High resolution imaging apparatus and method
Inventors
Corkum, Paul • Loboda, Alexander V.
Assignees
University of Ottawa • Standard Biotools Canada Inc
Publication Number
US-11264221-B2
Publication Date
2022-03-01
Expiration Date
2039-06-18
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Abstract
The present invention relates to the high resolution imaging of samples using imaging mass spectrometry (IMS) and to the imaging of biological samples by imaging mass cytometry (IMCTM) in which labelling atoms are detected by IMS. LA-ICP-MS (a form of IMS in which the sample is ablated by a laser, the ablated material is then ionised in an inductively coupled plasma before the ions are detected by mass spectrometry) has been used for analysis of various substances, such as mineral analysis of geological samples, analysis of archaeological samples, and imaging of biological substances. However, traditional LA-ICP-MS systems and methods may not provide high resolution. Described herein are methods and systems for high resolution IMS and IMC.
Core Innovation
The invention relates to high resolution imaging of samples using imaging mass spectrometry (IMS) and imaging mass cytometry (IMC™). Specifically, it addresses the use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and sputtering based ionisation techniques to improve lateral resolution in imaging elemental mass spectrometry of biological samples, achieving sub-micrometer spatial resolution.
The problem solved is the limitation of traditional LA-ICP-MS and IMC systems which do not provide sufficiently high resolution, primarily due to challenges of confining the sampling spot size to around 200 nm or less and ensuring a sufficient signal-to-noise ratio from the analyte in the ablated material. The invention aims to provide improved apparatus and methods overcoming these challenges by employing ultrathin sample sections, immersion lenses with high refractive indices, incorporation of electron microscopy, and advanced ionisation methods including laser post-ionisation coupled with charged particle sputtering.
The invention comprises broadly two systems: a sampling and ionisation system containing a sample chamber with a stage holding the sample, where sample material is removed and ionised (hard ionisation techniques used), and a detector system (for example, a mass detector) analyzing the ionised material. Key innovations include the use of immersion media between the objective lens and the sample stage to achieve numerical apertures greater than 1.0, thereby reducing laser spot size below 200 nm, down to 100 nm or less. The invention also combines electron microscopy and IMS/IMC in a single apparatus or workflow for enhanced structural and elemental imaging, and introduces charged particle beam sputtering coupled with laser ionisation (laser-SNMS) to improve subcellular resolution and ionisation efficiency.
Claims Coverage
The patent includes one independent claim detailing a method for analyzing a sample via imaging mass spectrometry involving specific sequencing of radiation and ionisation steps.
Method for high resolution mass spectrometry imaging with expanded material ionisation
Radiating a sample with a first energy source to release material, allowing the material to expand past significant charge neutralization, ionising the expanded material with an ionizing laser to form microplasma and generate elemental ions, followed by mass spectrometry analysis.
Partial vacuum expansion for improved material ionisation
Allowing expansion of the released material into a partial vacuum within the sample chamber, specifically in the range of 10-10,000 Pa, enhancing the formation of microplasma and elemental ion generation.
Use of specified laser parameters for ionising expanded material
Employing an ionizing laser which can be an IR or visible laser, operable as picosecond or femtosecond pulses, synchronised near the sample surface to efficiently ionise expanded material.
Integration with advanced mass spectrometry and cytometry techniques
Analyzing generated elemental ions using time-of-flight or magnetic sector mass spectrometry, and optionally performing imaging mass cytometry for high multiplexed biological samples.
Configured use of energy sources and optics for submicron resolution sampling
Utilizing first energy sources such as short-wavelength lasers with wavelengths below 300 nm, focused through objective lenses with numerical aperture of at least 0.9 to achieve laser spot sizes of 250 nm or less, and positioning the laser opposite the sample on the sample stage. Alternative energy sources include ion beams or electron beams.
The claim covers a comprehensive method utilizing controlled ion source radiation, material expansion under partial vacuum, precise laser ionisation forming microplasma, and advanced mass spectrometry detection to achieve high resolution and multiplexed imaging mass spectrometry and mass cytometry, particularly at submicron scales employing laser and charged particle sources.
Stated Advantages
Improved lateral resolution in imaging mass spectrometry down to 100 nm or less through use of immersion lenses with high refractive index media.
Enhanced signal-to-noise ratio via advanced ionisation techniques including laser post-ionisation coupled with charged particle sputtering (SNMS).
Capability to obtain ultrastructural images with electron microscopy integrated with IMS/IMC to refine imaging resolution beyond laser ablation limits.
Rapid scanning and ablation via laser scanning systems increasing acquisition speed and spatial resolution.
Enabling multiplexed analysis with many detectable labels simultaneously, facilitating high-content biological sample analyses.
Documented Applications
High resolution imaging of biological samples by imaging mass cytometry (IMC™) using elemental mass spectrometry.
Imaging of biological tissues and cells with submicron lateral resolution for detailed spatial analysis.
Use in pathological and clinical diagnosis through multiplexed detection of labelled biomolecules.
Combination of electron microscopy and IMS/IMC for structural and elemental imaging of ultrathin biological samples.
Analysis of biological samples labelled with mass tags comprising rare earth elements or other metal labels for molecular profiling.
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