SBIR-STTR Award

The goals of this project are twofold: a) to develop a new technique for nanoscale Mass spectrometry imaging based on AFM based tip enhanced IR ablation (nanoIR-MS). b) to apply this technique towards the application of Single cell imaging. Information on the chemical composition within a cell has implications in the understanding of cell metabolism, division, disease states, ecological effects etc. Given current technology limitations, most current analyses of biological systems are performed on groups of cells with the assumption that an ensemble average from the group will yield a useful result. However, this typically is not a valid assumption as cells of the same type exhibit diverse metabolic makeup depending on their phase in the cycle, history and interaction with the environment. Thus it is important that cells be analyzed individually in order to detect rare cells (e.g. circulating tumor cells), transient cell states, the influence of the cell environment on cells and states and aid in the understanding of differences in gene expression, protein levels, and small- molecule distributions at the single cell level. Cell heterogeneity is particularly significant in the "-omis" fields such as genomics, proteomics, lipidomics, and metabolomics that characterize biological systems at a molecular level. This significance led to the NIH launching a special focus program on Single cell Analysis Tools in late 2011. The size of mammalian cells is on the order of 10 ¿m and therefore the imaging of single cells requires imaging spatial resolution of at least 1¿m. The nanoIR-MS technology has a potential spatial resolution of at least 10x better than this or 100 nm which offers the possibility of the imaging of biomolecules in organelles. But to achieve an innovative and commercially successful product from this proposal, 1 ¿m spatial resolution would suffice. One of the Specific Aims of this proposal is to demonstrate that the nanoIR-MS technique can be applied for Single Cell Imaging. We will demonstrate this on two types of Single cells: Cells from Mouse brain and also to identify single Circulating Tumor cells (CTCs). As reiterated in our Letter of Support from our collaborator, Prof. Yeh who is an Oncology research surgeon, CTCs are the fundamental entities primarily responsible for spawning metastatic disease and there is a current lack of characterization technologies to identify them . To cure epithelial-based cancers-such as cancers of the breast, prostate, lung, colon and pancreas-therapies need to be directed towards those cells that cause metastases. However, the majority of metastatic lesions are never biopsied due to anatomic inaccessibility or associated morbidity of the procedure. CTCs offer a readily accessible means of studying the biology of metastatic cells throughout the course of disease and are often referred to as 'Liquid Biopsy'.

Public Health Relevance Statement:


Public Health Relevance:
The goals of this project are twofold: a) to develop a new technique for nanoscale Mass spectrometry imaging based on AFM based tip enhanced IR ablation (nanoIR-MS). b) To apply this technique towards the application of Single cell imaging. We will demonstrate the application of this platform technology on single mouse brain cells and to identify single cells of Circulatory Tumor cells.

Project Terms:
Ablation; American Type Culture Collection; Anatomy; base; Biochemical; Biocompatible Materials; biological systems; Biology; Biopsy; Blood; Brain; brain cell; brain tissue; Breast Cancer Cell; Buffers; Cancer Cell Growth; Cell Culture Techniques; Cells; cellular imaging; Chemicals; Citrates; Coagulation Process; Collection; Colon; Colorado; Culture Media; Deposition; Disease; Eagles; Environment; Epithelial; Exhibits; fetal bovine serum; Fluorescein; Fluorescence; Freezing; Gene Expression; Genomics; Glucose; Goals; Harvest; Heterogeneity; Image; Imagery; Imaging Techniques; Individual; innovation; ionization; ionization technique; Ions; Letters; Light; Liquid substance; Lung; malignant breast neoplasm; Malignant Neoplasms; Mammalian Cell; mass spectrometer; Mass Spectrum Analysis; MCF7 cell; Membrane; Metabolic; Metabolism; metabolomics; Metastatic Lesion; Microscope; Molecular; molecular mass; Molecular Weight; Morbidity - disease rate; Mus; nanoscale; Neoplasm Circulating Cells; Neoplasm Metastasis; neoplastic cell; oncology; Organelles; Oryctolagus cuniculus; Pancreas; Phase; Phosphate Buffer; Pituitary Gland; PKH67; prevent; Procedures; programs; Prostate; Proteins; Proteomics; public health relevance; Recording of previous events; Research; Resolution; Saline; Sampling; Scanning Probe Microscopes; Schools; Serum; single cell analysis; Slide; small molecule; Sodium Bicarbonate; sodium citrate; Solid; Solutions; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrometry, Mass, Secondary Ion; Staining method; Stains; Surgeon; Suspension substance; Suspensions; System; Techniques; Technology; tool; Trypsin; United States National Institutes of Health; Veterinary Medicine; Whole Blo

The goal of the proposed Phase II STTR research is to develop an imaging system capable of detecting and identifying biomolecules in cells and tissue sections with sub-cellular spatial resolution. The system combines an atomic force microscope (AFM) with mass spectrometry (MS) analysis and utilizes a unique tip-enhanced laser ablation (TELA) method developed in the Phase I portion of the research. The current technology limitation of imaging mass spectrometry is the fact that it is possible to obtain high-resolution images of small molecules and low-resolution images of large molecules, but it is not possible to both at once. The proposed instrument directly addresses this technology limitation. The TELA effect developed under Phase I as an off - line MS method will be developed under Phase II into an on-line instrument that integrates AFM and mass spectrometry imaging for both excellent spatial resolution and biomolecule mass spectrometry. The aims of the project are divided into 1) further refinement of TELA and development of the on-line TELA-MS interface, 2) integration of TELA and MS imaging, and 3) application to brain imaging. Refinement of the tip - enhanced laser ablation involves a study of the fundamental interaction between the pulsed ablation laser and the atomic force microscope tip. The optimized ablation system will be combined with an ultra-low flow electrospray ionization system in which charged particles from the electrospray source interact with the ablation plume to form biomolecule ions. Integration of the AFM and MS imaging systems will use four imaging modalities: spot-by-spot sampling, area sampling, area imaging, and depth profiling. The result is an AFM capable of both physical and chemical imaging of cells and tissue. The AFM TELA-MS device will be applied to detection of biomolecules in rat brain tissue. Spatial profiling of lipids, peptides, cocaine, and cocaine metabolites will be performed. The AFM TELA-MS system will be constructed by Anasys, based on past product development and manufacturing experience with nanoscale imaging systems such as atomic force microscopy and infrared spectroscopy chemical imaging using tunable pulsed laser sources. Mass spectrometry performance tests will be carried out on in the laboratory of Prof. Kermit Murray at Louisiana State University who is an expert in the development of laser ablation methods for the detection of biomolecules by mass spectrometry.

Public Health Relevance Statement:


Public Health Relevance:
The goal of this project is to develop an imaging instrument capable of identifying biological molecules on the scale of single cells. This device addresses the technology limitations of current imaging systems that lack either the ability to resolve singl cells or lack the ability to detect large biomolecules. This device is a critical tool for biomedicl research and will assist in the disease research, drug development, and in clinical diagnosis.

Project Terms:
Ablation; Address; Area; Atomic Force Microscopy; base; Benchmarking; Biological; Brain imaging; brain tissue; Cells; cellular imaging; Charge; Chemicals; clinical Diagnosis; Cocaine; Collaborations; Detection; Development; Devices; Disease; drug development; Electrospray Ionization; experience; Geometry; Goals; Image; Image Analysis; Imaging Device; imaging modality; imaging system; infrared spectroscopy; instrument; instrumentation; Ions; irradiation; Laboratories; Lasers; Lipids; Louisiana; Mass Spectrum Analysis; Methods; microscopic imaging; Modality; nano; nanoscale; National Institute of Drug Abuse; Online Systems; Outcome; particle; Peptides; performance tests; Phase; Physiologic pulse; product development; Progress Reports; public health relevance; Rattus; Research; Resolution; Sampling; Scanning Probe Microscopes; single cell analysis; Small Business Technology Transfer Research; small molecule; Source; Spottings; Staging; System; Technology; Testing; Tissues; tool; United States National Institutes of Health; Universities; user-friendly; Wood material; Work