We developed a spectrally-resolved multi-photon imaging system, based on a closed loop piezoelectric sample scanning stage, a transmission grating and an EMCCD. The method allows detailed analysis of the spectrally-resolved signals, including deconvolution of the resulting emission peaks, and precise differentiation of the resulting signals. The system also makes available the possibility of using point spread function analyses, such as pixel reassignment, Airy scan and full three-dimensional, spectral imaging. We present multi-photon induced fluorescence, second harmonic and fluorescence lifetime imaging analysis supporting the development of non-invasive photo-polymerizable scaffolds for treatment of peripheral artery disease. Currently, the technology, developed by Alumend, LLC, is undergoing clinical trials and is licensed by Alucent Medical. In this work we report a comparison of the second harmonic generation and two photon induced fluorescence imaging in drug-infused arteries, and compare these to fluorescence lifetime images obtained using our commercial (Picoquant) fluorescence lifetime imaging system. Primary signals from the endogenous fluorescence from the drug and the second harmonic generation, prevalent in collagen, are compared. Of particular interest, we observe the photo-chemical modification of the drug fluorescence emission energy and lifetime in the adventitia, composed primarily of collagen. The drug aids in cross-linking the compressed collagen fibrils in the arterial wall during the light activation step, which leads to formation of the stent.
Human epidermal growth receptor 2 (Her2) is a gene which plays a major role in breast cancer development. The quantification of Her2 expression in single cells is limited by several drawbacks in existing fluorescence-based single molecule techniques, such as low signal-to-noise ratio (SNR), strong autofluorescence and background signals from biological components. For rigorous genomic quantification, a robust method of orthogonal detection is highly desirable and we demonstrated it by two non-fluorescent imaging techniques -transient absorption microscopy (TAM) and second harmonic generation (SHG). In TAM, gold nanoparticles (AuNPs) are chosen as an orthogonal probes for detection of single molecules which gives background-free quantifications of single mRNA transcript. In SHG, emission from barium titanium oxide (BTO) nanoprobes was demonstrated which allows stable signal beyond the autofluorescence window. Her2 mRNA was specifically labeled with nanoprobes which are conjugated with antibodies or oligonucleotides and quantified at single copy sensitivity in the cancer cells and tissues. Furthermore, a non-fluorescent super-resolution concept, named as second harmonic super-resolution microscopy (SHaSM), was proposed to quantify individual Her2 transcripts in cancer cells beyond the diffraction limit. These non-fluorescent imaging modalities will provide new dimensions in biomarker quantification at single molecule sensitivity in turbid biological samples, offering a strong cross-platform strategy for clinical monitoring at single cell resolution.
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