Bx microscope

Cell samples (1 ml) were concentrated about 20x by centrifugation, prepared on an agar slab or a poly-lysine coated microscope slide and photographed within 30 min after sampling.
For the constructs FH4056, 4057, 4058, 4059, and 4060 pictures were taken on a Zeiss Axioplan microscope equipped with a Kappa DX 2 camera using an ImageBase capture program. For construct FH4035 an Olympus BX microscope was used in combination with programs ImageJ and MicroManager. Both microscopes had 100 × 1.3 oil immersion lens (PH3) giving a magnification of 15 pixels per μm. Four pictures of each field were obtained using the respective capture programs. Both microscopes had filters for detecting red, cyan, and green fluorescence.

Raman spectra acquisition during cell operation has been described in detail elsewhere and is schematically shown in Supplementary Figure 5 (ref. ^{39 (link)}). For the measurements, a special electrode was used which was prepared by mixing the active materials with super P and 5 wt% PTFE in water to obtain a homogenous slurry. The final content of the active materials was 80 wt%. The slurry was rolled into a thin film, which was then pressed onto a stainless-steel mesh. A delicate battery cell with a quartz window on the top was used (Supplementary Figure 5). The Raman spectra were recorded on a MicroRaman system (LabRAM HR spectrometer, Horiba) with an Olympus BX microscope and an argon ion laser (532.05 nm). Each spectrum was acquired for 20 s. The galvanostatic discharge of the cell was controlled by an electrochemical workstation (PARSTAT 4000). A similar battery cell to that used for the in operando Raman spectroscopy was designed, replacing the quartz window with a Kapton film window to perform the in situ XRD measurements (Supplementary Figure 8).

Zebrafish were euthanized in ice cold water according to the protocol outlined by Wilson and colleagues^{54 (link)}. The ventral abdomen of each fish was cut open to allow optimal fixation of organs. Fish were fixed whole in 10% neutral buffered formalin (NBF) for at least 24 h, embedded in paraffin, sectioned at 5um thickness, and stained with hematoxylin and eosin (H&E) or Periodic Acid-Schiff (PAS) at the NC State College of Veterinary Medicine (Raleigh, NC). Sagittal sections and step sections were analyzed for histopathology and morphometric studies. H&E staining was performed on four separate populations with an n = 3–10 for each diet depending on total population size. PAS staining was completed on n = 5 fish per diet across one population. For morphometry, H&E sagittal and step sections of pancreatic and subcutaneous fat were photographed at 10X objective using an Olympus BX microscope. The images of nine fish per diet were grouped by sex and specified fat depots were analyzed in Fiji (ImagJ version: 2.0.0-rc-64/1.51 s) following an adjusted version of the Parlee et al. protocol^{55 (link)}.

Heart tissues from all groups were collected, fixed in 4% formaldehyde, and embedded in paraffin. Then, thin 3 mm sections were prepared using a microtome and stained with hematoxylin and eosin (H&E) to examine the heart morphology. The morphology of the cardiac cells and the nucleus of myocardial fiber cells were compared using an optical microscope to evaluate the severity of cardiac damage (Olympus BX microscope and DP72 camera, Melville, FL, USA). The damage quantification from at least 10 areas corresponding to the myocardial tissue was graded using the following parameters: nuclear enlargement and inflammation based on a four-score evaluation system (0, histopathological changes = 1–25%; 1, histopathological changes = 26–50%; 2, histopathological changes = 51–75%; and 3, histopathological changes =76–100%). This procedure was conducted in at least 10 random areas in each heart section, in three animals from each group, at 400× magnification. The mean score for each parameter was calculated and subjected to statistical analysis. The cardiomyocyte cross-sectional size was estimated and evaluated using the H&E stained slides.

Liver paraffin sections prepared as above described, were used to detect cell apoptosis by the DeadEnd^TM Colorimetric TUNEL System (Promega, United States) according to the manufacturer’s protocol. The number of TUNEL-positive cells in 30 sections per slide was counted to estimate the level of liver tissue apoptosis at 200× magnification as described previously (Hu et al., 2013 (link)).
Liver paraffin sections were also used for immunohistochemistry staining as routinely processed (Zhou et al., 2016 (link)). The sections were blocked with 10% bovine serum for 30 min, and then incubated overnight at 4°C with cleaved caspase-3 (cat. no. 9664, Cell Signaling Technology, Inc., 1:100). Subsequently, the sections were incubated with goat Anti-Rabbit IgG H&L (Alexa Fluor^®488) (cat. no. ab150081; 1:2,000; Abcam). Finally, the immune-reactivity was visualized by staining with diaminobenzidine (DAB; D-5637, Sigma-Aldrich, Saint Louis, MO, United States) for 3 min and sections were counterstained with hematoxylin. All images were captured by using a light microscope (Olympus BX microscope, Shinzyuku, Tokyo, 200× magnification). Positively stained cells were observed and counted by the Image-Pro Plus image analysis management system (Media Cybernetics, Rockville, MD). Each slice was randomly selected five horizons to calculate positive cells.

A collimated light emitting diode (LED) with output power 650mW and peak emission wavelength of 470 nm (Thorlabs, Newton, New Jersey, USA) was mounted on the epi-illumination port of Olympus BX microscope. Light was passed through 40x Olympus objective. Field diaphragm in the epi-illumination pathway of the microscope was fully closed to achieve locality of stimulation. Analog output of Digidata 1440 ADC (Molecular Devices, USA) was fed either to the modulation input port of LED driver, or to the current input port of Dagan amplifier. Fluctuating voltage, generated by ADC output produced either fluctuation of light intensity, or fluctuation of the current passed through the cell membrane in experiments with intracellular current injection. For optically induced artificial EPSCs 2ms rectangular voltage pulse was applied to the modulation input port of LED driver.