Single-cell label-free photoacoustic flowoxigraphy in vivo

Abstract

Label-free functional imaging of single red blood cells (RBCs) in vivo holds the key to uncovering the fundamental mechanism of oxygen metabolism in cells. To this end, we developed single-RBC photoacoustic flowoxigraphy (FOG), which can image oxygen delivery from single flowing RBCs in vivo with millisecond-scale temporal resolution and micrometer-scale spatial resolution. Using intrinsic optical absorption contrast from oxyhemoglobin (HbO 2 ) and deoxyhemoglobin (HbR), FOG allows label-free imaging. Multiple single-RBC functional parameters, including total hemoglobin concentration (C Hb ), oxygen saturation (sO 2 ), sO 2 gradient (∇sO 2 ), flow speed (v f ), and oxygen release rate (rO 2 ), have been quantified simultaneously in real time. Working in reflection instead of transmission mode, the system allows minimally invasive imaging at more anatomical sites. We showed the capability to measure relationships among sO 2 , ∇sO 2 , v f , and rO 2 in a living mouse brain. We also demonstrated that single-RBC oxygen delivery was modulated by changing either the inhalation gas or blood glucose. Furthermore, we showed that the coupling between neural activity and oxygen delivery could be imaged at the single-RBC level in the brain. The single-RBC functional imaging capability of FOG enables numerous biomedical studies and clinical applications. Although individual parameters such as oxygen saturation (sO 2 ) (1, 2), partial oxygen pressure (pO 2 ) (3, 4), or blood flow speed (v f ) (4-7) may partially indicate tissue oxygenation, none of them can provide a complete description of oxygen transport. To quantify tissue oxygenation in vivo, three primary imaging modalities have been applied: positron emission tomography (PET) (8), functional magnetic resonance imaging (fMRI) (9, 10), and diffuse optical tomography (DOT) (11). Although these methods may provide deep functional imaging, they all suffer from millimeter-scale spatial resolutions. Recently, photoacoustic (PA) microscopy was proposed to measure the oxygen metabolic rate at feeding and draining blood vessels (12). However, the assessment is limited to a relatively large region; moreover, the feeding and draining blood vesselsespecially those surrounding a tumor-may be numerous and difficult to identify. Because micrometer-sized RBCs are the basic elements for delivering most of the oxygen to cells and tissues, the need for direct imaging of oxygen release from flowing single RBCs in vivo is imperative. A molecular imaging method based on the measurement of ground-state recovery time has been investigated for hemoglobin sensing (13), but in vivo measurement of oxygen saturation has not been implemented. Dual-wavelength spectrophotometry has been applied to measure oxygen release for decades (14), but transmission-mode imaging limits its application to very thin tissue. Many biomedical problems-for example, tumor or neuroscience studies-require minimally invasive imaging at various anatomical sites. A new method for in vivo imaging of single-RBC oxygen release...

Reflection-mode in vivo photoacoustic microscopy with subwavelength lateral resolution

“…Figure 4(b) shows the close-up image of those RBCs with estimated diameters of ~6-7 µm. Since subwavelength-resolution PAM enables well-resolved individual RBCs in vivo, the system, in conjunction with spectroscopic photoacoustic measurement, has the potential to provide important functional information in vivo, such as sickle cell disease, blood flow in capillaries, and even dynamic oxygen consumption at the single-cell level [20]. In photoacoustic imaging, the depth information is usually derived from the time of flight of the photoacoustic waves.…”

Adding fifth dimension to optoacoustic imaging: volumetric time-resolved spectrally enriched tomography

“…The pulsed laser fluence on the skin surface was kept below the exposure safety limits of 20 mJ cm 22 for the entire duration of the experiments. 25 The measurements were done in the wrist region, having large variety of vasculature of different sizes, depth and oxygenation levels, as well as a melanin-rich skin pigmentation, which presents characteristic spectral variations in addition to oxygenated and deoxygenated hemoglobin.…”

Simultaneous measurement of erythrocyte aggregarion and oxygen saturation under in vitro pulsatile blood flow by high-frequency photoacoustics

“…Over the past decade, SO 2 measurement methods have been significantly improved through advances in photoacoustic (PA) imaging [7,8]. However, these studies have focused on the PA measurement of the SO 2 in a micron-diameter vessels without considering hemodydnamic and hemorheological properties that control EA.…”