Infrared Laser Ablation for Biomolecule Sampling

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

An Inverse Finite Element Method For The Study Of Steady State Terrestrial Heat Flow Problems

In solving terrestrial heat flow problems, the complexity of the earth medium and boundary conditions calls for frequent use of numerical modeling techniques. The ill-posedness of the problems due to lack of perfect knowledge of the material properties and the boundary conditions requires that inverse theories be applied. Methods that incorporate both numerical techniques and inverse theories have therefore been gaining attentions in heat flow research.;In this study, an inverse finite element method is developed to solve 2-D steady state heat flow problems involving uncertain material properties and boundary conditions. The problems are first parameterized using an isoparametric finite element model, in which the field variables, the material properties and the boundary conditions are formulated as discrete parameters. Information on the parameters is described in the form of Gaussian probabilities. A nonlinear parameter estimation method of Bayesian type is then used to update our knowledge of the parameters. For computational efficiency, a gradient method is used in the parameter estimation procedure, and the gradients are derived analytically at the elemental level.;The method is applied to two types of conductive heat flow problems, namely the topographic correction and the downward continuation of heat flow data, and to the problem of coupled thermal and hydrological regimes of sedimentary basin scale. Numerical examples have shown that the method provides a rigorous treatment of uncertainities in these problems. In the case of the coupled problem, however, the power of the method is limited by the strong nonlinearity, and better a priori information is needed to constrain the solution