8.0., Oxford Instruments, 2021, Abingdon, UK) were used to construct three-dimensional (3D) image of the biofilms. More than three random fields from each sample were analyzed.

Approximately 20 gravid adults were dissected in M9 medium under a stereo microscope. Embryos were transferred to individual wells in a Thermo Scientific Nunc MicroWell 384-Well Optical-Bottom Plate (Thermo Scientific). Embryos were imaged using an Olympus spinning disk confocal based on an Olympus IX3 Series (IX83) inverted microscope, equipped with a dual-camera Yokogawa W1 spinning disk (Yokogawa Electric Corporation) and two ORCA-Flash 4.0 V3 Digital CMOS cameras (Hamamatsu). Each field was imaged using a 40×/0.75 NA (air) objective, 16 z-sections at 2 µm and conditions were as follows: bright-field (100% power 30 ms) 568 nm, (100% power, 500 ms). Image acquisition was performed using CellSense software (Olympus). Image processing and montages were created using Fiji and embryoCropUI^{78 (link)}.

The CLSM was performed to determine the biofilm depth, the presence of extracellular polysaccharides (EPS), and the amounts of live/dead bacteria. To label the EPS in the biofilms, 1 µl of a 1 mM Alexa Fluor 647-labeled Concanavalin A (ConA) conjugate solution (Molecular Probes, Life Technologies, Carlsbad, California, USA) was added to the samples during the incubation period with or without EGCG. After incubation, the biofilms were washed twice with 200 µl PBS and stained with 50 µl of live/dead (SYTO 9/propidium iodide (PI)) BacLight fluorescent dye (Molecular Probes, Life Technologies, Carlsbad, California, USA) for 20 min in the dark at RT. Live bacteria showed green fluorescence, while dead bacteria emitted red fluorescence. The stained biofilms were inspected under a Nikon Spinning Disk microscope (Nikon Corporation, Tokyo, Japan) connected to Yokogawa W1 Spinning Disk (Yokogawa Electric Corporation, Tokyo, Japan) [37 (link)]. Optical sections were acquired at spacing steps of 5 μm intervals from the surface through the depth of the biofilm. A three-dimensional image of the microbes and EPS distribution within the biofilms was constructed using the Nikon Imaging Software (NIS- Elements). The NIS elements software was used to quantify the fluorescence intensity in each biofilm layer.