High resolution imaging apparatus and method
Inventors
Corkum, Paul • Loboda, Alexander V.
Assignees
University of Ottawa • Standard Biotools Canada Inc
Publication Number
US-12278098-B2
Publication Date
2025-04-15
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 (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 relates to methods and systems for high resolution imaging of samples using imaging mass spectrometry (IMS) and imaging mass cytometry (IMC™). It particularly concerns overcoming challenges related to confining the sampling spot area to about 200 nm or less and ensuring sufficient signal-to-noise ratio from the ablated material. The apparatus combines a sampling and ionisation system and a detector system, wherein the sample is placed on a stage and material removed and ionised, then analysed by the mass detector.
A key improvement is the use of ultrathin biological sample sections of less than 100 nm thickness, enabling utilisation of immersion lenses with high refractive index media placed between the objective lens and the sample stage. This allows laser radiation to be focused to particularly small spots (ideally smaller than 200 nm), enhancing lateral resolution without damage to areas outside the targeted ablation spot. It also enables imaging at subcellular resolution beyond traditional systems.
The invention also discovers the possibility of performing electron microscopy on the same sample before IMS or IMC, combining high resolution structural images with elemental analysis, with components possibly integrated in a single apparatus. Furthermore, it provides methods using charged particle bombardment (ions or electrons) coupled with laser post-ionisation to achieve higher resolution due to tightly focused charged particle beams. Ion optics are used to direct charged particles to the sample and direct ionised sample to mass spectrometers.
Claims Coverage
The patent contains multiple independent claims covering methods and apparatuses for analysing biological samples, featuring aspects such as staining for electron microscopy, labelling with labelling atoms, laser radiation ablation, ionisation, charged particle based sampling, and high resolution laser optics.
Method combining electron microscopy and high resolution IMS/IMC imaging
A method comprising staining the biological sample with a contrast agent for electron microscopy; labelling the sample with labelling atoms; imaging by electron microscopy; directing laser radiation to ablate sample material; ionising the ablated plume; and detecting sample ions.
Apparatus using immersion medium for high numerical aperture laser focusing
An apparatus for analysing a biological sample comprising a sample stage, a laser source, focusing optics with objective lens directing laser radiation to the sample, and an immersion medium positioned between the objective lens and sample stage to achieve enhanced numerical aperture and improved spatial resolution.
Laser ablation-based sampling and ionisation system with laser scanning
A laser ablation sampling and ionisation system comprising a sample stage, a laser source, laser scanning system, and focusing optics with objective lens, optionally including an immersion medium, enabling rapid and precise ablation of sample regions.
Sputtering based sampling and ionising system with charged particle beam and laser post-ionisation
An apparatus comprising a source of charged particles and charged particle column to pass a charged particle beam to the sample stage, and a first laser source with focusing optics configured to direct laser beam to the sample stage to post-ionise sputtered neutral sample material.
Synchronised charged particle beam sputtering with subsequent laser ablation
An apparatus where a pulse of charged particles creates a sample ignition state at a location on the sample, followed by a synchronised pulse of laser beam directed to the same location to provide energy pumping leading to ablation and ionisation.
Two pulse laser ablation system for electron seeding and ablation with high resolution
An apparatus with a first laser source configured to seed electrons in the sample (pre-seeding) and a second laser source configured to ablate sample material pre-seeded with electrons by the first laser source, with both lasers directed to the same location and the second pulse synchronised shortly after the first pulse.
Two pulse laser ablation system with optional third laser source for post-ionisation
An apparatus comprising first and second laser sources as above, additionally comprising a third laser source configured to ionise plumes of sample material ablated from the sample, with pulse synchronisation such that the third pulse arrives shortly after ablation to provide post-ionisation.
The independent claims cover methods that combine electron microscopy with high resolution IMS/IMC imaging, apparatuses employing immersion media to increase numerical aperture for laser focusing, laser ablation systems with laser scanning, sputtering-based systems using charged particle beams with laser post-ionisation, synchronised charged particle and laser ablation, two pulse laser systems for improved resolution, and inclusion of a third laser for post-ionisation. Overall, the inventive features enable enhanced spatial resolution, signal quality, and multiplexed imaging capabilities in biological sample analysis.
Stated Advantages
Provides sub-micrometer resolution imaging by using immersion lenses and ultrathin biological sections.
Enables higher signal-to-noise ratios and improved detection sensitivity with charged particle bombardment combined with laser ionisation.
Combines electron microscopy with IMS/IMC to refine resolution beyond laser ablation limits.
Laser scanning system substantially increases ablation speed and pixel acquisition rate over traditional stage movement.
Use of immersion medium overcomes numerical aperture limitations in conventional IMS and IMC systems.
Two-pulse laser ablation reduces ablation spot size enabling higher resolution imaging.
Documented Applications
High resolution imaging of biological samples, including tissue sections and monolayers of cells, by IMS and IMC.
Imaging mass cytometry analysis of biological samples stained for electron microscopy and labelled with labelling atoms.
Analysis of biological samples using combined electron microscopy and IMS/IMC methods.
Multiplexed detection of various molecular targets in biological samples using mass-tagged antibodies and elemental labelling.
Analysis of metal-containing drugs and accumulation of heavy metals in biological samples.
Subcellular imaging of nucleic acids using mass-tagged oligonucleotides with amplification techniques.
Rapid high resolution biological imaging using laser scanning combined with high repetition rate lasers and immersion lenses.
Use of two pulse laser ablation methods for sample ignition and ablation to reduce spot size and improve resolution.
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