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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.

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Schematic diagram of intravital quantitative phase microscopy.(a) Mach-Zehnder interferometry recording holograms from various illumination angles scanned by a galvanomirror (GM). BS: beam splitter, and M: mirror. (b) The schematic diagram of live mouse mesentery staged on a heating plate. (c) Raw holograms recorded by a camera. (d) Retrieved optical phase delay maps from measured holograms in (c). (e) Optical phase delay maps subtracted by the first frame to eliminate surrounding adipocytes. (f) Final optical phase delay maps subtracted by the minimum values of sequential phase maps which correspond to the phase delay of red blood cells in the microvasculature of the mouse mesentery. Scale bar corresponds to 10 μm. Supplementary Movie 1 shows time-lapse images of raw hologram, optical phase map before and after applying the scattering reduction method as well as emulated images to be measured using typical microscopy such as bright field, phase contrast, and differential interference contrast (DIC) microscopy.
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f1: Schematic diagram of intravital quantitative phase microscopy.(a) Mach-Zehnder interferometry recording holograms from various illumination angles scanned by a galvanomirror (GM). BS: beam splitter, and M: mirror. (b) The schematic diagram of live mouse mesentery staged on a heating plate. (c) Raw holograms recorded by a camera. (d) Retrieved optical phase delay maps from measured holograms in (c). (e) Optical phase delay maps subtracted by the first frame to eliminate surrounding adipocytes. (f) Final optical phase delay maps subtracted by the minimum values of sequential phase maps which correspond to the phase delay of red blood cells in the microvasculature of the mouse mesentery. Scale bar corresponds to 10 μm. Supplementary Movie 1 shows time-lapse images of raw hologram, optical phase map before and after applying the scattering reduction method as well as emulated images to be measured using typical microscopy such as bright field, phase contrast, and differential interference contrast (DIC) microscopy.

Mentions: In order to perform intravital QPI of microvasculature, we used interferometric microscopy with a method to filter out static scattering signals. A Mach-Zehnder interferometric microscope was used to measure optical fields of a coherent laser beam passing through tissues (Fig. 1a). A diode-pumped solid-state laser (λ = 532 nm, 100 mW, Cobolt Co., Sweden) was used as an illumination source. After spatial filtering, the beam from the laser is divided into two arms by a beam splitter. One arm is used as a reference beam. The other beam impinges onto a sample with a long-working distance dry objective lens (LMPLFLN 50×, NA = 0.5, Olympus Inc., Japan) and a tube lens (f = 200 mm). For tomographic measurements, the angle of illumination is rotated by using a dual-axis scanning galvanometer (GVS012, Thorlabs, NJ, USA). Mouse mesentery was staged on a sample plane equipped with a heating plate (Fig. 1b). The diffracted beam from the sample is collected by either a high numerical aperture (NA) objective lens (UPLSAPO, 100×, oil immersion, NA = 1.4, Olympus Inc.) or a water-immersion objective lens (UPLSAPO, 60×, water immersion, NA = 1.2, Olympus Inc.). The beam is further magnified four times with an additional 4-f telescopic system so that total field-of-view becomes 35.0 μm × 35.0 μm (high NA objective lens) or 58.4 μm × 58.4 μm (water-immersion objective lens). The 2-D spatial resolution using the high NA objective lens and the water-immersion objective lens are calculated as 190 nm and 221.6 nm, respectively, which are defined as λ/(2NA). The beam diffracted from the sample interferes with the reference beam at an image plane, which generates spatially modulated holograms. The holograms are recorded by a high-speed CMOS camera (1024 PCI, Photron USA Inc., CA, USA) with a frame rate of 1,000 Hz and the exposure time of 1/5,000 sec.


Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice
Schematic diagram of intravital quantitative phase microscopy.(a) Mach-Zehnder interferometry recording holograms from various illumination angles scanned by a galvanomirror (GM). BS: beam splitter, and M: mirror. (b) The schematic diagram of live mouse mesentery staged on a heating plate. (c) Raw holograms recorded by a camera. (d) Retrieved optical phase delay maps from measured holograms in (c). (e) Optical phase delay maps subtracted by the first frame to eliminate surrounding adipocytes. (f) Final optical phase delay maps subtracted by the minimum values of sequential phase maps which correspond to the phase delay of red blood cells in the microvasculature of the mouse mesentery. Scale bar corresponds to 10 μm. Supplementary Movie 1 shows time-lapse images of raw hologram, optical phase map before and after applying the scattering reduction method as well as emulated images to be measured using typical microscopy such as bright field, phase contrast, and differential interference contrast (DIC) microscopy.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5015119&req=5

f1: Schematic diagram of intravital quantitative phase microscopy.(a) Mach-Zehnder interferometry recording holograms from various illumination angles scanned by a galvanomirror (GM). BS: beam splitter, and M: mirror. (b) The schematic diagram of live mouse mesentery staged on a heating plate. (c) Raw holograms recorded by a camera. (d) Retrieved optical phase delay maps from measured holograms in (c). (e) Optical phase delay maps subtracted by the first frame to eliminate surrounding adipocytes. (f) Final optical phase delay maps subtracted by the minimum values of sequential phase maps which correspond to the phase delay of red blood cells in the microvasculature of the mouse mesentery. Scale bar corresponds to 10 μm. Supplementary Movie 1 shows time-lapse images of raw hologram, optical phase map before and after applying the scattering reduction method as well as emulated images to be measured using typical microscopy such as bright field, phase contrast, and differential interference contrast (DIC) microscopy.
Mentions: In order to perform intravital QPI of microvasculature, we used interferometric microscopy with a method to filter out static scattering signals. A Mach-Zehnder interferometric microscope was used to measure optical fields of a coherent laser beam passing through tissues (Fig. 1a). A diode-pumped solid-state laser (λ = 532 nm, 100 mW, Cobolt Co., Sweden) was used as an illumination source. After spatial filtering, the beam from the laser is divided into two arms by a beam splitter. One arm is used as a reference beam. The other beam impinges onto a sample with a long-working distance dry objective lens (LMPLFLN 50×, NA = 0.5, Olympus Inc., Japan) and a tube lens (f = 200 mm). For tomographic measurements, the angle of illumination is rotated by using a dual-axis scanning galvanometer (GVS012, Thorlabs, NJ, USA). Mouse mesentery was staged on a sample plane equipped with a heating plate (Fig. 1b). The diffracted beam from the sample is collected by either a high numerical aperture (NA) objective lens (UPLSAPO, 100×, oil immersion, NA = 1.4, Olympus Inc.) or a water-immersion objective lens (UPLSAPO, 60×, water immersion, NA = 1.2, Olympus Inc.). The beam is further magnified four times with an additional 4-f telescopic system so that total field-of-view becomes 35.0 μm × 35.0 μm (high NA objective lens) or 58.4 μm × 58.4 μm (water-immersion objective lens). The 2-D spatial resolution using the high NA objective lens and the water-immersion objective lens are calculated as 190 nm and 221.6 nm, respectively, which are defined as λ/(2NA). The beam diffracted from the sample interferes with the reference beam at an image plane, which generates spatially modulated holograms. The holograms are recorded by a high-speed CMOS camera (1024 PCI, Photron USA Inc., CA, USA) with a frame rate of 1,000 Hz and the exposure time of 1/5,000 sec.

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.


Related in: MedlinePlus