Noninvasive label-free photoacoustic detection of circulating tumor cells was demonstrated directly in the bloodstream of patients with melanoma.

In vivo photoacoustic (PA) flow cytometry (PAFC) has already demonstrated a great potential for the diagnosis of deadly diseases through ultrasensitive detection of rare disease‐associated circulating markers in whole blood volume. Here, we demonstrate the first application of this powerful technique for early diagnosis of malaria through label‐free detection of malaria parasite‐produced hemozoin in infected red blood cells (iRBCs) as high‐contrast PA agent. The existing malaria tests using blood smears can detect the disease at 0.001–0.1% of parasitemia. On the contrary, linear PAFC showed a potential for noninvasive malaria diagnosis at an extremely low level of parasitemia of 0.0000001%, which is ∼10³ times better than the existing tests. Multicolor time‐of‐flight PAFC with high‐pulse repetition rate lasers at wavelengths of 532, 671, and 820 nm demonstrated rapid spectral and spatial identification and quantitative enumeration of individual iRBCs. Integration of PAFC with fluorescence flow cytometry (FFC) provided real‐time simultaneous detection of single iRBCs and parasites expressing green fluorescence proteins, respectively. A combination of linear and nonlinear nanobubble‐based multicolor PAFC showed capability to real‐time control therapy efficiency by counting of iRBCs before, during, and after treatment. Our results suggest that high‐sensitivity, high‐resolution ultrafast PAFC–FFC platform represents a powerful research tool to provide the insight on malaria progression through dynamic study of parasite–cell interactions directly in bloodstream, whereas portable hand‐worn PAFC device could be broadly used in humans for early malaria diagnosis. © 2016 International Society for Advancement of Cytometry

Clinical applications of photoacoustic (PA) flow cytometry (PAFC) for detection of circulating tumor cells in deep blood vessels are hindered by laser beam scattering, that result in loss of PAFC sensitivity and resolution. We demonstrate biocompatible and rapid optical clearing (OC) of skin to minimize light scattering and thus, increase optical resolution and sensitivity of PAFC. OC effect was achieved in 20 min by sequent skin cleaning, microdermabrasion, and glycerol application enhanced by massage and sonophoresis. Using 0.8 mm mouse skin layer over a blood vessel in vitro phantom we demonstrated 1.6-fold decrease in laser spot blurring accompanied by 1.6-fold increase in PA signal amplitude from blood background. As a result, peak rate for B16F10 melanoma cells in blood flow increased 1.7-fold. By using OC we also demonstrated the feasibility of PA contrast improvement for human hand veins.

Photoacoustic (PA) detection of single absorbing targets such as nanoparticles or cells can be limited by absorption background. We show here that this problem can be overcome by using the nonlinear photoacoustics based on the differences in PA signal dependences on the laser energy from targets and background. Among different nonlinear phenomena, we focused on laser generation of nanobubbles as more efficient PA signal amplifiers from strongly absorbing, highly localized targets in the presence of spatially homogenous absorption background generating linear signals only. This approach was demonstrated by using nonlinear PA flow cytometry platform for label-free detection of circulating melanoma cells in blood background in vitro and in vivo. Nonlinearly amplified PA signals from overheated melanin nanoclusters in melanoma cells became detectable above still linear blood background. Nonlinear nanobubble-based photoacoustics provide new opportunities to significantly (5–20-fold) increase PA contrast of single nanoparticles, cells, viruses and bacteria in complex biological environments.

In vivo photoacoustic flow cytometry (PAFC) has demonstrated potential for early diagnosis of deadly diseases through detection of rare circulating tumor cells, pathogens, and clots in nearly the entire blood volume. Before clinical application, this promising diagnostic platform requires verification and optimization using adequate preclinical models. We show here that this can be addressed by examination of large mouse blood vessels which are similar in size, depth and flow velocity to human vessels used in PAFC. Using this model, we verified the capability of PAFC for ultrasensitive, noninvasive, label-free, rapid malaria diagnosis. The time-resolved detection of delayed PA signals from deep vessels provided complete elimination of background from strongly pigmented skin. We discovered that PAFC’s sensitivity is higher during examination of infected cells in arteries compared to veins at similar flow rate. Our advanced PAFC platform integrating a 1060 nm laser with tunable pulse rate and width, a wearable probe with a focused transducer, and linear and nonlinear nanobubble-amplified signal processing demonstrated detection of parasitemia at the unprecedented level of 0.00000001% within 20 seconds and the potential to further improve the sensitivity 100-fold in humans, that is approximately 10⁶ times better than in existing malaria tests.

In vivo, label-free, high-speed (up to 10,000 with the potential for 40,000 frames per second), high-resolution (up to $300nm$) real-time continuous imaging with successive framing of circulating individual erythrocytes, leukocytes, and platelets in fast blood flow is developed. This technique, used in an animal model, reveals the extremely high dynamic deformability of erythrocytes in natural flow. Potential applications of this technique are discussed with focus on time-resolved monitoring of the cell deformation dynamics in the native biological environment, which may have diagnostic value for the early diagnosis of diseases.