Tcs sp8 mp flim

Cells were seeded onto glass coverslips and then treated with CuE as indicated concentrations for 6 h, fixed in 4% paraformaldehyde for 30 min, and then permeabilized with 0.1% Triton X-100 for 30 min. After washing with PBS, cells were blocked with 5% BSA for 1 h at room temperature and probed with primary antibodies overnight at 4 °C. Cells were incubated with secondary antibody conjugated to rabbit Dylight-594 for 2 h at room temperature. Images were captured (594/618 nm for rabbit Dylight-594) using a confocal laser scanning microscope (TCS SP8 MP FLIM, Leica, Germany).

Multiphoton FLIM imaging (2D 2PEF FLIM) of synthetic melanin and NHMK coculture was performed with a commercial microscope (Leica TCS SP8 MP FLIM, Leica, Germany) integrating a time-correlated single photon counting (TCSPC) PicoHarp 300 module (Picoquant, Germany) and an 80 MHz IR fs pulsed laser (Newport Chameleon Ultra II, 680–1080 nm). Images were acquired upon 760 nm excitation and detection in the 410–650 nm range using a 40×/1.1 NA water immersion objective. Images of 205 × 205 µm² (512 × 512 pixels × 3128 time channels; 4 ps/time channel, 0–12.5 ns time range, position of the peak of the intensity decay at 1.33 ns) were acquired with 1.2 µs pixel dwell time, 50 frame accumulation number at 2 mW for melanin solution, and respectively 200 for NHMK coculture at 5 mW.

Lucifer yellow CH (Sigma, St. Louis, MO, USA) was selected as a fluorescent tracer for in vivo measurements from a series of fluorescent tracers, including dextran-tetramethylrhodamine (Invitrogen, Carlsbad, CA, USA), acridine orange (Sigma), and Gd-DO3A-ethylthiourea-FITC. A concentration of 10 mmol/L Lucifer yellow CH was diluted with 154 mmol/L artificial cerebrospinal fluid; the procedure for stereotactic injection was similar to that used for Gd-DTPA. Rats were perfused transcardially with 300 mL physiological saline for 15 min, followed by 150 mL 4% paraformaldehyde. Brains were removed and post-fixed in 4% paraformaldehyde (6 h, 4°C). Brain slices were made along the connection direction of caudate nucleus and thalamus.
The Leica TCS SP8 MP FLIM new Merging function can store m × n single images during the process of scanning. The whole image of the sample in the designated area can then be obtained by image reconstruction after scanning. Experimental fluorescence images were collected by the LSCM Merging function. LSCM parameters: objective, 5× and 25×; LY wavelength: λEx = 488 nm, λEm = 530 ± 20 nm.

Lucifer yellow CH (Sigma, St. Louis, MO, USA) was selected from a series of fluorescent tracers [5 (link),6 (link)], including dextran-tetramethylrhodamine (Invitrogen, Carlsbad, CA, USA), acridine orange (Sigma), and Gd-DO3A-ethylthiourea-FITC, for in vivo measurements. The Leica TCS SP8 MP FLIM new merging function can store m × n single images during scanning. The whole image of the sample of a designated area can then be obtained by image reconstruction after scanning. Experimental fluorescence images were collected with the LSCM Merging function [6 (link),9 ].

The cells were seeded onto glass coverslips coated with poly-L-lysine for 24 h. Subsequently, the cells were transiently transfected with appropriate plasmids for 48 h. After transfection, the cells were treated with or without PTA (20 μM) as indicated. The cells were fixed in 4% paraformaldehyde for 20 min and then permeabilized with 0.5% Triton X-100 for 30 min. After washing with PBS, the cells were blocked with 5% BSA for 1 h at room temperature, washed 3 times with PBS, and probed with primary antibodies overnight at 4 ºC. The cells were incubated with the secondary antibody conjugated to rabbit Dylight-594 or mouse Alexa Flour-488 for 1 h at room temperature. Images were captured (594/618 nm for Rabbit Dylight-594; 488/519 nm for mouse Alexa Flour-488) using a confocal laser scanning microscope (TCS SP8 MP FLIM, Leica, Germany).

ImDCs and mDCs were cultured on poly L-lysine-treated coverslips and stained with rhodamine phalloidin as mentioned above. The nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI, Beyotime Biotech., Shanghai, China) for 5 min. After washing with PBS, the cells were observed under a laser scanning confocal microscope (Leica TCS SP8 MP FLIM, Wetzlar, Germany).