Towards instantaneous cellular level bio diagnosis: laser extraction and imaging of biological entities with conserved integrity and activity

Abstract

The prospect for spatial imaging with mass spectroscopy at the level of the cell requires new means of cell extraction to conserve molecular structure. To this aim, we demonstrate a new laser extraction process capable of extracting intact biological entities with conserved biological function. The method is based on the recently developed picosecond infrared laser (PIRL), designed specifically to provide matrix-free extraction by selectively exciting the water vibrational modes under the condition of ultrafast desorption by impulsive vibrational excitation (DIVE). The basic concept is to extract the constituent protein structures on the fastest impulsive limit for ablation to avoid excessive thermal heating of the proteins and to use strongly resonant 1-photon conditions to avoid multiphoton ionization and degradation of the sample integrity. With various microscope imaging and biochemical analysis methods, nanoscale single protein molecules, viruses, and cells in the ablation plume are found to be morphologically and functionally identical with their corresponding controls. This method provides a new means to resolve chemical activity within cells and is amenable to subcellular imaging with near-field approaches. The most important finding is the conserved nature of the extracted biological material within the laser ablation plume, which is fully consistent with in vivo structures and characteristics.

Homogenization of tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo composition of protein species as compared to mechanical homogenization

“…The soft ablation of tissue by PIRL is achieved under the conditions of desorption by impulsive excitation (DIVE) [21] in which the water molecules contained within the tissue are transferred into the gas phase in an ultrafast, explosive manner. DIVE results in intact cellular biomolecules blasting out of the sample with minimized heating or shock wave damage imposed on the tissue and biomolecules [22] , [23] , [24] , [25] . Kwiatkowski et al showed that the tissue proteome is present in the condensate of the DIVE-induced tissue aerosol.…”

Mass Spectrometry-Based Intraoperative Tumor Diagnostics

“…The gentle process of PIRL ablation was confirmed by a study of Ren et al . in which they showed that protein conformations are not significantly disturbed and even large macromolecular structures such as viruses are extracted intact without losing their biological activity [107]. They further showed that biological samples can be taken down to the level of individual cells using PIRL offering a finer resolution than the finest biopsy needles, thus having the potential to enable minimally invasive biopsies.…”

Digital interference microscopy and density reconstruction of picosecond infrared laser desorption at the water-air interface

“…At the same time, efficient energy redistribution into thermal modes is ensured by keeping the irradiance low enough to avoid nonlinear optical excitation of higher-lying molecular states. Picosecond infrared laser (PIRL) irradiation fulfilling these criteria has been shown to be well-suited for the extraction of large bio-molecules and even intact viruses for subsequent offline analysis, [20][21][22] as an atmospheric online ion source in combination with electrospray ionization (LAESI), 23 and has been suggested as a potential tool for sample-delivery in high-speed X-ray crystallography. 24 Moreover, the absence of ionizing radiation and minimal deposition of thermal and acoustic energy into the non-ablated sample surface make DIVE a valuable tool for precise material removal in surgical applications.…”