Ixon ultra 897

All images were captured using an inverted fluorescence microscope (Eclipse Ti-E, Nikon) equipped with either an electron-multiplying charge-coupled device camera (ImagEM C9100-13, Hamamatsu; or iXon Ultra 897, Andor) or a scientific complementary metal-oxide-semiconductor camera (ORCA-flash 4.0 C11440-22C, Hamamatsu). We used a light-emitting diode light source (SPECTRA X Light Engine, Lumencor) to provide illumination with filter sets (from Semrock or Nikon): (i) excitation (Ex), 390/40 nm; dichroic, 405 nm; emission (Em), 452/45 nm (for DiFMU); (ii) Ex, 427/10 nm; dichroic, 458 nm; Em, 483/32 nm (for mseCFP); (iii) Ex, 480/40 nm; dichroic, 505 nm; Em, 535/50 nm (for mNeonGreen, and fluorescein); (iv) Ex, 504/12 nm; dichroic, 515 nm; Em, 542/28 nm (for mVenus); (v) Ex, 554/23 nm; dichroic, 573 nm; Em, 609/54 nm (for tdTomato, and mRuby2); and (vi) Ex, 630/38 nm; dichroic, 655 nm; Em, 694/44 nm. A 60× objective lens (Plan Apo VC; numerical aperture, 1.4; Nikon) was used for imaging experiments.

We modified a commercial cryo-FM system (Cryo CLEM; Leica) equipped with a 50× 0.9-NA objective lens (Cryo CLEM Objective HCX PL APO 50×/0.9; Leica) for cryo-SOFI imaging by coupling lasers (iChrome MLE; Toptica) into the microscope body for fluorophore excitation and photoswitching. Laser light after the single-mode fiber was collimated using an achromatic lens with 19-mm focal length to achieve an illuminated area of ∼40-µm diameter in the object plane. A schematic illustration of additionally required hardware is in SI Appendix, Fig. S1. For all biological examples presented here, data were acquired using 488-nm laser illumination and the standard GFP filter cube of the microscope system (excitation: 470/40; dichroic: 495 low pass; emission: 525/50). Overview images where recorded using a standard CCD camera (DFC365 FX; Leica). Cryo-SOFI data were recorded with an electron multiplying CCD (EMCCD) camera (iXon Ultra 897; Andor) and additional 2× magnification to reach an overall magnification of 100× for matching the larger pixel size of the EMCCD camera. Typical camera settings for cryo-SOFI data acquisition were 50-ms integration time per frame (at a rate of 20 frames per second) for a time series of 2,000 images at an EM gain of 20–200.

In this study, DF microscopy imaging was used under a Nikon inverted microscope (ECLIPSE Ti-U). In DF mode, the microscope utilized a Nikon Plan Fluor 100× 0.5–1.3 oil iris objective and a Nikon DF condenser. An Andor iXonEM + CCD camera (iXon Ultra 897) was employed to record DF images of Ag@AuNRs. The collected images were analyzed with the Image J software. Furthermore, DF scattering spectra were acquired with an Andor spectrophotometer (SHAMROCK 303i, SR-303I-A) and an Andor CCD camera (Newton DU920P-OE). When recording a spectrum, the scanning stage moved the sample to the desired location was collected by the objective. The scattered light was directed to the entrance of the spectrometer, dispersed by a grating (300 L mm⁻¹) and detected by the Newton CCD camera. The background was measured at a region without nanoparticles. Data analysis on the experimental data was performed by Matlab programs specially designed for this study.