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Real-time high dynamic range laser scanning microscopy.

Vinegoni C, Leon Swisher C, Fumene Feruglio P, Giedt RJ, Rousso DL, Stapleton S, Weissleder R - Nat Commun (2016)

Bottom Line: In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes.We address reconstruction and segmentation performance on samples with different size, anatomy and complexity.Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Richard B. Simches Research Center, 185 Cambridge Street, Boston, Massachusetts 02114, USA.

ABSTRACT
In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

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In vivo intravascular dye kinetics.In vivo intravascular real-time quantification of the time–intensity variations demonstrating the vascular pharmacokinetics of a fluorescent probe across multiple regions of interests (ROIs). A bolus of 2 MDa FITC-Dextran was injected intravenously through the lateral tail vein (see ‘Methods' section) and vascular kinetics were captured by collecting a time sequence of real-time HDR images. (a) ROIs were selected in several blood vessels within a dorsal window chamber and time–intensity curves calculated as the mean of the signal within the ROI as a function of time. (b–d) Time–intensity curves are plotted for both (b,c) LDR and (d) HDR time sequences. LDR sequences have a limited ability to capture the full dynamic range of intensities, while HDR sequences present a high SNR and extended dynamic range demonstrating the ability to maintain imaging fidelity for kinetic quantification. Scale bar, 125 μm.
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f10: In vivo intravascular dye kinetics.In vivo intravascular real-time quantification of the time–intensity variations demonstrating the vascular pharmacokinetics of a fluorescent probe across multiple regions of interests (ROIs). A bolus of 2 MDa FITC-Dextran was injected intravenously through the lateral tail vein (see ‘Methods' section) and vascular kinetics were captured by collecting a time sequence of real-time HDR images. (a) ROIs were selected in several blood vessels within a dorsal window chamber and time–intensity curves calculated as the mean of the signal within the ROI as a function of time. (b–d) Time–intensity curves are plotted for both (b,c) LDR and (d) HDR time sequences. LDR sequences have a limited ability to capture the full dynamic range of intensities, while HDR sequences present a high SNR and extended dynamic range demonstrating the ability to maintain imaging fidelity for kinetic quantification. Scale bar, 125 μm.

Mentions: A bolus of 2 MDa FITC-Dextran was injected intravenously followed by a bolus of a 4 kDa FITC-dextran (see ‘Methods' section). The temporal resolution of the two-photon real-time acquisition was sufficient to capture the vascular kinetics of the 2 MDa probe (Fig. 10) and the extravasation of the 4 kDa probe into the interstitial tissue (Supplementary Fig. 24). Regions of interest were selected in the vascular (Fig. 10) and extravascular (Supplementary Fig. 24) compartments and time–intensity curves were calculated as the mean of the signal within the region of interest as a function of time.


Real-time high dynamic range laser scanning microscopy.

Vinegoni C, Leon Swisher C, Fumene Feruglio P, Giedt RJ, Rousso DL, Stapleton S, Weissleder R - Nat Commun (2016)

In vivo intravascular dye kinetics.In vivo intravascular real-time quantification of the time–intensity variations demonstrating the vascular pharmacokinetics of a fluorescent probe across multiple regions of interests (ROIs). A bolus of 2 MDa FITC-Dextran was injected intravenously through the lateral tail vein (see ‘Methods' section) and vascular kinetics were captured by collecting a time sequence of real-time HDR images. (a) ROIs were selected in several blood vessels within a dorsal window chamber and time–intensity curves calculated as the mean of the signal within the ROI as a function of time. (b–d) Time–intensity curves are plotted for both (b,c) LDR and (d) HDR time sequences. LDR sequences have a limited ability to capture the full dynamic range of intensities, while HDR sequences present a high SNR and extended dynamic range demonstrating the ability to maintain imaging fidelity for kinetic quantification. Scale bar, 125 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4821995&req=5

f10: In vivo intravascular dye kinetics.In vivo intravascular real-time quantification of the time–intensity variations demonstrating the vascular pharmacokinetics of a fluorescent probe across multiple regions of interests (ROIs). A bolus of 2 MDa FITC-Dextran was injected intravenously through the lateral tail vein (see ‘Methods' section) and vascular kinetics were captured by collecting a time sequence of real-time HDR images. (a) ROIs were selected in several blood vessels within a dorsal window chamber and time–intensity curves calculated as the mean of the signal within the ROI as a function of time. (b–d) Time–intensity curves are plotted for both (b,c) LDR and (d) HDR time sequences. LDR sequences have a limited ability to capture the full dynamic range of intensities, while HDR sequences present a high SNR and extended dynamic range demonstrating the ability to maintain imaging fidelity for kinetic quantification. Scale bar, 125 μm.
Mentions: A bolus of 2 MDa FITC-Dextran was injected intravenously followed by a bolus of a 4 kDa FITC-dextran (see ‘Methods' section). The temporal resolution of the two-photon real-time acquisition was sufficient to capture the vascular kinetics of the 2 MDa probe (Fig. 10) and the extravasation of the 4 kDa probe into the interstitial tissue (Supplementary Fig. 24). Regions of interest were selected in the vascular (Fig. 10) and extravascular (Supplementary Fig. 24) compartments and time–intensity curves were calculated as the mean of the signal within the region of interest as a function of time.

Bottom Line: In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes.We address reconstruction and segmentation performance on samples with different size, anatomy and complexity.Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

View Article: PubMed Central - PubMed

Affiliation: Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Richard B. Simches Research Center, 185 Cambridge Street, Boston, Massachusetts 02114, USA.

ABSTRACT
In conventional confocal/multiphoton fluorescence microscopy, images are typically acquired under ideal settings and after extensive optimization of parameters for a given structure or feature, often resulting in information loss from other image attributes. To overcome the problem of selective data display, we developed a new method that extends the imaging dynamic range in optical microscopy and improves the signal-to-noise ratio. Here we demonstrate how real-time and sequential high dynamic range microscopy facilitates automated three-dimensional neural segmentation. We address reconstruction and segmentation performance on samples with different size, anatomy and complexity. Finally, in vivo real-time high dynamic range imaging is also demonstrated, making the technique particularly relevant for longitudinal imaging in the presence of physiological motion and/or for quantification of in vivo fast tracer kinetics during functional imaging.

Show MeSH