Ff01 593 40

For multicolour luminescence imaging, five images were acquired with Semrock FF01-447/60 filter (for Nluc), Olympus BA460-510CFP (for CeNL), Semrock FF01-525/35 filter (for GeNL), Semrock FF01-562/40 (for OeNL) and Semrock FF01-593/40 (for ReNL). Five HeLa cells (expressing each fusion construct) were imaged with identical conditions to determine the coefficients for linear unmixing. The signals from NLuc, CeNL, GeNL, OeNL and ReNL were then separated by linear unmixing using these coefficients by PrizMage software (Molecular Devices).

FA measurements were performed on a home-built dual-channel confocal fluorescence microscope based on a TE2000 microscope (Nikon) equipped with a 532 nm solid-state laser (MLL-III-532, CNI) (42 (link)). The laser beam was vertically polarized by a polarizer and then focused inside the sample solution, through an oil immersion objective (NA 1.4, 100×, Nikon), 10 μm above the glass surface. The laser power was 300 μW. The fluorescence was separated from the excitation light by a dichroic mirror (Z532, Chroma). After being focused through a 30 μm pinhole, the fluorescence was separated into p-polarized and s-polarized components with a polarized beam splitter (PBS) (Daheng, China). Each component was detected by a photon-counting avalanche photodiode (APD) (SPCM-AQRH-14, Perkin-Elmer Optoelectronics) after passing through a filter (Semrock FF01-593/40). Fluorescence intensities were recorded with a photon counters card (PMS-400A, Becker & Hickl) in 100 ms bin time. The raw data was converted to r(t) with 1 s bin time using the following equation: where I_p(t) and I_s(t) are the fluorescence intensities of p-polarized and s-polarized components, respectively, in each 1 s bin.

A custom-built TIRF microscope was used, as described previously.^{19 (link)} Briefly, the beam from a 100 mW, 488 nm laser or 150 mW, 561 nm laser (Light HUB-6, Omicron, Germany) was expanded using a Galilean beam expander and focused at the back focal-plane of a high numerical aperture, oil-immersion, objective lens (PlanApo, 100×, NA 1.45, Olympus, Japan) using a small, aluminium-coated mirror (3 mm diameter, Comar Optics, UK) placed at the edge of the back-aperture of the objective lens. The average laser power at the specimen plane was adjusted to ∼0.5 μW μm⁻² and the incident laser beam angle was adjusted to ∼63° to create the evanescent field at the glass–aqueous medium interface. A second small mirror was placed at the opposite edge of the objective lens back-aperture to remove the returning (internally-reflected) laser beam from the microscope and a narrow band-pass emission filter FF01-525/50 or FF01-593/40 (Semrock, Rochester, NY) was used to block the scattered 488/561 nm laser light and other unwanted light. An EMCCD camera (iXon897BV, Andor, UK) captured video sequences at a rate of 20–50 fps, and the data were stored on a computer hard drive for analysis.