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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

Cell viability after live imaging with CARS. (A,D) Single z-plane DIC using a 1.27 NA water objective and a 1.4 NA oil condenser and (B,E) depth colour-coded images of CARS stacks at wavenumber 2850 cm−1, of an egg before and after in vitro maturation, showing that development can still occur after live imaging with CARS (n=40). (C,F) Histograms of the number of LDs making up clusters in these cells, demonstrating the change in LD distribution over time. 0.1×0.1 µm xy pixel size; 0.5 µm z-step; 0.01 ms pixel dwell time; ∼13 mW (∼9 mW) pump (Stokes) power at the sample. Scale bars: 10 µm. Colour bar shows depth colour-coding from –25 µm-25 µm (0 µm being the equatorial plane). Data from >5 trials, using 1-3 mice each.
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DEV129908F3: Cell viability after live imaging with CARS. (A,D) Single z-plane DIC using a 1.27 NA water objective and a 1.4 NA oil condenser and (B,E) depth colour-coded images of CARS stacks at wavenumber 2850 cm−1, of an egg before and after in vitro maturation, showing that development can still occur after live imaging with CARS (n=40). (C,F) Histograms of the number of LDs making up clusters in these cells, demonstrating the change in LD distribution over time. 0.1×0.1 µm xy pixel size; 0.5 µm z-step; 0.01 ms pixel dwell time; ∼13 mW (∼9 mW) pump (Stokes) power at the sample. Scale bars: 10 µm. Colour bar shows depth colour-coding from –25 µm-25 µm (0 µm being the equatorial plane). Data from >5 trials, using 1-3 mice each.

Mentions: We investigated in detail the excitation conditions in our CARS experiment that allowed for live-cell imaging. We found that immature GV oocytes matured to MII stage in vitro after CARS imaging of the whole cell under the excitation conditions as in Fig. 1 (40/47 oocytes matured). Fig. 3 shows a CARS image of a fully scanned GV oocyte prior to in vitro maturation (IVM), and the same cell after 18 h in culture, once it had reached a mature stage. Notably, before maturation (Fig. 3A-C) the GV oocyte had a widely dispersed LD spatial distribution, and after IVM (Fig. 3D-F) the same cell is seen as an MII egg with characteristically aggregated LDs confirmed by quantitative analysis (Fig. 3C,F), supporting the observations from Fig. 1, in this case on the same egg followed over time. Control eggs that were not imaged prior to IVM, along with MII eggs matured in vivo, are included in Fig. S3.Fig. 3.


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)

Cell viability after live imaging with CARS. (A,D) Single z-plane DIC using a 1.27 NA water objective and a 1.4 NA oil condenser and (B,E) depth colour-coded images of CARS stacks at wavenumber 2850 cm−1, of an egg before and after in vitro maturation, showing that development can still occur after live imaging with CARS (n=40). (C,F) Histograms of the number of LDs making up clusters in these cells, demonstrating the change in LD distribution over time. 0.1×0.1 µm xy pixel size; 0.5 µm z-step; 0.01 ms pixel dwell time; ∼13 mW (∼9 mW) pump (Stokes) power at the sample. Scale bars: 10 µm. Colour bar shows depth colour-coding from –25 µm-25 µm (0 µm being the equatorial plane). Data from >5 trials, using 1-3 mice each.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

DEV129908F3: Cell viability after live imaging with CARS. (A,D) Single z-plane DIC using a 1.27 NA water objective and a 1.4 NA oil condenser and (B,E) depth colour-coded images of CARS stacks at wavenumber 2850 cm−1, of an egg before and after in vitro maturation, showing that development can still occur after live imaging with CARS (n=40). (C,F) Histograms of the number of LDs making up clusters in these cells, demonstrating the change in LD distribution over time. 0.1×0.1 µm xy pixel size; 0.5 µm z-step; 0.01 ms pixel dwell time; ∼13 mW (∼9 mW) pump (Stokes) power at the sample. Scale bars: 10 µm. Colour bar shows depth colour-coding from –25 µm-25 µm (0 µm being the equatorial plane). Data from >5 trials, using 1-3 mice each.
Mentions: We investigated in detail the excitation conditions in our CARS experiment that allowed for live-cell imaging. We found that immature GV oocytes matured to MII stage in vitro after CARS imaging of the whole cell under the excitation conditions as in Fig. 1 (40/47 oocytes matured). Fig. 3 shows a CARS image of a fully scanned GV oocyte prior to in vitro maturation (IVM), and the same cell after 18 h in culture, once it had reached a mature stage. Notably, before maturation (Fig. 3A-C) the GV oocyte had a widely dispersed LD spatial distribution, and after IVM (Fig. 3D-F) the same cell is seen as an MII egg with characteristically aggregated LDs confirmed by quantitative analysis (Fig. 3C,F), supporting the observations from Fig. 1, in this case on the same egg followed over time. Control eggs that were not imaged prior to IVM, along with MII eggs matured in vivo, are included in Fig. S3.Fig. 3.

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