We describe a new method for delivering macromolecules into the target cells based on light-absorbing cationic colloidal
gold nanoparticles that are irradiated by focused femtosecond laser pulses. Cationic colloidal 15nm gold particles 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 two-photon fluorescence microscopy, and lifetime images of the same targets were taken by TCSPC
technique in order to verify the fluorescence of the marker and the luminescence of the gold particles.
A macromolecular 10k Dalton fluorescein isothiocyanate dextran (FITC-D), was added into the sample and the focused
femtosecond laser of two-photon fluorescence microscopy was employed to scan the target cells layer by layer. Typical
laser power level used in biological imaging is about 3-5 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.
Due to the unique optical properties, gold nanoparticles (NPs) can play a useful role in biological cellular imaging as
biological probes. Using multiphoton microscopy and fluorescence lifetime imaging (FLIM) system, we recorded the
images of Karpas 299 cells incubated without, or with gold NPs, and ACT1 antibodies conjugated with gold NPs. From
the FLIM, we can easily discriminate the difference among different experiment conditions due to the distinct lifetime
between cells and gold NPs. Our results present that nonconjugated gold NPs are accumulated inside cells, but
conjugated gold NPs bind homogeneously and specifically to the surface of cancer cells. For single Karpas 299 cells, the
signal is very week when the excitation power is about 10mw; while the power is approximately 28 mw, a very sharp cell
imaging can be obtained. For the Karpas 299 incubated with ACT1 conjugated gold NPs, while the excitation power is
10mw, gold NPs have clear fluorescence signal so that the profile of cells can be detected; Signal of gold NPs is very
strong when the power arrived in 20mw. These results suggest that the multiphoton lifetime imaging of antibody
conjugated gold NPs can support a useful method in diagnosis of cancer.