Degree

Doctor of Philosophy (PhD)

Department

Chemistry

Document Type

Dissertation

Abstract

In this research, an infrared laser at a wavelength of 3 µm was used to ablate material from tissue sections for biomolecule analysis. Pulsed infrared (IR) irradiation of tissue with a focused laser beam efficiently removed biomolecules, such as proteins, enzymes, DNA, and RNA from tissue sections for further analysis. In a proteomics project, matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was used to determine regions of interest (ROI) for laser ablation. The matrix was then washed off. By overlaying the MSI generated heat-map, the section was sampled using IR laser ablation and custom stage-control software. Two ROI were selected and ablated from the same tissue section after MALDI-MSI. More than 700 proteins were identified in each region. A comparison of molecular localization and activity of identified proteins from two regions was performed. IR laser ablation was used to transfer enzymes while retaining their enzymatic activity. Three different laser fluences were used for ablating two enzymes: trypsin and catalase. Approximately 75% of the enzyme was transferred for all the fluences tested. According to fluorescence quantification, around 35% of the captured trypsin and 51% of the captured catalase were active after laser ablation. Regions were ablated and captured from frontal cortex and cerebellum of rat brain tissue sections and catalase activity was measured from the ablated material without further sample preparation. The catalase activity in the two regions was consistent with previously published data, demonstrating transfer of active enzymes from tissue. IR-laser ablation was used for sampling DNA and RNA. To test ablation transfer of large DNA, a 3200 base pair plasmid was used and evaluation of DNA quality after laser ablation was accomplished by comparing the sequencing performance of samples obtained from laser ablation and a control plasmid. Consistent results for intact DNA were obtained when the laser fluence was below 24 kJ/m2. Regions 1 and 4 mm2 square were ablated from rat brain and kidney tissue sections. Ablated material was amplified using polymerase chain reaction (PCR) with four primers from two genes. For RNA sampling, human kidney total RNA was used. The integrity of the RNA after laser ablation was monitored by gel electrophoresis. Low and high energy thresholds were determined, indicating the range in which intact RNA transfer could be achieved at the highest efficiency. Areas 2 mm2 square were ablated from the rat brain tissue. After RNA purification and reverse transcription, mRNA was amplified and quantified using quantitative PCR with two genes.

Date

3-18-2019

Committee Chair

Murray, Kermit K.

DOI

10.31390/gradschool_dissertations.4876

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