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Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

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ABSTRACT

Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

No MeSH data available.


Quantitative analysis of the effect of LPS injection on live microvasculature.(a,b) Quantitative phase images of individual RBCs flowing through the microvasculature of the mouse mesentery before (top) and after (bottom panel) the injection of phosphate-buffered saline (PBS) solution (a) and lipopolysaccharide (LPS) solution (b). Green and blue triangles indicate the initial and final position of RBCs, respectively, along the microcapillaries for which center lines are indicated by solid black lines. Scale bars indicate 10 μm. See also Supplementary Movies 5 and 6. (c,d) Space-time phase images of RBCs before (top) and after (bottom panel) the injection of PBS solution (c) and LPS solution (d), respectively. Individual RBCs indicated as green and blue triangles in the phase images in (a,b) are marked as the same triangles. (e,f) Changes in relative volume flow rate (e) and haemoglobin (Hb) mass rate (f) over time before and after the injection of PBS solution (n = 5, grey boxes) and LPS solution (n = 4, red boxes). Values from individual microcapillaries are normalized by the value measured before the injection of the solution.
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f3: Quantitative analysis of the effect of LPS injection on live microvasculature.(a,b) Quantitative phase images of individual RBCs flowing through the microvasculature of the mouse mesentery before (top) and after (bottom panel) the injection of phosphate-buffered saline (PBS) solution (a) and lipopolysaccharide (LPS) solution (b). Green and blue triangles indicate the initial and final position of RBCs, respectively, along the microcapillaries for which center lines are indicated by solid black lines. Scale bars indicate 10 μm. See also Supplementary Movies 5 and 6. (c,d) Space-time phase images of RBCs before (top) and after (bottom panel) the injection of PBS solution (c) and LPS solution (d), respectively. Individual RBCs indicated as green and blue triangles in the phase images in (a,b) are marked as the same triangles. (e,f) Changes in relative volume flow rate (e) and haemoglobin (Hb) mass rate (f) over time before and after the injection of PBS solution (n = 5, grey boxes) and LPS solution (n = 4, red boxes). Values from individual microcapillaries are normalized by the value measured before the injection of the solution.

Mentions: To extend the applicability of intravital QPI for biological studies, we performed quantitative analysis of the change in blood flow rate in a mouse sepsis model. We intravenously injected lipopolysaccharides (LPS) solution (20 mg/kg), large molecules found in the outer membrane of gram-negative bacteria, to induce a severe septic condition which is a dysregulated systemic inflammatory response including increases in proinflammatory cytokines32. We measured time-lapse quantitative phase images before and after the LPS injection for an hour for every 15 minutes, and the same experiment was performed with the injection of PBS solution to compare the effect of the LPS injection on the change in blood flow. As shown in Fig. 3a and Supplementary Movie 5, the elapsed time for individual RBCs to pass through a microcapillary is maintained before (34 ms) and at 45 minutes after PBS injection (30 ms). However, the elapsed time (379 ms) for individual RBCs to pass through a microcapillary was much longer at 45 minutes after the LPS injection compared with that (19 ms) in the same trajectory of the identical capillary before the LPS injection (Fig. 3b and Supplementary Movie 6). The difference in the effect of PBS and LPS injection on blood flow is more clearly seen in the space-time phase images in Fig. 3c,d, where the slope of the phase image of individual RBCs indicates the flow velocity. The flow velocity of RBCs after LPS injection significantly decreases compared to the velocity before the injection, while the change in the flow velocity is minimal in the PBS injection.


Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice
Quantitative analysis of the effect of LPS injection on live microvasculature.(a,b) Quantitative phase images of individual RBCs flowing through the microvasculature of the mouse mesentery before (top) and after (bottom panel) the injection of phosphate-buffered saline (PBS) solution (a) and lipopolysaccharide (LPS) solution (b). Green and blue triangles indicate the initial and final position of RBCs, respectively, along the microcapillaries for which center lines are indicated by solid black lines. Scale bars indicate 10 μm. See also Supplementary Movies 5 and 6. (c,d) Space-time phase images of RBCs before (top) and after (bottom panel) the injection of PBS solution (c) and LPS solution (d), respectively. Individual RBCs indicated as green and blue triangles in the phase images in (a,b) are marked as the same triangles. (e,f) Changes in relative volume flow rate (e) and haemoglobin (Hb) mass rate (f) over time before and after the injection of PBS solution (n = 5, grey boxes) and LPS solution (n = 4, red boxes). Values from individual microcapillaries are normalized by the value measured before the injection of the solution.
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f3: Quantitative analysis of the effect of LPS injection on live microvasculature.(a,b) Quantitative phase images of individual RBCs flowing through the microvasculature of the mouse mesentery before (top) and after (bottom panel) the injection of phosphate-buffered saline (PBS) solution (a) and lipopolysaccharide (LPS) solution (b). Green and blue triangles indicate the initial and final position of RBCs, respectively, along the microcapillaries for which center lines are indicated by solid black lines. Scale bars indicate 10 μm. See also Supplementary Movies 5 and 6. (c,d) Space-time phase images of RBCs before (top) and after (bottom panel) the injection of PBS solution (c) and LPS solution (d), respectively. Individual RBCs indicated as green and blue triangles in the phase images in (a,b) are marked as the same triangles. (e,f) Changes in relative volume flow rate (e) and haemoglobin (Hb) mass rate (f) over time before and after the injection of PBS solution (n = 5, grey boxes) and LPS solution (n = 4, red boxes). Values from individual microcapillaries are normalized by the value measured before the injection of the solution.
Mentions: To extend the applicability of intravital QPI for biological studies, we performed quantitative analysis of the change in blood flow rate in a mouse sepsis model. We intravenously injected lipopolysaccharides (LPS) solution (20 mg/kg), large molecules found in the outer membrane of gram-negative bacteria, to induce a severe septic condition which is a dysregulated systemic inflammatory response including increases in proinflammatory cytokines32. We measured time-lapse quantitative phase images before and after the LPS injection for an hour for every 15 minutes, and the same experiment was performed with the injection of PBS solution to compare the effect of the LPS injection on the change in blood flow. As shown in Fig. 3a and Supplementary Movie 5, the elapsed time for individual RBCs to pass through a microcapillary is maintained before (34 ms) and at 45 minutes after PBS injection (30 ms). However, the elapsed time (379 ms) for individual RBCs to pass through a microcapillary was much longer at 45 minutes after the LPS injection compared with that (19 ms) in the same trajectory of the identical capillary before the LPS injection (Fig. 3b and Supplementary Movie 6). The difference in the effect of PBS and LPS injection on blood flow is more clearly seen in the space-time phase images in Fig. 3c,d, where the slope of the phase image of individual RBCs indicates the flow velocity. The flow velocity of RBCs after LPS injection significantly decreases compared to the velocity before the injection, while the change in the flow velocity is minimal in the PBS injection.

View Article: PubMed Central - PubMed

ABSTRACT

Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

No MeSH data available.