Imaging of cancer cells by multiphoton microscopy using gold nanoparticles and fluorescent dyes

Abstract

Due to their unique optical properties, optical probes, including metal nanoparticles (NPs) and fluorescent dyes, are increasingly used as labeling tools in biological imaging. Using multiphoton microscopy and fluorescence lifetime imaging (FLIM) at 750-nm excitation, we recorded intensity and FLIM images from gold NPs (30 nm) and the fluorescent dye Alexa 488 (A488) conjugated with monoclonal ACT-1 antibodies as well as Hoechst 33258 (H258) after incubation with the lymphoma cell line (Karpas-299). From the FLIM images, we can easily discriminate the imaging difference between cells and optical probes according to their distinct fluorescence lifetimes (cellular autofluorescence: 1 to 2 ns; gold NPs: <0.02 ns; A488: 3.5 ns; H258: 2.5 ns). The NP-ACT-1 and A488-ACT-1 conjugates were bound homogeneously on the surface of cells, whereas H258 stained the cell nucleus. We demonstrate that the emission intensity of gold NPs is about ten times stronger than that of the autofluorescence of Karpas-299 cells at the same excitation power. Compared with fluorescent dyes, stronger emission is also observed from gold NPs. Together with their high photostability, these observations suggest that gold NPs are a viable alternative to fluorescent dyes for cellular imaging and cancer diagnosis.

Nonlinear plasmonic imaging techniques and their biological applications

“…The contrasts are based on (D1) intensity, and (D2) fluorescence lifetime. In (D2), the gold nanoparticles (yellow) are clearly distinguishable from the dye (blue-green color) Reprinted with permission from ref [127]. (E) Near-field fluorescence imaging with a gold nano dimer as localized FWM photon source.…”

“…An interesting possibility is to adopt luminescence lifetime measurement, because, compared to cellular autofluorescence or dye molecule fluorescence, gold nanoparticles have much shorter PL lifetime. Figure 3D presents the two-photon intensity ( Figure 3D1) and fluorescence lifetime ( Figure 3D2) images of lymphoma cells labeled with gold nanoparticles (yellow color) and H258 dye (blue-green color) at the membrane and the nuclear region, respectively [127], manifesting their clear distinction in lifetime domain.…”

Non-linear processes in the interaction of atoms and molecules with intense EUV and X-ray fields from SASE free electron lasers (FELs)

“…This field is so well established that it is becoming applications directed and hugely important in biomedical 'theranostics' with real societal benefits such as oncological multiphoton imaging for cancer cell detection [102] and therapeutic intervention [103], not to mention its application in sub-cellular imaging [104]. This 'conventional non-linear optical' domain concerns only the response of valence or outer-shell electrons driven by intense and coherent optical laser fields -in effect non linear optics has to date been limited to accessing only weakly bound electrons.…”

Imaging Gold Nanorods by Plasmon-Resonance-Enhanced Four Wave Mixing

“…In the linear regime, plasmon scattering and absorption have been utilized for dark field microscopy 1,2 and photoacoustic tomography. [3][4][5] In the nonlinear regime, multiphoton luminescence has been recently observed from gold nanospheres, 6,7 nanorods, 8 nanoshells, 9 and nanowires. 10,11 Despite the brightness of the multiphoton luminescence, its spectral profile is generally broad and may overlap with fluorescence from other molecules in bioimaging study, which makes it hard to distinguish gold particles from other fluorescent molecules.…”