In Vitro Quantification of Single Red Blood Cell Oxygen Saturation by Femtosecond Transient Absorption Microscopy

Abstract

Hemoglobin, the oxygen carrying protein, ferries nearly all bodily oxygen from the lungs to cells and tissues in need. Blood oxygen saturation (sO2) thus plays an important role in maintaining energy homeostasis throughout the body. Clinical and research tools have been developed to monitor sO2 at a wide range of temporal and spatial scales. However, real-time quantification of sO2 at single red blood cell (RBC) resolution remains challenging. Such capability is critically important to study energy metabolism in heterogeneous tissues including brain and tumor tissue. In this work, we develop a ratiometric transient absorption microscopy technique to image hemoglobin sO2. By exploiting differences in transient lifetime kinetics between oxyhemoglobin and deoxyhemoglobin, we directly quantified the sO2 of single RBCs in real-time without the need for injection of exogenous agents. This simple and high-speed nonlinear optical imaging technique is well suited for in vitro and in vivo quantification of sO2.

In Vivo Simultaneous Nonlinear Absorption Raman and Fluorescence (SNARF) Imaging of Mouse Brain Cortical Structures

“…Another potential benefit of label-free TAM imaging compared to fluorescent tracers is that it is possible to acquire oxygen saturation information based on the difference in absorption between oxy-hemoglobin and deoxy-hemoglobin. 25 This approach could potentially increase the speed in measuring oxygen tension by three orders of magnitude compared to current oxygen probes 59 , thus enabling measurement of oxygen delivery change in response to both transient and persistent stalled flow.…”

“…Transient absorption microscopy (TAM) is another label-free chemical imaging technique that is potentially useful for in vivo brain imaging. Recent reports have used the intrinsic absorption of hemoglobin to quantify hemoglobin glycation, 24 oxygenation, 25 and concentration 26 ex vivo. Importantly, imaging of hemoglobin allows direct visualization of blood flow and relieves the need for intravenous fluorescent dye injections into the blood compartment, which are invasive and can suffer from quick clearing times.…”

Beyond intensity modulation: new approaches to pump-probe microscopy

“…However, some of these methods introduce a third wavelength that is different from the original pump and probe (or Stokes) beam, which could cause additional transient processes like fluorescence emissions, and would add additional layers of complexity to the microscope. In a similar approach, intensity modulation of two phase shifted pump pulse trains for pump-probe measurements of blood oxygenation have been shown [7].…”

“…Time-delay modulation can be used to reduce thermal effects and fluorescence backgrounds of radiative states. Instead of switching the pump on and off, the pump power is kept constant (𝑎𝑎 0 = 𝑎𝑎 1 ) and the inter-pulse delay is modulated [7][8][9]. Another imaging modality, polarization modulation, can be used to selectively enhance or suppress nonlinear processes that are sensitive to the relative polarization angle between pump and probe.…”

Direct Quantification of Single Red Blood Cell Hemoglobin Concentration with Multiphoton Microscopy

“…TAM is a multiphoton microscopy technique that is sensitive to numerous photophysical processes involving electronic excited states including, but not limited to, two-photon absorption (TPA), excited state absorption (ESA), and ground state bleaching (GSB) . Similar to SPM, TAM makes use of the absorption characteristics of hemoglobin to gain intrinsic contrast but with a significant advantage in that TAM is a three-dimensional imaging technique that allows imaging through scattering medium such as tissue. − Recently, we utilized the intrinsic differences between oxyhemoglobin and deoxyhemoglobin to achieve real-time quantification of oxygen saturation in RBCs …”