Confocal fluorescence microscope

A SuperK EXTREME EXB-6 supercontinuum white light laser with a SuperK SELECT wavelength selector (NKT Photonics, Birkerød, Denmark) was used as the excitation source. Four excitation wavelengths were selected: 465 nm, 488 nm, 560 nm, and 633 nm, for Eu(III), Tb(III) and F18, MitoTracker Red, and ATTO647N, respectively. The laser powers with 77.88 MHz repetition rate for each wavelength were 2 μW, 7.2 μW, 1.2 μW and 2.9 μW, respectively. Shortpass or bandpass filters (Table 1) were added to the excitation light path.

SP, short pass; LP, long pass; BP, band pass

The home-built scanning fluorescence confocal microscopy setup was based on an Olympus IX71 inverted microscope with a piezo-driven scanning stage (P5173CL, Physik Intrumente, Karlsruhe, Germany), controlled by a home-written software program (LabView, National Instruments), allowing for point-by-point imaging of the sample in a raster scanning fashion in a range up to 100 μm × 100 μm. Upon laser illumination, the emission signal from the sample was collected by the same 100× oil immersion objective (Olympus UPLFLN 100×, 1.3 NA). A 70/30 beamsplitter (XF122, Omega Filters) was used in microscope instead of a dichroic mirror.

The emission light was focused though a 50 μm pinhole, directed through optical long pass filters (Table 1) and detected in a CCD-based spectrometer (Princeton Instruments SPEC-10:100B/LN_eXcelon CCD camera, SP 2356 spectrometer with 1-030-500 grating 300 g/mm @ 500 nm, all controlled by the same LabView program that controls the scanner). The X axis of the emission spectra was calibrated using emission lines of a neon lamp (6032 neon lamp, Newport Corporation, Irvine, CA). The Y axis (Intensity) was not corrected for differences in optical transmission and detection efficiency.