High resolution imaging apparatus and method
Inventors
Corkum, Paul • Loboda, Alexander V.
Assignees
University of Ottawa • Standard Biotools Canada Inc
Publication Number
US-11967496-B2
Publication Date
2024-04-23
Expiration Date
2039-06-18
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 (IMC™) 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 discloses methods and apparatus for high resolution imaging of biological samples using imaging mass spectrometry (IMS) and imaging mass cytometry (IMC) where labelling atoms are detected by IMS. It addresses the challenge of achieving sub-micrometer resolution, specifically confining the sampling spot size to about 200 nm or less and ensuring sufficient analyte quantity for a high signal-to-noise ratio.
The invention comprises two main systems: a sampling and ionisation system, which removes material from the sample and converts it into ions, and a detector system, which analyses these ions. Improvements include the use of immersion media with refractive index greater than 1.0 between the objective lens and sample stage to achieve numerical apertures greater than 1.0, thereby reducing laser spot size below 200 nm. Biological samples are prepared as ultrathin sections (down to 30 nm) facilitating higher lateral resolution and potential for three-dimensional imaging by sequential sectioning or scanning.
Further innovations include integration or incorporation of electron microscopy components to obtain high-resolution structural images that complement IMS or IMC data for resolution beyond laser ablation limits. The invention also develops sputtering-based sampling systems using charged particle beams to sputter material, combined with laser post-ionisation either subsequent to sputtering or during sampling. Charged particle beams can be focused to smaller spot sizes than laser ablation, enabling increased resolution and ionisation efficiency.
Claims Coverage
Several independent claims focus on apparatus for analysing biological samples using a combination of charged particle beams and laser radiation to achieve high resolution sampling, ionisation, and detection.
Apparatus for high-resolution sampling using charged particles and laser radiation directed at opposite sides of the sample stage
An apparatus comprising a sample stage, a source of charged particles configured to pass a beam to a location on the sample stage, and a first laser source with focusing optics, where the charged particle beam and laser beam are directed towards opposite sides of the sample stage.
Synchronization of laser pulse with charged particle beam pulse
Focusing optics of the first laser source are configured to synchronise a pulse of laser beam to arrive directly after a pulse of charged particles at the sample location, facilitating ionisation of sputtered material.
Use of multiple laser sources for ionisation of sputtered material
Apparatus comprising a second laser source and focusing optics configured to synchronise a pulse from the second laser source to ionise plume material sputtered by charged particles.
Configuration of laser beams directed to same or opposite sides of sample stage
The first and second laser sources and their focusing optics can be configured to direct beams either to the same side or opposite sides of the sample stage.
Ionisation of sputtered material by avalanche ionisation
At least one of the laser sources is configured to ionise material via avalanche ionisation, enhancing ionisation efficiency.
Charged particle beam source variations
The source of charged particles can be a primary ion beam source or an electron beam source, optionally integrated within an electron microscope.
Pulsed charged particle beam and scanning capability
The charged particle beam is pulsed and the apparatus can include a charged particle beam scanning system to scan the beam across multiple locations on the sample stage.
Sample stage transparency and variable delay line
The sample stage may be transparent or have a cut-out portion and the apparatus can comprise a variable delay line to coordinate pulse timing.
Integration of electron microscope
The apparatus can further comprise an electron microscope, where the electron beam source is the source of charged particles.
Mass spectrometer detection
The apparatus includes a mass spectrometer configured to detect ionised sample material.
The claims cover apparatus for high-resolution analysis of biological samples combining charged particle beams and laser sources with precise spatial configurations and synchronization to enable sputtering, ionisation via avalanche or post-ionisation, scanning, and detection. These configurations leverage pulsed beams, electron microscopy integration, and mass spectrometry detection for improved resolution and sensitivity.
Stated Advantages
Improved lateral resolution of imaging mass cytometry and imaging mass spectrometry to sub-200 nm spot sizes by use of immersion media with refractive index greater than 1.0 increasing numerical aperture beyond 1.0.
Enhanced signal-to-noise ratio due to improved ionisation efficiency using laser post-ionisation coupled with charged particle beam sputtering.
Integration with electron microscopy enables combining high-resolution structural images with elemental images, improving resolution beyond laser ablation limits.
Rapid scanning capability via laser scanning systems reduces sample imaging time from days to hours or less.
The apparatus achieves subcellular or even ultrastructural resolution imaging in biological samples by using ultrathin sections and advanced sampling and ionisation techniques.
Configurations for minimizing detector overload and damage extend detector life and ensure quantitative data acquisition.
Documented Applications
High resolution imaging of biological samples including tissue sections, monolayer of cells, and individual cells using imaging mass cytometry and imaging mass spectrometry.
Analysis of biological samples labelled with specific binding pair members (e.g., antibodies, nucleic acids) conjugated to mass tags for multiplexed detection of targets including proteins and nucleic acids.
Imaging and analysis of ultrathin biological sections (down to 30 nm thick) suitable for electron microscopy and elemental imaging combining structural and elemental data.
Applications involving histochemical staining using metal-containing stains (e.g., osmium tetroxide, ruthenium red, phosphotungstic acid) compatible with imaging mass cytometry.
Use in analysis of metal-containing drugs localization and biodistribution in biological samples, including chemotherapeutic drugs such as cisplatin and other platinum complexes.
Single cell and subcellular analysis via targeted laser ablation or charged particle sputtering with multiplexed mass detection enabling cellular and organelle resolution.
Expansion microscopy sample preparation combined with imaging mass spectrometry for improved effective resolution through physical sample expansion.
Interested in licensing this patent?