Hal100

The SLIM add-on module is mounted to the output camera port of a commercial phase-contrast microscope. The module contains an SLM (Meadowlark, XY Series) and a camera (Hamamatsu, ORCA-Flash 4.0 V2). Measurements were conducted using an inverted microscope (Zeiss, Axio Observer Z1) with a halogen light source (Zeiss, HAL 100). Cells were imaged with an incubation system under ×63/1.4 NA, ×40/0.75 NA, and 10×0.3 NA objectives with matching phase-contrast illumination. The sampling was uniform in the x, y, and z directions. For example, for a ×40/0.75 NA objective, the distance is 0.14 μm, which is smaller than the diffraction-limited resolution of 0.4 μm.
The 40-ms reconstruction per frame is faster than the SLIM image acquisition rate of 180 ms, which requires 30 ms for SLM stabilization (Meadowlark XY Series), 10 ms for exposure (Hamamatsu, V2 Orca Flash), and 60 ms for z scanning. We expect the technique to be able to be implemented on faster hardware without modification.

The extent of apoptosis in the damaged tissues was assessed using the TUNEL assay. For this purpose, we used the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Chemicon, CA).
The rats were sacrificed and fixed by cardiac perfusion with 4% paraformaldehyde at 24 h after I/R modelling. The brain tissue was isolated and fixed in 4% paraformaldehyde for 3 days. The fixed brain tissues were then sectioned coronally (thickness, 3 μm) and mounted on prechilled glass slides coated with poly-L-Iysine. Tissue sections were incubated in a dry oven for 1 h at 60°C. The tissues were treated with a working solution containing reaction buffer and enzyme (7:3) mixture for 1 h at 37°C, followed by an anti-digoxigenine peroxidase conjugate for 30 min at room temperature. The tissues were then treated with the DAB substrate (1:50) for 5 min in a dark room, followed by haematoxylin for 2 minutes in the dark room for cell staining.
The TUNEL-stained sections were examined under a microscope (ZEISS, HAL100, 200× magnification) and photographed. A brown stain in the nucleus represents an apoptotic cell. The number of TUNEL-positive cells and total cells were counted using ImageJ software in 3 randomly chosen fields from the ischemic core and peri-infarct regions. The percentage of TUNEL-positive cells relative to the total cell count was used to evaluate the apoptosis rate.

The spatially resolved reflectance spectrum was measured by using an optical microscopic spectrum system, which included an optical microscope (Zeiss Axio Imager. A2m) equipped with a halogen lamp (Zeiss Hal 100, 12 V, and 100 W), and a spectrometer (Horiba Jobin Yvon Triax 320). One end of an optical fiber was placed at the image plane of the microscope to selectively couple part of the light signal there to the spectrometer. The diameter of the optical fiber is 9 μm.

After 14 days in differentiation medium, mutant and WT myotubes cultures in petri dishes were placed on a 37 °C heated stage (ThorLabs TC200) on an upright phase-contrast microscope (Zeiss Hal 100). Myotube contraction was elicited using silver electrode-driven, 1 V broad field stimulation at frequencies of 0.3, 0.5, 1.0, 2.0 and 4.0 Hz for 15 s. Myotube contractions were captured using a high-speed acquisition digital camera (Hamamatsu model C8484-05G) from a selected region of interest (ROI). This procedure is described in detail by Santhanam et al.^{57 (link)}. Contraction synchrony was calculated by dividing the number of contractions by the total number of expected contractions at a given frequency.

After euthanizing guinea pigs and calves, the muscle tissue of immunization sites, lung, and small intestinal tissue were collected and fixed in 10% buffered neutral formalin for 12 h. Depending on the tissue, different concentrations of ethanol (100%, 75%, 50%, and 25%) were used for dehydration treatment for 30 min, and the ethanol and xylene mixture (1:1) liquid was transparently treated. The tissue was treated overnight with paraffin and xylene mixture (1:1) embedded in paraffin. After cutting the tissue into 7 μm sections and staining with hematoxylin and eosin, the pathological and the morphological changes in each tissue were observed under ZEISS microscopy (ZEISS, HAL100, Gottingen, Germany).