Fv1000 mpe multiphoton laser scanning confocal microscope

All chemical reagents in the syntheses were purchased from Energy Chemical (Shanghai, China) Co., Ltd., and used without purification unless otherwise. Solvents were dried by standard methods prior to use. MitoTracker Green FM and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide were all obtained from Thermo-Fisher Biochemical Products (Beijing, China) Co., Ltd. ¹H NMR and ¹³C NMR spectra of synthetic intermediates and final probe were recorded on Agilent DD2 (Agilent Technologies, Santa Clara, CA, USA) (600 MHz, 150 MHz). Electrospray ionization mass spectrometry (ESI-MS) was operated on a Bruker maXis 4G mass spectrometer (Bruker Daltonik Gmbh, Bremen, Germany). pH measurements were made on a Sartorius PB-10 pH meter (Göttingen, Germany). UV-Vis absorption spectra were recorded on a SHIMADZU UV-1750 spectrophotometer (Kyoto, Japan). Fluorescence spectra were taken on a PerkinElmer LS-55 spectrofluorometric (Waltham, MA, USA). HeLa cells were cultured in a Thermo Forma, model 371 CO₂ incubator (Thermo Fisher Scientific, Waltham, MA, USA). The absorbance for an MTT assay was obtained using a Thermo Scientific Varioska Flash microplate reader (Waltham, MA, USA). Confocal microscopy fluorescence images and two-photon fluorescence images were taken using an Olympus FV1000-MPE multiphoton laser scanning confocal microscope (Olympus Co., Ltd., Nagano, Japan).

Pollen tubes were stained with the lipophilic dye FM4-64 (Invitrogen). The loading of pollen tubes with FM4-64 was achieved by direct addition of FM4-64 dye (5 μM in liquid pollen germination medium) on the surface of solid pollen germination medium. After incubation with FM4-64 solution for 15 min, images were captured with an Olympus FV1000MPE multiphoton laser scanning confocal microscope as described above. FM4-64 dye was excited with an argon laser at 546 nm, and the emission wavelength was set in a range of 600–650 nm.

Arabidopsis seedlings were imaged with an Olympus FV1000 MPE Multiphoton Laser Scanning Confocal Microscope (60× water-immersion objective; numerical aperture, 1.35). Both GFP and FM4–64 were excited using a 488-nm laser. The fluorescence emission spectra were separated with a 560LP dichroic mirror. GFP fluorescence was collected in the range of 495 to 540 nm, and that of FM4–64 was collected in the range of 570 to 650 nm. mRFP and mCherry were imaged by a 543-nm laser, and the emission fluorescence of mRFP was collected in the range of 580 to 620 nm and that of mCherry in the range of 600 to 650 nm. The colocalization analysis and determination of Pearson’s coefficient were done using the WCIF ImageJ intensity correlation analysis plugin (http://wwwfacilities.uhnresearch.ca/wcif/imagej) [25 (link)]. Images acquired were further processed using Adobe Photoshop, version 7.