Developing contrast-enhanced optical coherence tomography (OCT) techniques is important for specific imaging of tissue lesions, molecular imaging, cell-tracking, and highly sensitive microangiography and lymphangiography. Multiplexed OCT imaging in the second near-infrared (NIR-II) window is highly desirable since it allows simultaneous imaging and tracking of multiple biological events in high resolution with deeper tissue penetration in vivo. Here we demonstrate that gold nanobipyramids can function as OCT multiplexing contrast agents, allowing high-resolution imaging of two separate lymphatic flows occurring simultaneously from different drainage basins into the same lymph node in a live mouse. Contrast-enhanced multiplexed lymphangiography of a melanoma tumor in vivo shows that the peritumoral lymph flow upstream of the tumor is unidirectional, and tumor is accessible to such flow. Whereas the lymphatic drainage coming out from the tumor is multidirectional. We also demonstrate real-time tracking of the contrast agents draining from a melanoma tumor specifically to the sentinel lymph node of the tumor and the three-dimensional distribution of the contrast agents in the lymph node.
Background
A single base pair mutation in the genome can result in many congenital disorders in humans. The recent gene editing approach using CRISPR/Cas9 has rapidly become a powerful tool to replicate or repair such mutations in the genome. These approaches rely on cleaving DNA, while presenting unexpected risks.
Results
In this study, we demonstrate a modified CRISPR/Cas9 system fused to Cytosine DeAminase (Cas9-DA), which induces a single nucleotide conversion in the genome. Cas9-DA was introduced into sea urchin eggs with sgRNAs targeted for SpAlx1, SpDsh, or SpPks, each of which is critical for skeletogenesis, embryonic axis formation, or pigment formation, respectively. We found that both Cas9 and Cas9-DA edit the genome, and cause predicted phenotypic changes at a similar efficiency. Cas9, however, resulted in significant deletions in the genome centered on the gRNA target sequence, whereas Cas9-DA resulted in single or double nucleotide editing of C to T conversions within the gRNA target sequence.
Conclusion
These results suggest that the Cas9-DA approach may be useful for manipulating gene activity with decreased risks of genomic aberrations.
The
ability to detect multiple contrast agents simultaneously would greatly
enhance optical coherence tomography (OCT) images, providing nuanced
biological context to physiological structures. However, previous
OCT contrast agent work has been limited to scenarios where only a
single contrast agent could be robustly detected within each voxel.
We present a novel spectroscopic technique for demixing the spectral
signal from multiple OCT contrast agents within a single voxel. We
validate our technique in vitro and also demonstrate in vivo imaging of three spectrally distinct gold nanobipyramids,
trafficking within the lymphatic system of a live mouse.
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