Wide-field imaging and flow cytometric analysis of cancer cells in blood by fluorescent nanodiamond labeling and time gating

Abstract

Nanodiamonds containing high density ensembles of negatively charged nitrogen-vacancy (NV(-)) centers are promising fluorescent biomarkers due to their excellent photostability and biocompatibility. The NV(-) centers in the particles have a fluorescence lifetime of up to 20 ns, which distinctly differs from those (<10 ns) of cell and tissue autofluorescence, making it possible to achieve background-free detection in vivo by time gating. Here, we demonstrate the feasibility of using fluorescent nanodiamonds (FNDs) as optical labels for wide-field time-gated fluorescence imaging and flow cytometric analysis of cancer cells with a nanosecond intensified charge-coupled device (ICCD) as the detector. The combined technique has allowed us to acquire fluorescence images of FND-labeled HeLa cells in whole blood covered with a chicken breast of ~0.1-mm thickness at the single cell level, and to detect individual FND-labeled HeLa cells in blood flowing through a microfluidic device at a frame rate of 23 Hz, as well as to locate and trace FND-labeled lung cancer cells in the blood vessels of a mouse ear. It opens a new window for real-time imaging and tracking of transplanted cells (such as stem cells) in vivo.

Figure 1. Spectroscopic characterization of hemoglobin and fluorescent nanodiamond.

(a) Comparison between the excitation spectrum of 100-nm FND and the absorption spectra of hemoglobin (Hb) in three different forms, deoxyHb, oxyHb, and metHb. The emission of FND was collected at 700–800 nm to obtain its excitation spectrum. Two vertical black lines indicate the points of the excitation at 532 nm and 599 nm. (b) Comparison between the fluorescence spectra of FND (1 mg/mL) and metHb (150 mg/mL) excited at 532 nm in water.

Figure 2. The experimental setup.

(a) Schematic of the wide-field time-gated fluorescence microscope. BF: bandpass filter, BS: beam splitter, DM: dichroic mirror, ICCD: intensified charge-coupled device, L: lens, M: mirror, Obj: objective, PC: personal computer, and PD: photodiode. The drawing was created with Microsoft Office PowerPoint 2007. (b) Power dependence of the second-order Stokes Raman conversion of the 532-nm laser using a Ba(NO₃)₂ crystal as the Raman shifter. The maximum output energy is about 25 μJ/pulse. (c) Oscilloscope traces of the laser pulses before and after passing through the Raman shifter.

Figure 3. In vitro imaging of FND-labeled cells in blood.

(a, b) Wide-field fluorescence images of FND-labeled HeLa cells attached to a coverglass slide and immersed in human blood without (a) or with (b) time gating. The exposure times used for the fluorescence imaging with a 100× oil-immersion objective lens in (a) and (b) are 0.1 s and 0.3 s, respectively. (c) Intensity profiles along the black and red color lines denoted in (a) and (b), respectively.

Figure 4. In vitro imaging of FND-labeled cells covered with chicken breast.

(a, b) Wide-field fluorescence images of FND-labeled HeLa cells in blood on a glass slide covered with a thin layer (~0.1 mm thickness) of chicken breast (a) without and (b) with time gating. (c) Time-gated fluorescence images of the corresponding FND-labeled HeLa cells in (a) and (b) without chicken breast. The objective lens is 40×. (d) Intensity profiles along the black, red color, and green lines denoted in (a), (b), and (c), respectively.

Figure 5. Flow cytometric analysis of FND-labeled cells in a microchannel.

(a) Snapshots of the flow of a single HeLa cell labeled with 100-nm FNDs in a microchannel with human blood. The channel width is 50 μm, the frame rate is 23 Hz (i.e. 43.5 ms/frame), and the objective lens is 10×. The red scale bar corresponds to 50 μm. (b) Histogram of the FND-labeled cells flowing across the microchannel with a cell concentration of 1.7 × 10⁵ cells/mL and a pumping rate of 0.01 mL/h for 2155 s. The expected and counted numbers of the cells are 998 and 862, respectively.

Figure 6. In vivo imaging and tracking of FND-labeled cells in blood vessels.

(a) Bright-field image of a mouse ear tissue. The green arrow indicates the position of an FND-labeled lung cancer cell in the blood vessel of ~50 μm in diameter. (b) Enlarged view of the fluorescence image of the square green region in (a). The bright spot corresponds to the FND-labeled lung cancer cell. (c) Enlarged view of the fluorescence image of the rectangular green region in (b), showing the trajectory of the FND-labeled lung cancer cell moving in the vessel. The average speed of the cell movement is 0.4 μm/s. The frame rate is 2 Hz and the objective lens is 10×. The red scale bar corresponds to 100, 50, and 10 μm in (a), (b) and (c), respectively.