The microscopy samples were observed using an Elyra PS.1 SIM microscope (Zeiss) with objective lens alpha Plan-Apochromat 100x/1.46 oil. Chl, MitoTracker, and BODIPY were excited with 642, 561, and 488 nm laser, respectively, and fluorescence was acquired through a 655 nm longpass filter, a 570–620 nm bandpass filter, and a 495–550 nm bandpass filter, respectively. The image acquisition was done as fully controlled by ZEN software (Zeiss). One focal plane for each 3D-SIM image was obtained by sequential fluorescence image acquisitions via the excitation with the patterned light of 3 rotated angled, each of which contains 5 phases shifted. The z-interval distance was 101 nm. Raw images were processed to reconstruct 3D images using ZEN software. Extraction of the intensity data from each focal plane was performed using the SIMcheck plugin (Ball, et al. 2015 (link)) for ImageJ software (US National Institutes of Health). Peak fitting and FWHM measurements were done using the multipeak fit analysis packages in Igor Pro software (WaveMetrics).
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3D-SIM Microscopy Sample Imaging
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Corresponding Organization : University of California, Berkeley
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Variable analysis
independent variables
- Excitation wavelengths: 642 nm, 561 nm, 488 nm
- Fluorescence intensity
- Full width at half maximum (FWHM) of fluorescence peaks
- Microscope: Elyra PS.1 SIM (Zeiss)
- Objective lens: alpha Plan-Apochromat 100x/1.46 oil
- Fluorescence filters: 655 nm longpass, 570-620 nm bandpass, 495-550 nm bandpass
- Image acquisition: ZEN software (Zeiss)
- Image reconstruction: 3D-SIM with 3 rotated angled, 5 phase-shifted fluorescence image acquisitions, z-interval 101 nm
- Image analysis: SIMcheck plugin for ImageJ, multipeak fit analysis in Igor Pro
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