Limits...
Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

Bradley J, Pope I, Masia F, Sanusi R, Langbein W, Swann K, Borri P - Development (2016)

Bottom Line: Notably, it can be used in a way that does not compromise oocyte maturation or embryo development.We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling.These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos.

View Article: PubMed Central - PubMed

Affiliation: Cardiff School of Biosciences, The Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, UK.

No MeSH data available.


Related in: MedlinePlus

CARS and DIC images in living oocytes, eggs and early embryonic stages. (A-G) DIC images (single z-plane, except E, which is a maximum intensity projection) representative of populations of mouse eggs and embryos, from (A) immature GV stage (n=∼90), (B) MII eggs (n=∼70), (C) two-cell (n=∼65), (D) four-cell (n=∼60), (E) eight-cell (n=∼10), (F) morula (n=∼35) and (G) blastocyst stage (n=∼20) embryos using a 1.27 NA water objective and a 1.4 NA oil condenser. (H-N) Depth colour-coded images of CARS z-stacks at wavenumber 2850 cm−1 through the same eggs and embryos, showing LDs throughout these developmental stages. Inset in (I) shows a typical LD cluster seen at this stage. 0.1×0.1 µm xy pixel size; 0.5 µm z-step; 0.01 ms pixel dwell time; ∼14 mW (∼9 mW) pump (Stokes) power at the sample. Scale bars: 10 µm. Colour bar shows depth colour-coding from –25 µm-25 µm of 101 z-stacks (0 µm being the approximately equatorial plane of the egg or embryo), the brightness of each colour is the maximum intensity at each corresponding z-plane. Data from ≥2 trials, using 1-3 mice each.
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DEV129908F1: CARS and DIC images in living oocytes, eggs and early embryonic stages. (A-G) DIC images (single z-plane, except E, which is a maximum intensity projection) representative of populations of mouse eggs and embryos, from (A) immature GV stage (n=∼90), (B) MII eggs (n=∼70), (C) two-cell (n=∼65), (D) four-cell (n=∼60), (E) eight-cell (n=∼10), (F) morula (n=∼35) and (G) blastocyst stage (n=∼20) embryos using a 1.27 NA water objective and a 1.4 NA oil condenser. (H-N) Depth colour-coded images of CARS z-stacks at wavenumber 2850 cm−1 through the same eggs and embryos, showing LDs throughout these developmental stages. Inset in (I) shows a typical LD cluster seen at this stage. 0.1×0.1 µm xy pixel size; 0.5 µm z-step; 0.01 ms pixel dwell time; ∼14 mW (∼9 mW) pump (Stokes) power at the sample. Scale bars: 10 µm. Colour bar shows depth colour-coding from –25 µm-25 µm of 101 z-stacks (0 µm being the approximately equatorial plane of the egg or embryo), the brightness of each colour is the maximum intensity at each corresponding z-plane. Data from ≥2 trials, using 1-3 mice each.

Mentions: The CH2 symmetric stretch vibration at ∼2850 cm−1 is abundant in the acyl chain of FAs, and is an ideal target for single-frequency CARS microscopy of LDs. Living mouse oocytes, eggs and early embryos were imaged label-free using differential interference contrast (DIC) and CARS microscopy. Fig. 1A-G shows DIC images in a single, approximately equatorial z-plane of all developmental stages, from germinal vesicle (GV) stage through to a blastocyst embryo, in a set of representative specimens, each showing stage-specific characteristics. Accompanying each DIC image is a corresponding 3D CARS image of the same sample, acquired immediately after DIC imaging. These are shown as depth colour-coded CARS z-stack projections, measured at the 2850 cm−1 CH2 symmetric stretch vibrational resonance, and enabled us to determine the LD spatial distribution throughout the measured volume at each stage. LDs in a GV oocyte have a homogenous spatial distribution throughout the cell cytoplasm, but not within the GV itself (Fig. 1H). The distribution of LDs in metaphase II (MII) eggs is noticeably different, with many clusters of LDs seen throughout the cytoplasm, and fewer dispersed singular droplets (Fig. 1I). CARS images of early embryo stages (two-cell, four-cell embryos) show that LDs fill most of the cytoplasm as the cells divide, with little or no difference in distribution between blastomeres (Fig. 1J,K). LDs appear of uniform size, ∼0.5 µm diameter (see Fig. S1); however, by the eight-cell stage, larger droplets are seen, and by the later early-embryo stages (morula and blastocyst) fewer, but much larger (>1 µm) LDs are present, varying in size, suggesting fusion of existing droplets as development continues through these stages (Fig. 1L-N).Fig. 1.


Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

Bradley J, Pope I, Masia F, Sanusi R, Langbein W, Swann K, Borri P - Development (2016)

CARS and DIC images in living oocytes, eggs and early embryonic stages. (A-G) DIC images (single z-plane, except E, which is a maximum intensity projection) representative of populations of mouse eggs and embryos, from (A) immature GV stage (n=∼90), (B) MII eggs (n=∼70), (C) two-cell (n=∼65), (D) four-cell (n=∼60), (E) eight-cell (n=∼10), (F) morula (n=∼35) and (G) blastocyst stage (n=∼20) embryos using a 1.27 NA water objective and a 1.4 NA oil condenser. (H-N) Depth colour-coded images of CARS z-stacks at wavenumber 2850 cm−1 through the same eggs and embryos, showing LDs throughout these developmental stages. Inset in (I) shows a typical LD cluster seen at this stage. 0.1×0.1 µm xy pixel size; 0.5 µm z-step; 0.01 ms pixel dwell time; ∼14 mW (∼9 mW) pump (Stokes) power at the sample. Scale bars: 10 µm. Colour bar shows depth colour-coding from –25 µm-25 µm of 101 z-stacks (0 µm being the approximately equatorial plane of the egg or embryo), the brightness of each colour is the maximum intensity at each corresponding z-plane. Data from ≥2 trials, using 1-3 mice each.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4920167&req=5

DEV129908F1: CARS and DIC images in living oocytes, eggs and early embryonic stages. (A-G) DIC images (single z-plane, except E, which is a maximum intensity projection) representative of populations of mouse eggs and embryos, from (A) immature GV stage (n=∼90), (B) MII eggs (n=∼70), (C) two-cell (n=∼65), (D) four-cell (n=∼60), (E) eight-cell (n=∼10), (F) morula (n=∼35) and (G) blastocyst stage (n=∼20) embryos using a 1.27 NA water objective and a 1.4 NA oil condenser. (H-N) Depth colour-coded images of CARS z-stacks at wavenumber 2850 cm−1 through the same eggs and embryos, showing LDs throughout these developmental stages. Inset in (I) shows a typical LD cluster seen at this stage. 0.1×0.1 µm xy pixel size; 0.5 µm z-step; 0.01 ms pixel dwell time; ∼14 mW (∼9 mW) pump (Stokes) power at the sample. Scale bars: 10 µm. Colour bar shows depth colour-coding from –25 µm-25 µm of 101 z-stacks (0 µm being the approximately equatorial plane of the egg or embryo), the brightness of each colour is the maximum intensity at each corresponding z-plane. Data from ≥2 trials, using 1-3 mice each.
Mentions: The CH2 symmetric stretch vibration at ∼2850 cm−1 is abundant in the acyl chain of FAs, and is an ideal target for single-frequency CARS microscopy of LDs. Living mouse oocytes, eggs and early embryos were imaged label-free using differential interference contrast (DIC) and CARS microscopy. Fig. 1A-G shows DIC images in a single, approximately equatorial z-plane of all developmental stages, from germinal vesicle (GV) stage through to a blastocyst embryo, in a set of representative specimens, each showing stage-specific characteristics. Accompanying each DIC image is a corresponding 3D CARS image of the same sample, acquired immediately after DIC imaging. These are shown as depth colour-coded CARS z-stack projections, measured at the 2850 cm−1 CH2 symmetric stretch vibrational resonance, and enabled us to determine the LD spatial distribution throughout the measured volume at each stage. LDs in a GV oocyte have a homogenous spatial distribution throughout the cell cytoplasm, but not within the GV itself (Fig. 1H). The distribution of LDs in metaphase II (MII) eggs is noticeably different, with many clusters of LDs seen throughout the cytoplasm, and fewer dispersed singular droplets (Fig. 1I). CARS images of early embryo stages (two-cell, four-cell embryos) show that LDs fill most of the cytoplasm as the cells divide, with little or no difference in distribution between blastomeres (Fig. 1J,K). LDs appear of uniform size, ∼0.5 µm diameter (see Fig. S1); however, by the eight-cell stage, larger droplets are seen, and by the later early-embryo stages (morula and blastocyst) fewer, but much larger (>1 µm) LDs are present, varying in size, suggesting fusion of existing droplets as development continues through these stages (Fig. 1L-N).Fig. 1.

Bottom Line: Notably, it can be used in a way that does not compromise oocyte maturation or embryo development.We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling.These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos.

View Article: PubMed Central - PubMed

Affiliation: Cardiff School of Biosciences, The Sir Martin Evans Building, Museum Avenue, Cardiff CF10 3AX, UK.

No MeSH data available.


Related in: MedlinePlus