Yulian A. Menyaev

In vivo high-speed imaging of individual cells in fast blood flow

In vivo, label-free, high-speed (up to 10,000 with the potential for 40,000 frames per second), high-resolution (up to 300 nm) 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.

Optical clearing in photoacoustic flow 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.

Nonlinear photoacoustic signal amplification from single targets in absorption background.

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 for early malaria diagnosis

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.

In Vivo Long-Term Monitoring of Circulating Tumor Cells Fluctuation during Medical Interventions.

The goal of this research was to study the long-term impact of medical interventions on circulating tumor cell (CTC) dynamics. We have explored whether tumor compression, punch biopsy or tumor resection cause dissemination of CTCs into peripheral blood circulation using in vivo fluorescent flow cytometry and breast cancer-bearing mouse model inoculated with MDA-MB-231-Luc2-GFP cells in the mammary gland. Two weeks after tumor inoculation, three groups of mice were the subject of the following interventions: (1) tumor compression for 15 minutes using 400 g weight to approximate the pressure during mammography; (2) punch biopsy; or (3) surgery. The CTC dynamics were determined before, during and six weeks after these interventions. An additional group of tumor-bearing mice was used as control and did not receive an intervention. The CTC dynamics in all mice were monitored weekly for eight weeks after tumor inoculation. We determined that tumor compression did not significantly affect CTC dynamics, either during the procedure itself (P = 0.28), or during the 6-week follow-up. In the punch biopsy group, we observed a significant increase in CTC immediately after the biopsy (P = 0.02), and the rate stayed elevated up to six weeks after the procedure in comparison to the tumor control group. The CTCs in the group of mice that received a tumor resection disappeared immediately after the surgery (P = 0.03). However, CTC recurrence in small numbers was detected during six weeks after the surgery. In the future, to prevent these side effects of medical interventions, the defined dynamics of intervention-induced CTCs may be used as a basis for initiation of aggressive anti-CTC therapy at time-points of increasing CTC number.

Real-Time Label-Free Embolus Detection Using In Vivo Photoacoustic Flow Cytometry.

Thromboembolic events are one of the world’s leading causes of death among patients. Embolus or clot formations have several etiologies including paraneoplastic, post-surgery, cauterization, transplantation, or extracorporeal circuits. Despite its medical significance, little progress has been made in early embolus detection, screening and control. The aim of our study is to test the utility of the in vivo photoacoustic (PA) flow cytometry (PAFC) technique for non-invasive embolus detection in real-time. Using in vivo PAFC, emboli were non-invasively monitored in the bloodstream of two different mouse models. The tumor-free mouse model consisted of two groups, one in which the limbs were clamped to produce vessel stasis (7 procedures), and one where the mice underwent surgery (7 procedures). The melanoma-bearing mouse model also consisted of two groups, one in which the implanted tumor underwent compression (8 procedures), and one where a surgical excision of the implanted tumor was performed (8 procedures). We demonstrated that the PAFC can detect a single embolus, and has the ability to distinguish between erythrocyte–rich (red) and leukocyte/platelet-rich (white) emboli in small vessels. We show that, in tumor-bearing mice, the level of circulating emboli was increased compared to tumor-free mice (p = 0.0013). The number of circulating emboli temporarily increased in the tumor-free control mice during vessel stasis (p = 0.033) and after surgical excisions (signed-rank p = 0.031). Similar observations were noted during tumor compression (p = 0.013) and after tumor excisions (p = 0.012). For the first time, it was possible to detect unlabeled emboli in vivo non-invasively, and to confirm the presence of pigmented tumor cells within circulating emboli. The insight on embolus dynamics during cancer progression and medical procedures highlight the clinical potential of PAFC for early detection of cancer and surgery-induced emboli to prevent the fatal thromboembolic complications by well-timed therapy.

Preclinical photoacoustic models: application for ultrasensitive single cell malaria diagnosis in large vein and artery.

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 PAFCs 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.

Fluctuation of probe beam in thermolens schematics as potential indicator of cell metabolism, apoptosis, necrosis and laser impact

Recently we discovered that in developed integrated flow cytometry using combination high resolution transmission and two beam (pump-probe) of photothermal microscopy with flow module the temporal fluctuation of probe beam even without pump laser pulse are very sensitive to functional states of single cells (e.g. with 2-3 differences in average fluctuation amplitude for live and dead cells). In this paper we are focusing on theoretical and experimental studies of these new phenomena using thermolens schematics with highly stabilized continuous-wave He-Ne laser and photodiode/photomultiplier with pinhole. This scheme allows to study both random and laser-induced thermolens effects in passive and active mode including short and long term monitoring of scattered light fluctuation in trans-illumination mode at single cellular and even sub-cellular levels in vitro, and in vivo. We demonstrate these schematics has some potential to monitor intracellular dynamics including change in cell metabolism, and necrosis, especially, under pump laser impacts in vitro in stationary condition. Besides, we present some relevant data obtained with other similar schematics, including evaluation of bacterial motility by dynamic light scattering. We discuss further potential developing this approach for flow cytometry with fast speed photodetector and digital camera and the multi- wavelength statistical and correlation analysis of speckle-related signals in applications to analysis of complex motions and functional properties of cells in bioflows including the evaluation of the rotational and translational components of the motion of individual live and dead cells.

Parametrical Tuning of Twisting Generators

Generators of uniformly distributed random numbers are broadly applied in simulations of stochastic processes that rely on normal and other distributions. In a point of fact, the uniform random numbers are actively used for applications that range from, modeling different phenomena such as theoretical mathematics and technical designing, to evidence-based medicine. This paper proposes a novel approach which consists of a combination of global twister with circular technique and initial congruential generation with complete stochastic sequences. It has been experimentally confirmed that for complete sequences this type of generation provides uniformity in distribution of random numbers. The offered program codes include the tuning methods for the generation technique where random numbers may take any bit length. Moreover, the automatic switching of generator parameters such as initial congruential constants depending on intervals for generated numbers is considered as well. Demonstrated results of testing confirm the uniformity of distribution without any repeated or skipped generated elements.

Photoacoustic Flow Cytometry for Single Sickle Cell Detection In Vitro and In Vivo

Control of sickle cell disease (SCD) stage and treatment efficiency are still time-consuming which makes well-timed prevention of SCD crisis difficult. We show here that in vivo photoacoustic (PA) flow cytometry (PAFC) has a potential for real-time monitoring of circulating sickled cells in mouse model. In vivo data were verified by in vitro PAFC and photothermal (PT) and PA spectral imaging of sickle red blood cells (sRBCs) expressing SCD-associated hemoglobin (HbS) compared to normal red blood cells (nRBCs). We discovered that PT and PA signal amplitudes from sRBCs in linear mode were 2–4-fold lower than those from nRBCs. PT and PA imaging revealed more profound spatial hemoglobin heterogeneity in sRBCs than in nRBCs, which can be associated with the presence of HbS clusters with high local absorption. This hypothesis was confirmed in nonlinear mode through nanobubble formation around overheated HbS clusters accompanied by spatially selective signal amplification. More profound differences in absorption of sRBCs than in nRBCs led to notable increase in PA signal fluctuation (fluctuation PAFC mode) as an indicator of SCD. The obtained data suggest that noninvasive label-free fluctuation PAFC has a potential for real-time enumeration of sRBCs both in vitro and in vivo.