High spatial resolution genomics analysis is important because individual cells heterogeneity results in relevant differences in gene regulation, proteins expression, and metabolites levels that can be missed in analysis of bulk material. While in-situ techniques can be employed to analyze genomic material directly on a tissue surface, the tissue matrix may limit applicability of these techniques, thus making necessary an extraction step. Existing techniques such as cell sorting or manual picking are either sacrificing localization information or are limited in precision and throughput. Laser techniques, such as laser capture microdissection, can sample precisely from arbitrarily chosen location on a tissue section, but the resolution is limited to few microns by the size of the laser spot. We developed a technique called tip-enhanced laser ablation, which utilizes an atomic force microscopy (AFM) is coupled with a visible wavelength optical parametric oscillator (OPO) laser to sample cells and tissue at a lateral resolution of ~1 µm. The laser is directed onto a gold-coated silicon AFM tip to achieve tip-enhanced laser ablation at sub μm spatial resolution using the optical antenna effect. To test the integrity of laser-ablation transferred DNA, a 3200 base-pair plasmid was ablated and captured. Both AFM imaging and tip-enhanced laser ablation were performed in tapping mode. The captured material was quantified and it was found that 0.5 – 10 fg material could be captured intact from 10 ablated spots. The laser wavelength and pulse energy dependence of the ablation was studied. In ongoing experiments, DNA and RNA material ablated and captured from tissue samples is captured for sequencing and PCR quantification.

Tip-enhanced laser ablation capture for single cell genomics

255th National Meeting (2018)

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