The experimental set-up is schematically shown in . Picosecond diode pumped Nd:YVO4 laser (Laser 1 in ), picoTRAIN IC-10000 1064nm (HighQ Laser) with the pulse width ~10 ps and a repetition rate of 76MHz was used both as a source for ν1 incident wave. It also was used for synchronous pumping of Laser 2 - a tunable (781–923 nm) optical parametric oscillator Levante (APE) to produce another incident wave ν2 of ~10 ps pulse duration. Laser 1 and Laser 2 have been used separately for SHG signal generation at the sample. SFG and FWM signal generation require coherent mixing of ν1 and ν2 incident waves. For the coherent mixing process computer controlled delay line provided the temporal synchronization of picosecond pulses of the Nd:YVO4 laser and the OPO with a zero time jitter, and adjustable telescopes T1 and T2 ensured the beams focal point conjugation at the plane of the microscope specimen. Picosecond outputs of Laser 1 and Laser 2, coinciding in time and in space were directed to an inverted microscope TE2000-S (Nikon). A computer-controlled XY galvano scanner VM1000 (GSI Lumonics) insured fast laser beams scan along the sample in the lateral (XY) focal plane of the water-immersion objective O1, UPLSAPO 60XW, NA=1.2 (Olympus). The objective O1 was mounted on a computer-controlled piezo-stage (Piezosystem Jena) for an axial laser beam Z-scanning through the sample, with the minimum step of 0.1 nm. Splitting power ratio of Laser1 output between the pump power of Laser2 and the ν1 incident wave was controlled by the half-wave (λ/2) waveplate WP1. Polarizations of Laser 1 and Laser 2 were computer-controlled by rotating Glan-Thomson polarizers P1 and P2 and half-wave plate WP2. The SHG/SFG signals generated in the specimen plane were detected by a photomultiplier tube R928 (Hamamatsu Photonics), PMT1, in the reflection geometry; the narrow-bandpass barrier filter F1 cuts the fundamental frequencies ν1 and ν2 and separate 2ν1/2ν2 (SHG) or ν1 + ν2 (SFG) signals. The nonlinear FWM response at the frequency 2ν2 − ν1, generated by ZnO nanocrystals dispersion in forward direction, spectrally separated from ν1 and ν2 by a dichroic mirror, M11, and a barrier filter, F2, was detected by a photomultiplier tube R5108 (Hamamatsu Photonics), PMT2. A consistent operation of the optical scanner and acquisition system ensures digitization of the nonlinear signal at new frequencies 2ν1, 2ν2, (ν1+ν2) and (2ν2 − ν1) and generates the nonlinear optical images.
Figure 3 Optical setup of the laser scanning SHG/SFG imaging system. Laser 1 and Laser 2 are picosecond lasers with the outputs at ν1 and ν2, respectively; T1 and T2 are lens telescopes; WP1 and WP2 –λ/2 waveplates; M1 – (more ...)
Human nasopharyngeal epidermal carcinoma (KB) cells were chosen for imaging. The cell culture was plated overnight in 35 mm glass bottom cell dishes in a minimum essential medium (MEMα). with 10 % FBS and appropriate antibiotic, according to the manufacturers instructions (American Type Culture Collection). Next day, with the cells at a confluency of 70 %, the overnight media was aspirated and replaced with a fresh medium (2 mL/dish). To study the uptake, 200 µL of the aqueous dispersion of the ZnO nanoparticles (with and without incorporated folic acid) were added, mixed by gentle swirling, and replaced in the incubator at 37°C with, 5% CO2 (VWR Scientific, 2400). After 1 and 3 hours of incubation, the cells were rinsed with PBS and directly imaged.
In general, the light induced nonlinear polarization for a medium can be expressed by [14
is the induced polarization, χ(n)
is the nth
order of nonlinear susceptibility, and E
is the electric field vector of the incident light. The first term, χ(1)E1
, describes normal absorption and reflection of light, the second term , χ(2)E2
, describes SHG, SFG, and difference frequency generation, and the third term, χ(3)E3
, describes THG and TPEF, as well as FWM, including vibrational Coherent anti-Stokes Raman Scattering. In this article we present a demonstration of successful application of the second order SHG and SFG, and the third order FWM nonlinear phenomena in an aqueous dispersion of phospholipid micelle-encapsulated ZnO single nanocrystals for cellular bioimaging.
For nonlinear spectroscopy of ZnO nanocrystals, a sealed aqueous suspension of ZnO nanoparticles (~0.3% wt) between a glass slide and a cover slip sealed by Eppendorf* in situ Frame was used as a specimen. Two laser beams with required combinations of the laser frequencies ν1 and/or ν2 were introduced into the microscope. Laser 1 and Laser 2 were adjusted to approximately equal average power of 150 mW at the frequencies ν1 and ν2 in the focal plane of microscope objective O1. Computer controlled laser shutters and tunable OPO wavelength varied the combination of the frequencies at the specimen. Nonlinear signals, generated by ZnO nanoparticles suspension, were picked up on the location of detectors PMT1 and PMT2 by the input fiber of the spectrometer SpectraPro® 2500i (Acton Research) equipped with Spec-10:100B CCD (Princeton Instruments). Four types of nonlinear optical response of ZnO nanocrystals were detected by the spectrometer. The experimental data for the combination of incident wavelength λ1 = 1064 nm and λ2 = 850.6 nm are presented in . Narrow linewidth high efficient second harmonic output at 532 nm (2ν1) from Laser 1 () and at 425.3 nm (2ν2) from Laser 2 () were derived from the sample when the laser shutters were opened alternatively for Laser 1 or Laser 2. High intensive narrow line SFG signal at 472.7 nm (ν1 + ν2) was detected in the backward propagating direction () and FWM signal at 708.5 nm (2ν2 − ν1) was detected in the forward direction () when both shutters were opened. The second order susceptibility χ(2) characterizes the first three new wavelengths of SHG (532 nm, 425.3nm) and SFG (472.7 nm) output intensity emissions. The FWM intensity signal at 708.5 nm is characterized by the third order susceptibility χ(3) of ZnO nanocrystals. To compare the efficiency of these nonlinear outputs, a square area of the ZnO nanoparticles homogeneous suspension was scanned and the average image intensity for SHG, SFG and FWM was measured. The experimental signal intensities are shown in the . It is noteworthy, that all four new generated signals are intense enough to generate microscopy images. Moreover, the SFG/SHG output (SHG and SFG were not separated) is about twice higher than that of SHG when both laser shutters were opened and laser pulses were delayed in time for ~ 5 ps. Thus, SFG signal from ZnO nanoparticles can provide an opportunity for high contrast bioimaging by tuning on both beams through the near IR spectral region of maximum biological transparency.
Spectral distribution of SHG (a, b), SFG (c), and FWM (d) signals generated by the water-dispersed ZnO nanocrystals (λ1 = 1064 nm and λ2 = 850.6 nm).
Signal intensity distribution for SHG/SFG and FWM output.
The confocal SFG images of KB cells treated with the aqueous dispersion of ZnO and ZnO-FA (targeted by folic acid), for 1 and 3 hours, are shown in . It can be seen that while for the non-targeted ZnO the intracellular uptake is poor in the case of, 1 and 3 hours of treatment (), robust intracellular SFG signal is observed in the case of cells treated with ZnO-FA for 3 hours (). The nanoparticles seem to be distributed throughout the cytoplasm. The localized narrow spectrum, a laser like line, confirms the observed SFG from internalized nanoparticles. Besides SFG response, intensive FWM output was generated by ZnO nanoparticles in the forward direction of the incident beams. The corresponding images of FWM and SFG signals in the same scan are shown in and . We could clearly identify FWM emission (arrows in ) exactly from the same location where SFG signal was detected (arrows in ). However, in contrast to the SFG signals generated by ZnO nanoparticles, the FWM nonlinear response is generated both by internalized nanoparticles and cell compartment materials such as lipids, membranes, proteins, etc. This reduces the contrast of FWM imaging of ZnO nanocrystals and favors SFG for bioimaging application. To the best of our knowledge, this experiment is the first demonstration of targeted intracellular delivery of ZnO nanocrystals and of non-linear optical bioimaging using these nanoparticles. During our experiments with the maximum laser peak intensity of ~ 6 GW/cm2, scanning speed of ~ 0.1 m/sec and scanned area ~ 70 ×70 µm2, there was no evidence of photodamage of the cells found even after 50 sequential image scans. Using a cell viability (MTS) assay, no indication of cytotoxicity could be observed at the experimental dosage (data not shown), thus further highlighting the potential of ZnO nanocrystals as non-toxic nonlinear optical probes for diagnostic imaging.
Figure 5 The SFG and FWM nonlinear optical images of treated KB cells. The SFG images of KB cells treated by the ZnO nanoparticles non-targeted (a,c) and targeted with folic acid (b,d), after 1 (a,b) and 3 (c,d) hours of incubation. The intensity-coded SFG images (more ...)