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Breaking the spatial resolution barrier via iterative sound-light interaction in deep tissue microscopy.

Si K, Fiolka R, Cui M - Sci Rep (2012)

Bottom Line: Random scattering causes the ballistic focus, which is conventionally used for image formation, to decay exponentially with depth.Optical imaging beyond the ballistic regime has been demonstrated by hybrid techniques that combine light with the deeper penetration capability of sound waves.This development opens up practical high resolution fluorescence imaging in deep tissues.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, Virginia 20147, USA.

ABSTRACT
Optical microscopy has so far been restricted to superficial layers, leaving many important biological questions unanswered. Random scattering causes the ballistic focus, which is conventionally used for image formation, to decay exponentially with depth. Optical imaging beyond the ballistic regime has been demonstrated by hybrid techniques that combine light with the deeper penetration capability of sound waves. Deep inside highly scattering media, the sound focus dimensions restrict the imaging resolutions. Here we show that by iteratively focusing light into an ultrasound focus via phase conjugation, we can fundamentally overcome this resolution barrier in deep tissues and at the same time increase the focus to background ratio. We demonstrate fluorescence microscopy beyond the ballistic regime of light with a threefold improved resolution and a fivefold increase in contrast. This development opens up practical high resolution fluorescence imaging in deep tissues.

No MeSH data available.


Related in: MedlinePlus

(a) Direct widefield image of the fluorescent structure without tissue phantoms.(b) Direct widefield image of the fluorescent structure surrounded by 2 mm thick tissue phantoms (μs  = 7.63 /mm, g factor = 0.9013). (c) Image acquired with the first round of ultrasound pulse guided DOPC. (d) Image acquired with five iterations. (e) Image acquired with nine iterations. (f) 2D convolution with a 2D Gaussian function (FWHM: 12 microns). Scalebar: a, b: 100 microns, c: 20 microns. Colorbar in arbitrary units.
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f3: (a) Direct widefield image of the fluorescent structure without tissue phantoms.(b) Direct widefield image of the fluorescent structure surrounded by 2 mm thick tissue phantoms (μs = 7.63 /mm, g factor = 0.9013). (c) Image acquired with the first round of ultrasound pulse guided DOPC. (d) Image acquired with five iterations. (e) Image acquired with nine iterations. (f) 2D convolution with a 2D Gaussian function (FWHM: 12 microns). Scalebar: a, b: 100 microns, c: 20 microns. Colorbar in arbitrary units.

Mentions: To demonstrate imaging of a complex fluorescent structure deep inside highly scattering media, we fabricated a c-shaped pattern consisting of fluorescent microspheres of 6 microns in diameter, completely embedded in the middle of a 4 mm thick tissue phantom (scattering coefficient: 7.63 /mm, g factor: 0.9013). In Fig. 3 a, a widefield microscopy image of the c-shaped fluorescence pattern is shown before it was embedded in the tissue phantom. Figure 3 b shows a widefield image taken through the tissue phantom. The shape information is completely lost due to the strong scattering. In Fig. 3 c, an image obtained with the first DOPC iteration is shown. The scanning step size was 6 microns and the raw data was re-sampled with linear interpolation. The structure can now be localized, but the shape of the object is not resolved. In Fig. 3 d, an image obtained after five DOPC iterations is shown. At this stage, the c-shape is already recognizable. After nine DOPC iterations the c-shaped structure is clearly resolved owing to the increased lateral resolution (Fig. 3 e). For comparison, we re-sampled the widefield image in Fig. 3 a to the same pixel size as in Fig. 3 c–e and convolved it with a Gaussian-shaped PSF (FWHM: 12 microns). The resulting image is shown in Fig. 3 f. An additional imaging experiment using sparsely distributed fluorescent beads is shown in Supplementary Fig. 2.


Breaking the spatial resolution barrier via iterative sound-light interaction in deep tissue microscopy.

Si K, Fiolka R, Cui M - Sci Rep (2012)

(a) Direct widefield image of the fluorescent structure without tissue phantoms.(b) Direct widefield image of the fluorescent structure surrounded by 2 mm thick tissue phantoms (μs  = 7.63 /mm, g factor = 0.9013). (c) Image acquired with the first round of ultrasound pulse guided DOPC. (d) Image acquired with five iterations. (e) Image acquired with nine iterations. (f) 2D convolution with a 2D Gaussian function (FWHM: 12 microns). Scalebar: a, b: 100 microns, c: 20 microns. Colorbar in arbitrary units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3475990&req=5

f3: (a) Direct widefield image of the fluorescent structure without tissue phantoms.(b) Direct widefield image of the fluorescent structure surrounded by 2 mm thick tissue phantoms (μs = 7.63 /mm, g factor = 0.9013). (c) Image acquired with the first round of ultrasound pulse guided DOPC. (d) Image acquired with five iterations. (e) Image acquired with nine iterations. (f) 2D convolution with a 2D Gaussian function (FWHM: 12 microns). Scalebar: a, b: 100 microns, c: 20 microns. Colorbar in arbitrary units.
Mentions: To demonstrate imaging of a complex fluorescent structure deep inside highly scattering media, we fabricated a c-shaped pattern consisting of fluorescent microspheres of 6 microns in diameter, completely embedded in the middle of a 4 mm thick tissue phantom (scattering coefficient: 7.63 /mm, g factor: 0.9013). In Fig. 3 a, a widefield microscopy image of the c-shaped fluorescence pattern is shown before it was embedded in the tissue phantom. Figure 3 b shows a widefield image taken through the tissue phantom. The shape information is completely lost due to the strong scattering. In Fig. 3 c, an image obtained with the first DOPC iteration is shown. The scanning step size was 6 microns and the raw data was re-sampled with linear interpolation. The structure can now be localized, but the shape of the object is not resolved. In Fig. 3 d, an image obtained after five DOPC iterations is shown. At this stage, the c-shape is already recognizable. After nine DOPC iterations the c-shaped structure is clearly resolved owing to the increased lateral resolution (Fig. 3 e). For comparison, we re-sampled the widefield image in Fig. 3 a to the same pixel size as in Fig. 3 c–e and convolved it with a Gaussian-shaped PSF (FWHM: 12 microns). The resulting image is shown in Fig. 3 f. An additional imaging experiment using sparsely distributed fluorescent beads is shown in Supplementary Fig. 2.

Bottom Line: Random scattering causes the ballistic focus, which is conventionally used for image formation, to decay exponentially with depth.Optical imaging beyond the ballistic regime has been demonstrated by hybrid techniques that combine light with the deeper penetration capability of sound waves.This development opens up practical high resolution fluorescence imaging in deep tissues.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, Janelia Farm Research Campus, 19700 Helix Drive, Ashburn, Virginia 20147, USA.

ABSTRACT
Optical microscopy has so far been restricted to superficial layers, leaving many important biological questions unanswered. Random scattering causes the ballistic focus, which is conventionally used for image formation, to decay exponentially with depth. Optical imaging beyond the ballistic regime has been demonstrated by hybrid techniques that combine light with the deeper penetration capability of sound waves. Deep inside highly scattering media, the sound focus dimensions restrict the imaging resolutions. Here we show that by iteratively focusing light into an ultrasound focus via phase conjugation, we can fundamentally overcome this resolution barrier in deep tissues and at the same time increase the focus to background ratio. We demonstrate fluorescence microscopy beyond the ballistic regime of light with a threefold improved resolution and a fivefold increase in contrast. This development opens up practical high resolution fluorescence imaging in deep tissues.

No MeSH data available.


Related in: MedlinePlus