Plant Model System
Tomato seeds were surface-sterilized, germinated, and grown on sterile Murashige and Skoog (MS) medium as previously described (6). Thirty-day-old tomato plants (cv. Micro-Tom) with an average stem length of 5 to 10 cm were used in all experiments.
The kinetic of QD-CNT uptake from the water was monitored in vivo in intact tomato plants transferred from agar medium to a tube with a regular water. First group (3 plants) remained in the water throughout the entire experiment (control). A second group (9 plants) was used to monitor the flow of QD-CNTs inside the plants tissues (experimental plants). Control PA signals (in the absence of QD-CNTs) were acquired from control plants and from experimental plants before the introduction of nanoparticles. After the control recordings, 200 μl of 25 μg/ml QD-CNT solution were added to the water in a tube containing the plant (Fig. 1B) and PA signals were recorded for 1 h from the mid-vein of a leaf or from the main stem. Experiments were repeated for each tomato plant serving as independent biological replicate.
Accumulation of QD-CNTs in tomato plants was studied with a focus on leaf examination in 30-day-old plants grown for 10 days on a MS medium supplemented with QD-CNTs (at a concentration of 50 μg/ml). Leaves of tomato plants grown on a standard MS medium were used as a control (total six leaves from three different plants). For each leaf from one to three areas with a size of 500 × 500 μm were analyzed to estimate background PT/PA signals. A tomato leaf was prepared for microscope calibration by local injection of 30 μl of a 0.5 mg/ml QD-CNT solution into the leaf tissues. For imaging convenience whole tomato leaves were detached from a stem and deposited for observation on a glass slide under a coverslip (Fig. 3C).
Figure 3. In vivo integrated flow and scanning cytometry with PT, PA, and fluorescent detection schematics. (A) Schematic of the integrated setup. (B) Customized optical fiber tip for laser radiation delivery to plants. (C) Scanning cytometry in a leaf with the microscope schematic. (D) Optical fiber-based flow cytometry. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary. com.]
Download figure to PowerPoint
Integrated Plant Cytometer
The prototype of a plant cytometer (Fig. 3A) was built on the technical platform of an Olympus invert IX81 microscope (Olympus America, Inc., Center Valley, PA) with incorporated scanning PT/PA setup, PA flow cytometer, and conventional fluorescent wide-field imaging module.
Scanning PT/PA microscope-cytometer implemented a tunable optical parametric oscillator (OPO, Opolette HR 355 LD, OPOTEK, Carlsbad, CA) with the following parameters: tunable wavelength range, 420 to 2,500 nm; pulse width, 5 ns; pulse repetition rate, 100 Hz; and beam diameter on sample, 1.2 μm. XY translation stage (H117 ProScan II, Prior Scientific, Rockland, MA) was used for raster scanning of the plant sample (Fig. 3C). The PT (also referred to as thermal-lens) effect was manifested by defocusing of a probe He-Ne laser beam (wavelength, 633 nm; power, 1.4 mW; model 117A, Spectra-Physics, Santa Clara, CA) at a photodetector (PDA36A, 40 dB amplification, ThorLabs, Newton, NJ) pinhole plane after the sample. The PT signal had the linear positive asymmetric component associated with fast heating and slower cooling effects and a nonlinear sharp negative peak associated with nano-bubble formation around the overheated zones (Fig. 4C) (22). Laser-induced acoustic waves in the sample were detected by an ultrasound transducer (XMS-310, Panametrics-NDT, Olympus NDT, Center Valley, PA) and amplified (preamplifier model 5662B; bandwidth, 50 kHz–5 MHz; gain 54 dB; Panametrics NDT, Olympus NDT Inc., Center Valley, PA). The transducer was placed directly onto a sample with water layer used to improve acoustic coupling. The PA signal had a classic bipolar shape transformed into a pulse train due to reflections and diffraction effects (Fig. 4D).
Figure 4. In vitro detection and imaging of individual QDs, CNTs, and QD-CNTs. (A) Transmission, PT, and fluorescent imaging of QD-CNTs. Calibration graph represents PT signals and fluorescence intensity for QD-CNTs aggregates of various sizes. Laser parameters: energy fluence of 0.05 J/cm2, wavelength 903 nm, scan step 1 μm. Scale bar: 5 μm. (B) Line profile for fluorescent intensity of 5 μm QD and QD-CNT clusters. (C) PT and (D) PA signals from QD, CNTs, and QD-CNT clusters. Laser parameters: 903 nm, 0.05 J/cm2. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Download figure to PowerPoint
In vivo PA plant flow cytometer was equipped with a high pulse rate Yb-doped fiber laser (MOPA-M-10, MultiWave Photonics, Portugal) with the following parameters: wavelength, 1,064 nm; pulse width, 10 ns; pulse repetition rate, 10 kHz to 0.5 MHz; energy fluence range on sample surface, 0.01 to 1.0 J/cm2. Laser radiation was delivered either through the microscope based setup described above or through a 400 μm multimode fiber (M28L05, Thorlabs, Newton, NJ) having a custom miniature tip with cylindrical optics (Fig. 3B). Both the transducer and the optical-fiber tip were fixed in a holder and were gently touching the plant stem (Fig. 3D). For both the microscope based setup and for the fiber tip the laser beam spot in the sample had linear shape with the dimensions of 200 × 150 and 50 × 400 μm, respectively.
The analysis of PT and PA signals was performed by a PC (Dell Precision 690) equipped with a high-speed (200 MHz) analog-to-digital converter board PCI-5124 (National Instruments, Austin, TX), which was used to acquire signals from the transducer and photodiode. Control of the setup and signal acquisition/procession was realized via custom software module (LabView 8.5 complex, National Instruments, Austin, TX). PT/PA images were constructed by plotting PT/PA signals in a XY coordinate plane with the grey shading. All the PT/PA signals with amplitudes not exceeding 5σ level (five times the standard deviation of the background signal in the absence of the excitation beam) were plotted in a black color.
Fluorescent wide-field imaging of the QDs was performed with the following filter: excitation 450 ± 40 nm, emission 600 ± 8 nm, and a single band dichroic mirror 510 nm (Semrock, Rochester, NY). Exposure time of fluorescent imaging was 200 ms for leaves and 5 s for roots. For leaves this exposure time corresponded to a “black” image of a control plant leaf (the maximal image pixel intensity below 5/256 level in a greyscale mode). This exposure duration was selected experimentally as the shortest one from a set of leaf images taken from five different control plants. The fluorescent spectra of the samples were obtained by spectrophotometer (USB4000, Ocean Optics, Dunedin, FL) with a fluorescent filter featuring a long-pass emission filter transmitting light with wavelengths longer than 600 nm (Semrock, Rochester, NY). The optical transmission and fluorescent images of selected plant parts were obtained with a color CCD camera (DP-72, Olympus, Center Valley, PA).