Photoacoustic and photothermal detection of circulating tumor cells, bacteria and nanoparticles in cerebrospinal fluid in vivo and ex vivo

Abstract

Circulating cells, bacteria, proteins, microparticles, and DNA in cerebrospinal fluid (CSF) are excellent biomarkers of many diseases, including cancer and infections. However, the sensitivity of existing methods is limited in their ability to detect rare CSF biomarkers at the treatable, early-stage of diseases. Here, we introduce novel CSF tests based on in vivo photoacoustic flow cytometry (PAFC) and ex vivo photothermal scanning cytometry. In the CSF of tumor-bearing mice, we molecularly detected in vivo circulating tumor cells (CTCs) before the development of breast cancer brain metastasis with 20-times higher sensitivity than with current assays. For the first time, we demonstrated assessing three pathways (i.e., blood, lymphatic, and CSF) of CTC dissemination, tracking nanoparticles in CSF in vivo and their imaging ex vivo. In label-free CSF samples, we counted leukocytes, erythrocytes, melanoma cells, and bacteria and imaged intracellular cytochromes, hemoglobin, melanin, and carotenoids, respectively. Taking into account the safety of PAFC, its translation for use in humans is expected to improve disease diagnosis beyond conventional detection limits.

Figure 1

Integrated technical platform for testing CSF in vivo and ex vivo. (a) Schematic of multicolor PAFC with the example of real-time two-color PAFC tracings from CSF. (b) The heating stereotaxic table for in vivo PAFC and CSF sampling.

Figure 2

Ex vivo examination of normal CSF collected from healthy animals. (a) High resolution optical imaging; (b) optical spectroscopy.

Figure 3

Detection and enumeration of S. aureus in CSF sample (15 µm × 15 µm × 120µm) using photothermal scanning cytometry. (a) PBS; (b) normal CSF; and (c) CSF with bacteria; high-resolution optical imaging in the insert (×100, oil immersion) confirmed the signals related to single bacteria. Laser wavelength and energy fluence are 710 nm and 100 mJ/cm², respectively.

Figure 4

Label-free optical and photothermal images of the leukocyte (a), erythrocyte (b) and melanoma cell (c). Laser wavelength is 530 nm; laser energy fluence is 400, 50, and 100 mJ/cm², respectively.

Figure 5

Detection of GNRs in CSF. (a) Photothermal images of GNRs color-coded by blue in CSF plasma (black). (b) Optical (left) and photothermal (right) images of the CSF sample with breast cancer cell labeled with GNRs. Laser wavelength and fluence are 530 nm and 50 mJ/cm², respectively

Figure 6

In vivo real-time tracing of CSF after injection of GNRs₆₇₀ in cisterna magna. Patterns I, II and III are related to the different time points at the same position of laser beam over CSF. Laser wavelength is 670 nm; laser energy fluence is 30 mJ/cm².

Figure 7

In vivo photoacoustic detection and counting of CTCs in CSF of tumor-bearing mice. (a) Dynamic of CTCs in blood circulation during tumor development measured by fluorescent flow cytometry. (b) Intravital whole-body imaging of primary tumor at week 10 after tumor inoculation. Black dashed lines indicate two photoacoustic scans (670 nm; 30 mJ/cm²) presented in (c). (d) Photoacoustic scanning of the sentinel lymph node; high-amplitude signals color-coded by yellow and red are associated with objects ≤15 µm. (e) two-color (670 nm and 820 nm) PAFC of CSF in vivo through scull. (f) In vivo two-color PAFC of CSF in cisterna magna after scull removing.