Rat Brain Tissue Sections Research Articles

Linalool acts as a chemical chaperone by inhibiting amyloid-β aggregation

Linalool is a neuroprotective monoterpene found in essential oils from aromatic plants. Linalool's effectiveness in AD animal models has been established previously, but its mechanisms of action remain unclear. Therefore, this study aims to investigate whether linalool binds directly to the amyloid beta (Aβ) fibrils to understand it's role in preventing neurodegeneration. The anti-aggregation ability of Linalool was determined using Dithiothreitol (DTT), and thermal aggregation assays followed by Thioflavin T (ThT) binding assay. AD animals were treated with Linalool, and Thioflavin T staining was used to check the binding of linalool to Aβ fibrils in rat brain tissue sections. Preliminary studies revealed the anti-aggregation potential of linalool under the thermal and chemical stimulus. Further, in ThT binding assay Linalool inhibited Aβ aggregation, binding directly to Aβ fibrils. The reduced fluorescence intensity of ThT in AD brain tissues following linalool administration, highlights its neuroprotective potential as a therapeutic agent for AD.

Improved Detection of Tryptic Peptides from Tissue Sections Using Desorption Electrospray Ionization Mass Spectrometry Imaging.

DESI-MSI is an ambient ionization technique used frequently for the detection of lipids, small molecules, and drug targets. Until recently, DESI had only limited use for the detection of proteins and peptides due to the setup and needs around deconvolution of data resulting in a small number of species being detected at lower spatial resolution. There are known differences in the ion species detected using DESI and MALDI for nonpeptide molecules, and here, we identify that this extends to proteomic species. DESI MS images were obtained for tissue sections of mouse and rat brain using a precommercial heated inlet (approximately 450 °C) to the mass spectrometer. Ion mobility separation resolved spectral overlap of peptide ions and significantly improved the detection of multiply charged species. The images acquired were of pixel size 100 μm (rat brain) and 50 μm (mouse brain), respectively. Observed tryptic peptides were filtered against proteomic target lists, generated by LC-MS, enabling tentative protein assignment for each peptide ion image. Precise localizations of peptide ions identified by DESI and MALDI were found to be comparable. Some spatially localized peptides ions were observed in DESI that were not found in the MALDI replicates, typically, multiply charged species with a low mass to charge ratio. This method demonstrates the potential of DESI-MSI to detect large numbers of tryptic peptides from tissue sections with enhanced spatial resolution when compared to previous DESI-MSI studies.

Imaging Mass Spectrometry of Isotopically Resolved Intact Proteins on a Trapped Ion-Mobility Quadrupole Time-of-Flight Mass Spectrometer.

In this work, we demonstrate rapid, high spatial, and high spectral resolution imaging of intact proteins by matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on a hybrid quadrupole-reflectron time-of-flight (qTOF) mass spectrometer equipped with trapped ion mobility spectrometry (TIMS). Historically, untargeted MALDI IMS of proteins has been performed on TOF mass spectrometers. While advances in TOF instrumentation have enabled rapid, high spatial resolution IMS of intact proteins, TOF mass spectrometers generate relatively low-resolution mass spectra with limited mass accuracy. Conversely, the implementation of MALDI sources on high-resolving power Fourier transform (FT) mass spectrometers has allowed IMS experiments to be conducted with high spectral resolution with the caveat of increasingly long data acquisition times. As illustrated here, qTOF mass spectrometers enable protein imaging with the combined advantages of TOF and FT mass spectrometers. Protein isotope distributions were resolved for both a protein standard mixture and proteins detected from a whole-body mouse pup tissue section. Rapid (∼10 pixels/s) 10 μm lateral spatial resolution IMS was performed on a rat brain tissue section while maintaining isotopic spectral resolution. Lastly, proof-of-concept MALDI-TIMS data was acquired from a protein mixture to demonstrate the ability to differentiate charge states by ion mobility. These experiments highlight the advantages of qTOF and timsTOF platforms for resolving and interpreting complex protein spectra generated from tissue by IMS.

A multimodal pipeline for image correction and registration of mass spectrometry imaging with microscopy

The integration of matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) and histology plays a pivotal role in advancing our understanding of complex heterogeneous tissues, which provides a comprehensive description of biological tissue with both wide molecule coverage and high lateral resolution. Herein, we proposed a novel strategy for the correction and registration of MALDI MSI data with hematoxylin & eosin (H&E) staining images. To overcome the challenges of discrepancies in spatial resolution towards the unification of the two imaging modalities, a deep learning-based interpolation algorithm for MALDI MSI data was constructed, which enables spatial coherence and the following orientation matching between images. Coupled with the affine transformation (AT) and the subsequent moving least squares algorithm, the two types of images from one rat brain tissue section were aligned automatically with high accuracy. Moreover, we demonstrated the practicality of the developed pipeline by projecting it to a rat cerebral ischemia-reperfusion injury model, which would help decipher the link between molecular metabolism and pathological interpretation towards microregion. This new approach offers the chance for other types of bioimaging to boost the field of multimodal image fusion.

Design and characterizing of robust probes for enhanced mass spectrometry imaging and spatially resolved metabolomics

As for the emerging and cut edge spatially resolved metabolomics, mass spectrometry imaging (MSI) is a powerful tool that can map thousands of metabolites from bio-tissue sections without chemical labels. However, the stability, sensitivity and spatial resolution of MSI are always limited by the performance of its ionization probe. Herein, two types of probes (fine probe (P-100) and large probe (P-200)) were designed and characterized to perform air-flow assisted desorption electrospray ionization (AFA-DESI) MSI analysis for spatially resolved metabolomics. It was determined that the spray introduced by P-100 was homogenous and stable under the spray solvent at a flow rate of 5-10 µL/min, while P-200 can endure a high flow rate of up to 10-30 µL/min. Moreover, the MSI images were acquired by AFA-DESI-MSI with P-100 from rat brain tissue section and with P-200 from whole-body tissue section of mouse, and these results presented unambiguous tissue structure with the distribution information of numerous metabolites. Furthermore, the spatially resolved metabolomic analysis of tumor tissue was successfully realized to discover the tumor associated biomarkers. As the key parts of AFA-DESI-MSI system, it has been demonstrated that the designed probs have excellent performance for spatially resolved metabolomics, and it will further promote its application in life science, and drug research and development.

Increased blood-brain barrier permeability correlate with microglial activation at hippocampal CA1 region in acute and chronic bilateral common carotid artery ligation in rats

Inflammatory processes might play a key role in the pathogenesis of post-stroke epilepsy. The activation of microglia and release of vascular cell adhesion molecule-1 (VCAM1) might induce blood-brain barrier (BBB) disintegration. However, the influence of such pathomechanisms in the generation of post-stroke epilepsy is still not clear. We investigated whether cerebral ischemia exerts effects on inflammation in the hippocampus by measuring the hippocampal injury score, expression of a microglial marker, and expression of VCAM1 in rats. A total of 24 Sprague Dawley rats were randomized into four groups with 6 rats in eachgroup i.e. sham operation (SO) as control, carotid ligation 1 (GCL1) as an acute model, carotid ligation 3 (GCL3) as a subacute model, and carotid ligation 7 (GCL7) as a chronic model. Immunostaining for microglia marker (CD68) was measured in rat brain tissue sections. The VCAM1 expression was evaluated by reverse transcription-polymerase chain reaction (RT-PCR). Cerebral ischemia increased the amount of microglial immunostaining and expression of VCAM1. The hippocampal injury score and microglial immunopositivity were significantly correlated with the duration of brain ischemia. We conclude that cerebral ischemia is correlated with neuroinflammatory reaction and disturbance of BBB permeability, and the correlation of those molecular impairments with the generation of post-stroke epilepsy remains to be elucidated.

Infrared Laser Ablation Microsampling for Small Volume Proteomics.

Infrared (IR) laser ablation was used to remove localized tissue regions from which proteins were extracted and processed with a low volume sample preparation workflow for bottom-up proteomics by liquid chromatography tandem mass spectrometry (LC-MS/MS). A polytetrafluoroethylene (PTFE) coated glass slide with 2 mm diameter microwells was used to capture ablated rat brain tissue for in situ protein digestion with submicroliter solution volumes. The resulting peptides were analyzed with LC-MS/MS for protein identification and label-free quantification. The method was used to identify an average of 600, 1350, and 1900 proteins from ablation areas of 0.01, 0.04, and 0.1 mm2, respectively, from a 50 μm thick rat brain tissue section. Differential proteomics of 0.01 mm2 regions captured from cerebral cortex and corpus callosum was accomplished to demonstrate the capabilities of the approach.

Capillary array electrophoresis imaging of biochemicals in tissue sections.

It is of great significance to reveal the molecular distribution images in biological tissues, which has led to the bloom of mass spectrometry imaging. Unfortunately, its application is encountering the resistance of high technical barriers and equipment cost, as well as the inability to image substances that cannot be desorbed or ionized, or cannot be separated by their mass-to-charge ratios. Herein presented is a complementary and cost-effective method called capillary array electrophoresis (CAE) imaging. To have the information of molecules and their spatial location, a gridding cutter was fabricated to orderly dissect a tissue section into a leakproof array of micro wells enclosed by the grid-blade arrays. After in situ extraction and fluorophore-labeling of analytes, the samples in the wells were directly subjected to CAE-LIF (laser-induced fluorescence), and the molecular distribution images were depicted with the separated peaks. The practicability was demonstrated by CAE imaging of rat brain tissue sections with amino acid neurotransmitters (e.g., glutamine, 4-aminobutyric acid, alanine, glutamic acid and aspartic acid) as targets. The resultant images showed the global differences of molecular distributions, with a spatial resolution of 1000μm that was presently determined by the well width but ultimately by the bore size of capillary (down to 10-50μm). CAE imaging can hence be promising for its low cost, low technical barriers and abundant mechanisms to separate the charged and non-charged, chiral and non-chiral substances.

Multimodal Mass Spectrometry Imaging of Rat Brain Using IR-MALDESI and NanoPOTS-LC-MS/MS.

Multimodal mass spectrometry imaging (MSI) is a critical technique used for deeply investigating biological systems by combining multiple MSI platforms in order to gain the maximum molecular information about a sample that would otherwise be limited by a single analytical technique. The aim of this work was to create a multimodal MSI approach that measures metabolomic and proteomic data from a single biological organ by combining infrared matrix-assisted laser desorption electrospray ionization (IR-MALDESI) for metabolomic MSI and nanodroplet processing in one pot for trace samples (nanoPOTS) LC-MS/MS for spatially resolved proteome profiling. Adjacent tissue sections of rat brain were analyzed by each platform, and each data set was individually analyzed using previously optimized workflows. IR-MALDESI data sets were annotated by accurate mass and spectral accuracy using HMDB, METLIN, and LipidMaps databases, while nanoPOTS-LC-MS/MS data sets were searched against the rat proteome using the Sequest HT algorithm and filtered with a 1% FDR. The combined data revealed complementary molecular profiles distinguishing the corpus callosum against other sampled regions of the brain. A multiomic pathway integration showed a strong correlation between the two data sets when comparing average abundances of metabolites and corresponding enzymes in each brain region. This work demonstrates the first steps in the creation of a multimodal MSI technique that combines two highly sensitive and complementary imaging platforms. Raw data files are available in METASPACE (https://metaspace2020.eu/project/pace-2021) and MassIVE (identifier: MSV000088211).

Deep-ultraviolet laser ablation sampling for proteomic analysis of tissue

Deep-ultraviolet laser ablation with a pulsed 193 nm ArF excimer laser was used to remove localized regions from tissue sections from which proteins were extracted for spatially resolved proteomic analysis by liquid chromatography tandem mass spectrometry (LC-MS/MS). The ability to capture intact proteins by ablation at 193 nm wavelength was verified by matrix-assisted laser desorption ionization (MALDI) of the protein standard bovine serum albumin (BSA), which showed that BSA was ablated and captured without fragmentation. A Bradford assay of the ablated and captured proteins indicated 90% efficiency for transfer of the intact protein at a laser fluence of 3 kJ/m2. Rat brain tissue sections mounted on quartz microscope slides and ablated in transmission mode yielded 2 μg protein per mm2 as quantified by the Bradford assay. Tissue areas ranging from 0.06 mm2 to 1 mm2 were ablated and the ejected material was collected for proteomic analysis. Extracted proteins were digested and the resulting peptides were analyzed by LC-MS/MS. The proteins extracted from the ablated areas were identified and the average number of identified proteins ranged from 85 in the 0.06 mm2 area to 2400 in the 1 mm2 area of a 50 μm thick tissue. In comparison to infrared laser ablation of equivalent sampled areas, both the protein mass and number of proteins identified using DUV laser ablation sampling were approximately four times larger.

Multimodal chemical imaging of a single brain tissue section using ToF-SIMS, MALDI-ToF and immuno/histochemical staining.

Cluster ion beam ToF-SIMS and/or MALDI-ToF mass spectrometry imaging (using 1,5-DAN matrix via sublimation) of a single coronal rat brain tissue section followed by classical- or immuno- histochemical staining faclilated a new multimodal chemical imaging workflow allowing complementary correlation of the lipid molecular ion images with the immuno/histological features within cerebellum region of the same brain tisue section.

Integration of 3D-printing for a desorption electrospray ionization source for mass spectrometry.

The field of ambient ionization mass spectrometry has witnessed the development of many novel and capable methods for the analysis and imaging of surfaces, with desorption electrospray ionization being a prominent technique that has been commercialized. The adaptation of this technique to existing mass spectrometry platforms requires a laboratory-built solution manufactured with the capability of fine, stable adjustments of the electrospray emitter for liquid or solid sampling purposes. The development, fabrication, and machining require tens of hours of labor for many custom solutions. Herein described is a highly modifiable alternative approach for the fabrication of a desorption electrospray ionization source, using computer-aided design and fused deposition modeling to three-dimensionally print a source platform that utilizes standard accessories of a commercial Bruker Daltonics mass spectrometer. Three-dimensional printing allows for the inexpensive, rapid development of highly modifiable plastic parts, with the total printing time of the apparatus requiring a singular day and only a few dollars of material using a consumer grade printer. To demonstrate the utility of this printed desorption electrospray ionization source, it was fitted on an unmodified Fourier transform ion cyclotron resonance mass spectrometer for a lipid fingerprint analysis in serial sections of rat brain tissue, with the acquisition of line scans of dye-coated slides for the demonstration of serial acquisition.

MALDI-2 on a Trapped Ion Mobility Quadrupole Time-of-Flight Instrument for Rapid Mass Spectrometry Imaging and Ion Mobility Separation of Complex Lipid Profiles.

Matrix-assisted laser desorption/ionization combined with laser-induced postionization (MALDI-2) is a recently introduced method for enhanced mass spectrometry imaging of numerous classes of biomolecules, including phospho- and glycolipids in tissue sections at high lateral resolution. Here we describe the first adaptation of the technology to a Bruker timsTOF fleX mass spectrometer. Upon use of a 1 kHz postionization laser, MALDI-2 produces a sizable increase in the number of detected features as well as in ion signal intensities. This enhancement is similar to that described previously for low repetition rate MALDI-2 systems, but now enables substantially enhanced measurement speeds. In our proof-of-concept study, we furthermore demonstrate, on examples of rat brain and testis tissue sections, that the combination of MALDI-2 with the trapped ion mobility spectrometry (TIMS) functionality of the instrument can crucially support unravelling the complex molecular composition of the lipidome. Numerous isomeric/isobaric ion species are successfully separated upon using the collisional cross section (CCS) as additional specific physical property. With the possibilities of high data acquisition speed or high separation powers in combination with the increased sensitivity of MALDI-2 available in one instrument, the described methodology could be a valuable tool in many areas of biological and medical research.

Simultaneous mass spectrometry imaging of multiple neuropeptides in the brain and alterations induced by experimental parkinsonism and L-DOPA therapy

Neuropeptides are important signalling molecules in the brain and alterations in their expression levels have been linked to neurological disorders such as Parkinson's disease. It is challenging to map neuropeptide changes across and within brain regions because of their low in vivo concentrations and complex post-translational processing. Consequently, the role of neuropeptides in Parkinson's disease is not well understood. Thus, we have developed and evaluated a method to image multiple neuropeptides simultaneously in both rat and primate brain tissue sections by matrix-assisted laser desorption/ionisation mass spectrometry imaging at high lateral resolution. Using a unilateral 6-hydroxydopamine rat model of Parkinson's disease, we imaged changes in enkephalins, dynorphins, tachykinins and neurotensin associated with the dopaminergic denervation and L-DOPA treatment in multiple brain regions. L-DOPA administration significantly affected neuropeptides in the globus pallidus, while neuropeptides in the caudate-putamen were mostly affected by dopamine depletion. Using high lateral resolution imaging, we observed an increase of neurotensin in the dorsal sub-region of the globus pallidus after dopamine depletion. This study highlights the capacity of mass spectrometry imaging to elucidate the dynamics of neuropeptide signalling during Parkinson's disease and its treatment.

Enhancement of Lipid Signal in MALDI MS imaging obtained from Formalin Fixed Human Brain Tissue

Matrix assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is used to perform mass spectrometric analysis directly on biological samples providing accurate visual/anatomical spatial information of molecules within the tissue. A current limitation of MALDI‐IMS is that it is largely performed on fresh frozen tissue whereas clinical tissue samples stored long term are fixed in formalin. It has been shown that fresh frozen tissue sections applied with an ammonium formate (AF) wash prior to matrix application in the MALDI‐IMS procedure display an increase in observed signal intensity and sensitivity for lipid molecules detected in the brain while maintaining the special distribution of molecules throughout the tissue. In this work we investigate the effectiveness of this AF wash on post‐fixed rat and human brain tissue sections in an effort to increase the viability of formalin fixed tissue imaging in a clinical setting. Results herein demonstrate that the AF wash significantly improved MALDI‐IMS spectra for gangliosides, including GM1 in fresh frozen rat brain, formalin‐fixed rat brain and formalin fixed human brain samples. AF wash also demonstrated improvements in MALDI‐IMS image quality while retaining the spatial distribution of molecules. Results indicate that this method will allow analysis of gangliosides from formalin‐fixed clinical samples, which can open additional avenues for neurodegenerative disease research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RNA sampling from tissue sections using infrared laser ablation

RNA was obtained from discrete locations of frozen rat brain tissue sections through infrared (IR) laser ablation using a 3-μm wavelength in transmission geometry. The ablated plume was captured in a microcentrifuge tube containing RNAse-free buffer and processed using a commercial RNA purification kit. RNA transfer efficiency and integrity were evaluated based on automated electrophoresis in microfluidic chips. Reproducible IR-laser ablation of intact RNA was demonstrated with purified RNA at laser fluences of 3–5 kJ/m2 (72 ± 12% transfer efficiency) and with tissue sections at a laser fluence of 13 kJ/m2 (79 ± 14% transfer efficiency); laser energies were attenuated ∼20% by the soda-lime glass slides used to support the samples. RNA integrity from tissue ablation was >90% of its original RIN value (∼7) and the purified RNA was sufficiently intact for conversion to cDNA and subsequent qPCR assay.

Molecular depth profiling on rat brain tissue sections prepared using different sampling methods.

Brain imaging using time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been reported to produce the distorted biomolecular distributions due to the cholesterol-induced matrix effect when cholesterol migrates to the surface, particularly in white matter, which contains a high level of cholesterol. Frozen-hydrated analysis has been used to inhibit the movement of cholesterol in the brain. In this paper, the authors propose new sample preparation and drying methods that can be used to obtain accurate biomolecular images at room temperature, instead of frozen-hydrated analysis using liquid-nitrogen, which must be continuously supplied to maintain the sample at -160 °C during the experiment. The rat brain prepared by the tape-supporting method on a precooled (-20 °C) stainless steel plate was freeze-dried in a load-lock chamber of ToF-SIMS for about an hour and moved directly to the main chamber. Using this preparation method, the authors found that cholesterol did not migrate to the surface in the corpus callosum (white matter) of the rat brain and sulfatide-related signals obtained from the cerebellum were not reduced in white matter. Our tape-supporting and freeze-drying sampling method for brain tissues could be a useful tool to study important metabolites of neurodegenerative diseases.

A novel animal model of hypoxic-ischemic encephalopathy established by tracheal clamping

Objective To establish a simple, reliable and reproducible animal model of neonatal hypoxic-ischemic encephalopathy (HIE) with similar clinical pathological process to neonates. Methods Seven days after birth, 180 Sprague-Dawley (SD) rats were randomly divided into six groups: blank control group, experimental control group and four hypoxia groups (8, 10, 12 and 14 min hypoxia groups). Those in the experimental groups were locally anesthetized with 5% lidocaine to separate their tracheas through blunt dissection, followed by tracheal clamping with vascular clamp for 8, 10, 12 and 14 min, respectively. Rats in the experimental control group were only treated with blunt dissection of trachea. No intervention was given to the blank control group. Due to significant reduction in rat survival rate after 14 min of hypoxia, no further morphological or behavioral examination was performed in this group. Rat brain tissue sections were stained with hematoxylin-eosin (HE) 12 h after modeling. Three days after modeling, the rat brain was weighted and the apoptosis of neural cells was detected with terminal deoxynucleotidyl transferase(TdT) mediated dUTP nick end labeling (TUNEL). Morris water maze was used to screen cognitive impairment in these rats at the age of two months. One-way analysis of variance was used for statistical analysis. SNK test and Dunnett 's T3 test were performed to compare homogeneous and non-homogeneous data between groups. Results Systemic cyanosis, loss of consciousness, paled body, urinary and fecal incontinence, twitching of the limbs and tail and other abnormal behavior were induced by hypoxia. Ischemic necrosis, bleeding, nucleus shrinkage in a large number of neurons and hyperchromatic nuclei were observed in the 8, 10 and 12 min hypoxia groups. Three days after modeling, brain weights of rats in the 8, 10 and 12 min hypoxia groups were lower than those of the blank control group and experimental control group [(1.16±0.07), (1.04±0.06), (0.97±0.12), (1.31±0.06) and (1.28±0.09) g, F=36.437, P<0.001]. However, the numbers of apoptotic cortical [(22.83±4.52), (30.25±3.02), (39.18±5.04), (7.96±2.24) and (8.86±2.49)/400 scope field, F=164.532, P<0.001] and hippocampal CA3 neurons of that three hypoxia groups were higher than those of the two control groups [(14.63±2.26), (20.25±3.02), (24.81±1.98), (4.75±2.66) and (6.67±1.78)/400 scope field, F=141.026, P<0.001]. At the age of two months, rats in the 8, 10 and 12 min hypoxia groups had longer escape latency [(17.99±6.48), (23.07±9.90), (38.94±32.46), (14.37±6.06) and (12.78±7.21) s, F=26.912, P<0.001] and fewer times of platform crossings than those in the control group and experimental control group [(5.00±1.41), (4.90±1.29), (3.75±1.83), (7.57±1.16) and (7.14±1.15) times, F=14.336, P<0.001]. Conclusions Pathological changes in brain tissues and behaviors of rats after modeling are in line with the characteristics of classic animal model of HIE and similar to the clinical pathology and physiology of HIE, and this could be a new, simple, reliable and reproducible animal model of HIE, being capable of controlling the duration of hypoxia accurately. Key words: Hypoxia-ischemia, brain; Disease models, animal; Trachea; Constriction

Infrared laser ablation sample transfer of tissue DNA for genomic analysis.

Infrared (IR) laser ablation was used to remove material from tissue sections mounted on microscope slides, with subsequent capture in a solvent-containing microcentrifuge tube. Experiments conducted with a 3200-bp double-stranded plasmid DNA template demonstrated IR-laser ablation transfer of intact DNA. The transfer efficiency and the molecular integrity of the captured DNA were evaluated using Sanger sequencing, gel electrophoresis, and fluorimetric analysis. The plasmid DNA was reproducibly transferred with an efficiency of 59±3% at laser fluences of between 10 and 20kJ/m2 at a wavelength of 3μm. IR laser ablation sample transfer was then used to ablate and capture DNA from 50-μm-thick rat brain and kidney tissue sections. DNA was extracted from the captured material using five commercial DNA extraction kits that employed significantly divergent methodologies, with all kits recovering sufficient DNA for successful amplification by polymerase chain reaction (PCR). Four sets of primers were employed, targeting one region of the CYP 11b2 gene (376bp) and three different regions of the Snn1g gene (298, 168, and 281bp). The PCR results were not consistently reliable when using unpurified ablation samples; however, after extraction, all samples produced PCR products of the expected size. This work expands the sampling capabilities of IR laser ablation, demonstrating that DNA can be isolated from tissue samples for genomic assays. Due to the small size of the ablation regions (1mm2), this technique will be useful for sampling discrete cell populations from tissue sections. Graphical abstract Infrared laser ablation transfer of intact DNA from a tissue section.

(Invited) Modification and Analysis of Carbon Based Structures By Metal Nanoparticulate Ions

Our collaborative work to create and soft land ionized gas phase single crystalline metal nanoparticulate ions onto substrates was motivated by Robert Hauge’s wish to produce templates for carbon nanotube carpet growth. Ionwerks created a machine to do this but, as is often the case, funding was found first for alternative applications. One such has been low energy (500 eV) injection of 8 nm silver nanoparticulate ions (AgNP-) into the near surface of bio-polymers. A uniform layer (70 e15 Ag atoms/cm2) of these implanted particles serve to assist the laser desorption of intact biomolecules from a brain tissue sample into the gas phase. Some of these desorbed molecules are ionized and can be transported into a mass spectrometer and uniquely identified by their exact mass to charge (m/z). By scanning a 50 micron focused laser across the surface of an AgNP implanted rat brain tissue sections, a complete mass spectrum can be acquired at each of several hundred thousand laser locations. Images of specific biomolecules can be derived from these spectra. Putative molecular biomarkers can then be identified by inter-comparing the numerous molecular images from each animal grouping within an animal study. Images from the same brain levels between injured (or diseased) and uninjured rats define histological micro domains in which elevated or decreased molecular ion intensities correlate with other measures of injury/disease state severity. Imaging of these biomarker ions is becoming useful in preclinical screening of drug candidates and should ultimately enter clinical settings. The AgNP enhanced mass spectral imaging has been also applied to the surface analysis of synthetic polymers, fullerenes, and nanotubes. A third application uses 30 keV, 15 nm AgNP ion impacts to form otherwise unattainable controlled nano-sized holes in graphene—especially “rebar” graphene. Alternatively, low energy soft landing at lower energy can form nanostructures upon the graphene which have implications for device formation. However, before departure, Bob made it clear that we needed to get back to carpet growth and other ideas which he outlined in extensive and explicit discussions—mostly at outdoor cafes and coffee shops. His dictates and imprimatur remain.