Two-photon fluorescence imaging of living cells' anionic sites labeled by cationic colloidal gold and its nano-scale microeffect

Cationic colloidal gold (CCG) nanoparticles were used for labeling on the anioinic sites of living cells under two-photon fluorescence (TPF) microscope, and for delivering macromolecules into the target cells when irradiated by focused femtosecond laser pulses. 15 nm CCG nanoparticles which were made by conjugation with poly-L-Lysine, were attached on the anionic sites, especially on the membrane, of CHO-K1 cells because of their strong positive charge at physiological pH. Target cells labeled with cationic gold nanoparticles were imaged under TPF microscope, and lifetime images of the same targets were taken by time correlated single photon counting (TCSPC) technique in order to verify the fluorescence of the marker and the luminescence of the gold particles. The results shown that CCG nanoparticles first accumulated on the negatively charged sites of the membrane, then entered via endocytic pathway and attached anionic sites in plasma. A macromolecular 10 ku fluorescein isothiocyanate dextran (FITC-D) was added into the sample and the focused femtosecond laser of TPL microscope was employed to scan the target cells layer by layer. Typical laser power level used in biological imaging is about 3 similar to 5 mW. Here the laser power of scanning was below 5 mW in order to prevent photochemical damage of the fs-pulses alone and to localize effects to the nanoparticles on a nano-scale. After scanning the target cells under stack mode, macromolecular fluoresceins surrounding the cells was observed to cross the membrane and to diffuse in the cytoplasma. Comparing with the images before scanning, the two-photon fluorescence and fluorescence lifetime images revealed the delivery of FITC-D into target cells. Photothermal effects, which may be responsible for the permeabilisation, are highly localized in nanoscale and are not expected to cause damage exceeding the cell membrane. After extensive of laser scanning also cell death occurred. The ratio of the uptake of FITC-D and cellular death under different conditions were measured by flow cytometer. The results shown: with the increased scanning times or ratio of particles to cells, transfer efficiency increased first and decreased afterwards, but the ratio of cellular death went up all along.