WO2018026910A1

WO2018026910A1 - Sample imaging apparatus and method

Info

Publication number: WO2018026910A1
Application number: PCT/US2017/045087
Authority: WO
Inventors: Alexander V. Loboda
Original assignee: Fluidigm Canada Inc.
Priority date: 2016-08-02
Filing date: 2017-08-02
Publication date: 2018-02-08

Abstract

Improved mass spectrometry-based apparatus and methods of analysis for analysing samples. A laser sampling system can be adapted to emit pulses of laser radiation at two or more wavelengths, wherein a first wavelength targets a first material and a second wavelength targets a second material. Atoms of the first and second materials removed by laser ablation are ionized by an ionisation system to form elemental ions, which can be detected by a mass spectrometer component. Laser ablation can be performed at multiple locations to form a plurality of plumes, which can be individually subjected to ionisation and spectrometry. Ablating at known locations or recording locational information for each location can permit construction of an image of the sample(s) based on detecting labelling atoms.

Description

SAMPLE IMAGING APPARATUS AND METHOD

FIELD OF THE INVENTION

The present invention relates to the imaging of samples using imaging mass spectrometry (IMS) following laser ablation and the imaging of biological samples by imaging mass cytometry (IMC).

BACKGROUND

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 [1].

For biological samples, single-cell measurements and multiplexed quantitative detection of molecular targets can provide insights on the state and behaviour of individual cells. Techniques which have been used for single-cell analysis include microscopy in combination with specific labelling techniques (e.g. using immunocytochemistry), single-cell imaging mass spectrometry, surface-enhanced Raman scattering spectroscopy, and LA-ICP-MS.

Some of these techniques can readily be extended to in situ imaging of tissues (e.g. using immunohistochemistry), but others cannot. For instance, approaches such as immunofluorescence microscopy (IFM) are useful for imaging down to nanometer resolutions, but are limited in practice to measuring seven or fewer targets molecules in a sample simultaneously. In contrast, LA-MS offers highly multiplexed quantitative analysis of antigen expression.

Imaging of biological samples by FMC has previously been reported for imaging at a cellular resolution [2,3,4]. Detailed imaging at a sub-cellular resolution has also recently been reported [5].

It is an object of the invention to provide further and improved apparatus and techniques for imaging of samples.

DETAILED DESCRIPTION

The apparatus of the invention, such as imaging mass spectrometer or an imaging mass cytometer, typically comprises three components. The first is a laser ablation system for the generation of plumes of vaporous and particulate material from the sample for analysis. Before the atoms in the plumes of ablated sample material (including any detectable labelling atoms as discussed below) can be detected by a mass spectrometer component (MS component; the third component), the sample must be ionised (and atomised). Accordingly, the apparatus comprises a second component which is an ionisation system that ionises the atoms to form elemental ions to enable their detection by the MS component based on mass/charge ratio (some ionisation of the sample material may occur at this point, but space charge effects result in the neutralisation of the charges). Thus in operation, the sample is taken into the apparatus, is ablated to generate vaporous/particular material, which is ionised by the ionisation system, and the ions of the sample are passed into the MS component. Although the MS component can detect many ions, most of these will be ions of the atoms that naturally make up the sample. In some applications, for example analysis of minerals, such as in geological or archaeological applications, this may be sufficient.

In some cases, for example when analysing biological samples, the native element composition of the sample may not be suitably informative. This is because, typically, all proteins and nucleic acids are comprised of the same main constituent atoms, and so while it is possible to tell regions which contain protein/nucleic acid from those that do not contain such proteinaceous or nucleic acid material, it is not possible to differentiate a particular protein from all other proteins. However, by labelling the sample with atoms not present in the material being analysed under normal conditions, or at least not present in significant amounts, (for example certain transition metal atoms, such as rare earth metals; see section on labelling below for further detail), specific characteristics of the particle sample can be determined. In common with IHC and FISH, the detectable labels can be attached to specific targets on or in the sample (such as fixed cells or a tissue sample on a slide), inter alia through the use of affinity reagents such as antibodies or nucleic acids targeting molecules on or in the sample. In order to detect the ionised label, the MS component is used, as it would be to detect ions from atoms naturally present in the sample. By linking the detected signals to the known positions of the laser ablations which gave rise to those signals it is possible to build-up an image of the atoms present at each position, both the native elemental composition and any labelling atoms (see e.g. references 2, 3, 4, 5). The technique allows the analysis of many labels in parallel, which is a great advantage in the analysis of biological samples.

Systems and methods known in the art employ lasers emitting light at a single wavelength in the laser ablation system. The inventors have now made improvements in IMS and IMC methods and apparatus to improve the use of the technique for the analysis of samples, particularly heterogeneous samples such as biological samples, through the use of multiple wavelengths of laser radiation to ablate the sample.

Laser sampling system

In brief summary, the components of a laser sampling system include a laser source that emits a beam of laser radiation that is directed upon a sample. The sample is typically positioned on a translation stage that is movable within a chamber in the laser sampling system (the sampling chamber), so that the sample can be moved relative to the beam of laser radiation, thereby enabling different locations on the sample to be sampled for analysis (typically by ablation). A gas is flowed through the sampling chamber, and the flow of gas carries away the plumes of aerosolised material generated when the laser source ablates the sample, for analysis and construction of an image of the sample. As explained further below, the laser system of the laser sampling system can also be used to desorb material from the sample.

For biological samples in particular, the sample is often heterogeneous (although heterogeneous samples are known in other fields of application of the invention, i.e. samples of a non-biological nature). A heterogeneous sample is a sample containing regions composed of different materials, and so some regions of the sample can ablate at lower threshold fluence at a given wavelength than the others. The factors that affect absorption thresholds are the absorbance coefficient of the material and mechanical strength of material. For biological tissues, absorbance coefficient will have a dominant effect as it can vary with the irradiation wavelength by several orders of magnitude (the mechanical strength of the different regions in a biological sample does not vary enough to be of significance).

For a biological sample, a region that contains proteinaceous material will absorb more readily in the 200-230nm wavelength range, while a region containing predominantly DNA will absorb more readily in the 260-280nm wavelength range. This situation is illustrated in Figure 6 by a graph showing absorption spectra for different protein/DNA mixtures (in which absorption is a logarithmic value of the absorbance coefficient). Thus, at an ablation wavelength of 220nm, a sample of pure DNA will have an ablation threshold that differs by more than 1 order of magnitude from that of a sample of pure protein. Thus ablating with wavelength of 220nm at constant fluence (energy density) will ablate pure protein differently from pure DNA.

In many cases it is highly desirable to conduct laser ablation at a fluence near the ablation threshold of the sample material. Ablating in this manner often improves aerosol formation which in turn can help improve the quality of data obtained by LA-MS. In addition, the ablation crater produced in the sample by the pulse of the laser source depends on the ratio between the fluence applied and the threshold ablation fluence: the greater the extent to which the fluence applied exceeds the threshold fluence, the larger the resulting crater. Often to obtain the smallest crater, to maximise the resolution of the resulting image, a Gaussian beam is employed. A cross section across a Gaussian beam records an energy density profile that has a Gaussian distribution. In that case, the fluence of the beam changes with the distance from the centre. As a result, the diameter of the ablation spot size is a function of two parameters: (i) the Gaussian beam waist (1/e²), and (ii) the ratio between the fluence applied and the threshold fluence.

Thus, in order to ensure consistent removal of a reproducible quantity of material in each laser shot, and thus maximise the quality of the imaging data, it is useful to maintain a consistent ablation diameter which in turn means adjusting the ratio of the energy supplied by the laser shot at the target to the ablation threshold energy of the material being ablated. This requirement represents a problem when ablating a heterogeneous sample where the threshold ablation energy varies across the sample, such as a biological tissue where the ratio of DNA and protein material varies, or in a geological sample, where it varies with the particular composition of the mineral in the region of the sample. To solve this problem, more than one wavelength of laser radiation is focused onto the same ablation location on a sample, to more effectively ablate the sample based on the composition of the sample at that location.

Accordingly, the invention provides an apparatus, for example an imaging mass cytometer and/or imaging mass spectrometer, comprising:

(i) a laser sampling system, wherein the laser system is adapted to emit pulses of laser radiation at two or more wavelengths, and wherein a first wavelength targets a first material (material type), and a second wavelength targets a second material (material type);

(ii) an ionisation system adapted to receive material removed from the sample by the laser system and to ionise said material to form elemental ions; and

(iii) a mass spectrometer to receive elemental ions from said ionisation system and to analyse said elemental ions.

For a biological sample, for example, a first wavelength at 213nm would target ablation of proteinaceous material, while a second wavelength can be chosen at 266nm, to target ablation of the DNA material. The invention also provides a method of analysing a sample comprising:

(i) performing laser ablation of the sample using laser radiation of two or more wavelengths, wherein the ablation is performed at multiple known locations to form a plurality of plumes; and

(ii) subjecting the plumes individually to ionisation and mass spectrometry, whereby detection of ions from atoms in the plumes permits construction of an image of the sample.

The sample analysed may be a biological sample. The method of analysis may be mass cytometry.

The invention also provides a method of performing mass cytometry on a sample comprising a plurality of cells, the method comprising:

(i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms, to provide a labelled sample;

(ii) performing laser ablation of the sample using laser radiation of two or more wavelengths, wherein the ablation is performed at multiple known locations to form a plurality of plumes; and

(iii) subjecting the plumes individually to ionisation and mass spectrometry, whereby detection of atoms in the plumes permits construction of an image of the sample.

Laser system of the laser sampling system

The laser system of the apparatus of the invention is set up to produce multiple wavelengths of laser radiation. Typically, the wavelengths of laser radiation discussed refer to the wavelength which has the highest intensity (the "peak" wavelength). The different wavelengths produced can be used for different purposes, for example, for targeting different materials in a sample (by targeting here is meant that the wavelength chosen is one which is absorbed well by a material).

In some embodiments, at least two of the two or more wavelengths of the laser radiation are discrete wavelengths. Thus when a first laser source emits a first wavelength of radiation that is discrete from a second wavelength of radiation, it means that no, or a very low level of radiation of the second wavelength is produced by the first laser source in a pulse of the first wavelength, for example, less than 10% of the intensity at the first wavelength, such as less than 5%, less than 4%), less than 3%, less than 2%, or less than 1%. Typically, when different wavelengths of laser radiation are produced by harmonics generation, or other non-linear frequency conversion processes, then when a specific wavelength is referred to herein, it will be understood by the skilled person that there will be some degree of variation about the specified wavelength in the spectrum produced by the laser. For example, a reference to X nm encompasses a laser producing a spectrum in the range X±10nm, such as X±5nm, for example X±3nm.

Lasers

In some embodiments, the laser system that emits laser radiation of two or more wavelengths of laser radiation comprises two or more laser sources, wherein each laser source is adapted to emit laser radiation at a wavelength that differs from the wavelength of laser radiation emitted be the other laser source(s) in the laser system.

Thus, the laser system may comprise a first laser source that emits laser radiation at a wavelength of 213nm, and a second laser source that emits laser radiation at 266nm (so that the first laser source ablates principally proteinaceous material, and the second ablates principally DNA material). If ablation at a third wavelength of laser radiation is desired, a third laser source is used in the laser system, and so on. Thus in some embodiments the laser system for emitting multiple wavelengths of laser radiation comprises three or more laser sources, such as four or more laser sources, five or more laser sources, six or more laser sources, seven or more laser sources, eight or more laser sources, nine or more laser sources, or ten or more laser sources. In some embodiments, each laser source is adapted to emit laser radiation at a single wavelength that differs from the wavelength of laser radiation emitted by the other laser(s) in the system.

In some embodiments, the laser system for emitting multiple wavelengths of laser radiation comprises a single laser source adapted to emit multiple wavelengths of laser radiation {i.e. one laser emits multiple wavelengths of laser radiation; the laser system may include further laser sources). Some laser sources emit laser radiation at a desired wavelength using wavelength conversion methods such as harmonics generation, by an optical parametric oscillator (OPO) technique, or by a combination of several techniques, as standard in the art. For instance, an Nd-YAG laser generates laser radiation at 1064nm wavelength, which is called its fundamental frequency. This wavelength can be converted into shorter wavelengths (when needed) by the method of harmonics generation. The 4^th harmonic of that laser radiation would be at 266nm (1064nm ÷ 4) and the 5^th harmonic would be at 213nm. Thus, the 4^th harmonic can target the optical band of high absorption for DNA material while the 5^th harmonic would target the band of high absorption for proteins. In many laser arrangements generation of the 5^th harmonic is based on the generation of the 4^th harmonic. Thus the 4^th harmonic will be already present in the laser generating the 5^th harmonics output, although often the lower harmonics (with longer wavelength) are filtered out in the laser. Removal of the appropriate filters thus enables the emission of multiple wavelengths of laser radiation. Examples of such lasers are commercially available from Coherent, Inc, RP Photonics, Lee Laser etc.

Accordingly, in some embodiments, the laser system of the laser sampling system for emitting multiple wavelengths of laser radiation comprises a laser source adapted to emit laser radiation at a wavelength of 266nm and at a wavelength of 213nm.

Another useful pair of harmonic frequencies is the 4^th and the 3^rd harmonics of a laser with a fundamental wavelength at around 800nm. The 4^th and the 3^rd harmonics here would have wavelengths of 200nm and 266nm respectively. Examples of such lasers are commercially available (Coherent, Inc., Spectra Physics). Again these would target protein absorption and DNA absorption bands. Accordingly, in some embodiments, the laser system for emitting multiple wavelengths of laser radiation comprises a laser source adapted to emit laser radiation at a wavelength of 266nm and at a wavelength of 200nm.

Sometimes, the laser system comprises features from both of the embodiments discussed above. Accordingly, in some embodiments, the laser system for emitting multiple wavelengths of laser radiation comprises two or more laser sources, wherein one or more of the laser sources adapted to emit multiple wavelengths of laser radiation and wherein at least one of the wavelengths emitted by one of the laser sources is not emitted by the other laser source(s) in the laser system.

In some embodiments, one of the two or more wavelengths of laser radiation emitted by the laser system is selected to preferentially ablate nucleic acid over protein - i.e. the wavelength is selected from the range of wavelengths at which the absorbance coefficient for nucleic acid is higher than for protein. In some embodiments, one of the two or more wavelengths of laser radiation emitted by the laser system is between about 250nm to about 270nm, such as about 255nm to about 266nm, optionally about 260nm or about 266nm.

In some embodiments, one of the two or more wavelengths of laser radiation emitted by the laser system is selected to preferentially ablate protein i.e. the wavelength is selected from the range of wavelengths at which the absorbance coefficient for protein is higher than for nucleic acid. In some embodiments, one of the two or more wavelengths of laser radiation emitted by the laser system is between about 185nm to about 235nm, such as about 195nm to about 225nm, optionally about 193nm or about 213nm.

In some embodiments, a first of the two or more wavelengths of laser radiation emitted by the laser system is selected to preferentially ablate nucleic acid and a second of the two or more wavelengths of laser radiation emitted by the laser system is selected to preferentially ablate protein. In some embodiments, the first of the two or more wavelengths of laser radiation emitted by the laser system is between about 250nm to about 270nm, such as about 255nm to about 266nm, optionally about 260nm or about 266nm and the second of the two or more wavelengths of laser radiation emitted by the laser system is between about 185nm to about 235nm, such as about 195nm to about 225nm, optionally about 193nm or about 213nm.

In some embodiments, the laser system is adapted to emit laser radiation at 213nm and 266nm, for example wherein the laser system is adapted to emit laser radiation at 213nm, 266nm and 355nm.

In some embodiments, the first wavelength of laser radiation is more than 5nm different from the second wavelength of laser radiation, such as at least about lOnm, at least 15nm, at least 20nm, at least 25nm, at least 30nm, or at least 40nm different.

In some embodiments, where the first wavelength of laser radiation and the second wavelength of laser radiation are produced by the same laser, the wavelengths are not produced via harmonics, but from a laser with a broad emission spectrum. In some embodiments, the emission spectrum of the laser is at least lOnm, such as at least 30nm, at least 50nm or at least lOOnm. In some embodiments, the laser is a white light laser or a supercontinuum laser.

The laser beam used for ablation in the laser systems discussed herein may have a spot size of ΙΟΟμπι or less, such as 50μπι or less, 25μπι or less, 20μπι or less, 15μπι or less, ΙΟμπι or less, 5μπι or less, 2μπι or less, or Ι μπι or less. The distance referred to as spot size corresponds to the longest internal dimension of the beam, e.g. for a circular beam it is a beam of diameter 2μπι, for a square beam corresponds to the length of the diagonal between opposed corners, for a quadrilateral it is the length of the longest diagonal etc. (the diameter of a circular beam with a Gaussian distribution is 1/e²).

When used for analysis of biological samples, in order to analyse individual cells the spot size of laser beam used will depend on the size and spacing of the cells. For example, where the cells are tightly packed against one another (such as in a tissue section) one or more laser sources in the laser system can have a spot size which is no larger than these cells. This size will depend on the particular cells in a sample, but in general the laser spot will have a diameter of less than 4 μπι e.g. within the range 0.1-4 μπι, 0.25-3 μπι, or 0.4-2 μπι. Thus a laser spot can have a diameter of about 3 μπι or less, about 2 μπι or less, about 1 μπι or less, about 0.5 μπι or less than 0.5 μπι, such as about 400nm or less, about 300nm or less, about 200nm or less, about lOOnm or less than lOOnm. In order to analyse cells at a subcellular resolution the invention uses a laser spot size which is no larger than these cells, and more specifically uses a laser spot size which can ablate material with a subcellular resolution. Sometimes, single cell analysis can be performed using a spot size larger than the size of the cell, for example where cells are spread out on the slide, with space between the cells. Here, a larger spot size can be used and single cell characterisation achieved, because the additional ablated area around the cell of interest does not comprise additional cells. The particular spot size used can therefore be selected appropriately dependent upon the size of the cells being analysed. In biological samples, the cells will rarely all be of the same size, and so if subcellular resolution imaging is desired, the ablation spot size should be smaller than the smallest cell, if constant spot size is maintained throughout the ablation procedure. Small spot sizes can be achieved using demagnification of wider laser beams and near-field optics. A laser spot diameter of 1 μπι corresponds to a laser focus point (i.e. the diameter of the laser beam at the focal point of the beam) of 1 μπι, but the laser focus point can vary by +20% or more due to spatial distribution of energy on the target (for instance, Gaussian beam shape) and variation in total laser energy with respect to the ablation threshold energy. For example, using a 25 μπι diameter laser beam, and subjecting this to 25 -fold demagnification onto the sample will give a spot size with a 1 μπι diameter.

For rapid analysis of a sample a high frequency of ablation is needed, for example more than 20

Hz (i.e. more than 20 ablations per second, giving more than 20 plumes per second). In some embodiments the frequency of ablation by the laser system is at least 40Hz, such as at least

50Hz, or at least 100Hz. In some embodiments the frequency of ablation by the laser system is within the range 40-2000 Hz, within the range 40-1500 Hz, within the range 40-500 Hz, within the range 40-200 Hz, within the range 40-150 Hz, or within the range 75-150 Hz. An ablation frequency of more than 40 Hz allows imaging of typical samples to be achieved in a reasonable time. The frequency with which laser pulses can be directed at a spot on the sample (assuming full ablation of the material at that spot) and still be individually resolved determines how quickly the pixels of the image can be obtained. Accordingly, if the duration of laser pulse required to ablate the material at a point means that only less than 5 pulses can be directed at a sample per second, the time taken to study a 1mm x 1mm area with ablation at a spot size of

Ιμπι would be over two days. With a rate of 40Hz, this would be around 6-7 hours, with further reductions in the analysis time for further increases in the frequency of pulses. At these frequencies the instrumentation must be able to analyse the ablated material rapidly enough to avoid substantial signal overlap between consecutive ablations, if it is desired to resolve each ablated plume individually. It is preferred that the overlap between signals originating from consecutive plumes is <10% in intensity, more preferably <5%, and ideally <2%. The time required for analysis of a plume will depend on the washout time of the ablation chamber (see ablation chamber section below), the transit time of the plume aerosol to and through the laser ionisation system, and the time taken to analyse the ionised material. Each laser pulse can be correlated to a pixel on the image of the sample that is subsequently built up, as discussed in more detail below.

In some embodiments, the laser is a femtosecond laser. In some embodiments, the laser is a picosecond laser. In some embodiments, the laser is a nanosecond laser.

Timing of the laser pulses of different wavelength

If both wavelengths are emitted by the same laser source, synchronisation between the pulses of each wavelength happens automatically. In embodiments employing multiple laser sources in the laser sampling system the laser pulses can be synchronised, if this is desired. Accordingly, in some embodiments where the laser system comprises two or more laser sources, the laser system further comprises a laser pulse timing controller, adapted to synchronise pulses from at least two of the two or more laser sources.

In some embodiments, where a single laser source emits multiple wavelengths of laser radiation and/or where two or more laser sources are used to emit multiple wavelengths laser radiation, there is a time delay between the pulses of different wavelength. When there are two or more laser sources, a delay can be achieved by firing the laser sources at different timepoints. Accordingly, in some embodiments where the laser system comprises two or more laser sources, the laser system further comprises a laser pulse timing controller, adapted to generate asynchronous pulses from at least two of the two or more laser sources. Sometimes, the asynchronous pulses are achieved by the use of a delay generator, which introduces a delay into the electrical trigger pulses which cause the pulses of laser radiation to be emitted by the laser sources.

When the laser pulses of different wavelength are emitted by the same laser source {i.e. a single pulse from the laser source comprises multiple wavelengths of light), then asynchronicity of the pulses of different wavelengths can be achieved by a number of different techniques (e.g. optical delay lines). When asynchronous pulses of different wavelengths of laser radiation are used, the plume resulting from each individual pulse can be analysed separately. Alternatively, the two pulses, although asynchronous, may be sufficiently close in time to produce a single plume, thus meaning the material from the two pulses is analysed as a single event in the mass spectrometer component of the apparatus of the invention. One technique to introduce a delay into the pulses of different wavelengths emitted by the same laser source is to direct the pulses of different wavelengths on different optical paths (see figure 1 A as discussed in more detail below). The pulse which is directed along the longer optical path will take longer to reach the sample, and so will be delayed with respect to the pulse of the wavelength which has the shorter optical path. The optical path of the wavelength can be lengthened through the use of a reflector arrangement. If the reflector arrangement is adjustable, so that the distance between the reflectors can be varied, this in turn varies the path length along which the laser pulse must travel before it hits the sample, and so permits control of the delay between the pulses of the wavelength directed along the shorter optical path versus the longer optical path. The separation of one wavelength from the one or more other wavelengths can be achieved, for example, by the use of a selectively reflective reflector (e.g. a dichroic filter) or by the use of other beam splitters such as prisms etc. Accordingly, in some embodiments, the laser system comprises an arrangement of selectively reflective reflectors and reflectors, arranged to introduce a delay into pulses of a second wavelength of light with respect to pulses of a first wavelength of light.

Another technique is to place an optical delay line, such as a variable optical delay line, between the laser source and the stage holding the sample to be ablated. Optical delay lines are commercially available from Oz Optics, Thorlabs, Newport, RF Optic etc. By introducing a delay into one of the wavelengths of laser radiation relative to the other, asynchronous delivery of the different wavelengths is achieved from an originally synchronous pulse (i.e. a controlled time delay is introduced into the pulse of one wavelength with respect to the pulse of the other wavelength). Accordingly, in some embodiments, the laser sampling system comprises a stage for supporting a sample and a laser system adapted to emit multiple wavelengths of laser radiation, wherein the laser system comprises a laser source adapted to emit multiple wavelengths of laser radiation, further comprising a variable optical delay line in the optical path of a wavelength of laser radiation between the laser source and the stage. In some embodiments, the laser system comprises two or more variable optical delay lines, arranged so that each variable optical delay line is used to delay one wavelength of laser radiation. Adapting the delay lines appropriately can be used to control the length of delay.

A delay between pulses of different wavelength can be exploited to improve the quality of laser ablation further. A burst of pulses with the same wavelength is known to be capable of having a different effect on ablation processes than a single shot with the summed energy. A similar difference should be observed for two pulses at different wavelengths. This phenomenon can be used to facilitate more uniform ablation of a heterogeneous biological sample. For example, in some instances, it might be beneficial to send a laser pulse targeting the protein band first. Since it is a deep UV light (e.g. 213nm) it can excite the protein molecules, which are ablated, and is capable of preparing the DNA sample for better absorption of the longer wavelength pulse (e.g. 266nm). In other instances, it might be advantageous to ablate the DNA material using the longer wavelength laser pulse first, before the shorter wavelength pulse ablates the remaining (principally proteinaceous) material.

An arrangement of components for introducing asynchronicity between the pulses of different wavelengths emitted as a synchronous pulse is illustrated in Figure 1A. Here, a laser source (100) emits a laser pulse comprising two wavelengths of laser radiation (110), for example the wavelength of different harmonics of the laser source. The laser radiation (110) hits a selectively reflective reflector (120), which reflects laser radiation of a first wavelength (111) but transmits laser radiation of a second wavelength (112). The first wavelength of laser radiation is directed off a reflector (121) and then a second selectively reflective reflector (122) onto the sample (140) to ablate it. After passing through the selectively reflective reflector (120), the second wavelength of laser radiation (112) optionally passes through a variable optical delay line (130), which retards the laser pulse (in some instances the longer path for the second wavelength of light may in itself introduce sufficient a time delay) between the pulses of the first and second wavelengths). The second wavelength of laser radiation (112) is then directed off a reflector (121) and through the second selectively reflective reflector (122) onto the sample (140) to ablate it, at a later point in time than the first wavelength.

Laser system optics for direction of laser radiation of different wavelengths

When two or more wavelengths of laser radiation are used in laser ablation pulses, the spatial overlap of ablation spots for different wavelengths is a parameter that may vary. For example, the ablation spot for the first wavelength can simply be misaligned from the ablation spot of the second wavelength i.e. the pulses will interrogate the different areas on the specimen, because the same optical elements differentially manipulate the wavelengths of laser radiation. This problem can be solved by the appropriate alignment of the optical components that direct the two wavelengths to the sample. Accordingly, in some embodiments, the laser system comprises an arrangement of optical components arranged to align the spots, for example the centre of the spots, of the laser radiation of different wavelengths (for example, wherein the radiation is collimated). Even if the alignment of the centre of the spots is precise, the spots can still vary in size. For instance, the diffraction limit of the focused laser radiation is related to the wavelength of laser radiation and the numerical aperture (NA) of the objective used for focusing. Thus, the spot produced by the longer wavelength can be bigger than the spot produced by the shorter wavelength. In some instances, for example where the longer wavelength pulse (i.e. larger spot size) hits the sample first, it may alter the properties of the sample across the larger spot and make the whole area illuminated more amenable to the ablation by the second pulse of shorter wavelength (which, although having a smaller spot size, still ablates a crater of material with the diameter of the spot size of the first pulse, as a result of the material being made more amenable to ablation by the first pulse). In other words the first pulse will predominantly define the size of the ablation area. In some applications, this may be advantageous and the phenomenon deliberately applied.

In another embodiments, when a Gaussian beam is utilised the spot size is also controlled by the ratio between the fluence supplied in the centre and the threshold fluence. Thus, the matching of ablation areas for the two different materials (such as DNA and protein) can be accomplished by independent adjustment of the laser energies at the different wavelengths. Accordingly, in some embodiments, the laser sampling system comprises a laser pulse power controller, programmed to control the fluence of laser pulses of differing wavelength. This can be achieved, for example, by employing independent channels of optical attenuation, one for each wavelength of laser radiation, which can be controlled by the laser pulse power controller. The power controller thus can be used to control absolute fluence of the laser pulses of each wavelength (and so can be used to maintain the fluence near the ablation threshold) and the relative fluence of the pulses of different wavelengths to ensure the same diameter of ablation crater is produced by pulses of each ablating wavelength. The energy to be used for each material at each wavelength can be adjusted offline, based on prior knowledge of ablation of the sample, or a similar sample under known conditions. Alternatively, the apparatus can comprise a controller module which monitors ablation, and adjusts energy of the pulses to maintain constant ablation craters.

Yet another technique to match the ablation spot size for two wavelengths is to employ optics where the NA for the first wavelength is different from the NA for the second wavelength. This may simply mean that the shorter wavelength laser radiation will be less expanded before arriving to the objective to reduce the NA for this wavelength. Accordingly, in some embodiments, the laser system comprises a beam expander and or a beam condenser, to control the diameter of the beam of a wavelength of laser radiation. In some embodiments, this can be achieved by using different lenses, so that the laser system comprises a first objective lens of a first NA for focussing laser radiation of a first wavelength onto the sample, and a second objective lens of a second NA for focussing laser radiation of a second wavelength onto the sample.

As an alternative to the Gaussian beam, beam shaping and beam masking can be employed to provide the desired ablation spot. For example, in some applications, a square ablation spot with a top hat energy distribution can be useful (i.e. a beam with near uniform fluence as opposed to a Gaussian energy distribution). This arrangement reduces the dependence of the ablation spot size on the ratio between the fluence at the peak of the Gaussian energy distribution and the threshold fluence. Accordingly, in some embodiments the laser system comprises beam masking and/or beam shaping components, such as a diffractive optical element, arranged to emit a laser beam of uniform or near-uniform fluence, such as a fluence that varies across the beam by less than ±25%, such as less than ±20%, ±15%, ±10% or less than ±5%. In some embodiments, the laser beam has a square cross-sectional shape. In some embodiments, the beam has a top hat energy distribution.

In order to construct an image, as noted above, plumes are generated from different locations on the sample. This is achieved by moving the sample relative to the laser source. Typically, the position of the laser source is fixed, and a translation stage holding the sample is movable within the ablation chamber of the laser sampling system. In order to produce images of a sample within a reasonable timeframe (e.g. imaging a ΙΟΟμπι x ΙΟΟμπι area within a few hours, and ablating with a laser spot size of Ι μηι), the system is required to shift the sample stage to the next location to generate the next pixel in the image rapidly.

In some instances, the time between incremental movements of the stage in order to keep up a reasonable speed of imaging is shorter than the time that is desired between pulses of a first wavelength and second wavelength hitting the same location on the sample (for example because a certain length of time is required for the sample material at that location to become most suitable for ablation with the second pulse). Here, therefore, to compensate for the movement of the stage, the first and second pulses will be directed to different locations relative to the fixed optical setup (see Figure IB) (although the movement of the stage permits the same location on the sample to be ablated with both wavelengths of laser radiation). Thus in some embodiments, the laser system comprises an optical arrangement configured so that pulses of different wavelengths are received asynchronously at the same location on a moving sample. Control of the ablation positions of the laser pulses of different wavelengths is simply a matter of appropriate positioning of mirrors/reflectors, and is well within the abilities of the skilled person, and is achievable both in lasers systems comprising multiple laser sources emitting different wavelengths and when a single laser source is adapted to emit multiple wavelengths of laser radiation.

An arrangement for directing laser radiation of different wavelengths to different locations is illustrated in Figure IB for a system comprising a single laser source. Here, a laser source (100) emits a laser pulse comprising two wavelengths of laser radiation (110), for example the wavelength of different harmonics of the laser source. The laser radiation (110) hits a selectively reflective reflector (120), which reflects laser radiation of a first wavelength (111) and so directs it onto the sample (140) on a sample carrier (150) at a first location, but transmits laser radiation of a second wavelength (112). The second wavelength is then directed onto a different location on the sample (140) by a second reflector (121). The diagram is not to scale, but illustrates how different wavelengths can be separately directed to different locations on the sample. If the spot size of the laser beam is of the order of Ι μπι, then the relative distance between the first and second locations would generally be of a few μπι (even though the spot ablated on the sample would be the same, because the sample would have been moved by the translation stage so that the spot on the sample ablated by the first wavelength would be the spot at which the second laser pulse is directed). The second wavelength of laser radiation (112) passes through a variable optical delay line (130), and so the pulse of the second wavelength of laser radiation hits the sample at a later point in time than the pulse of the first wavelength of laser radiation. If the variable optical delay line does introduce a sufficiently long delay (e.g. a delay longer than nanoseconds is required), then alternative components can be used to control the delay, for example a pulse picker can be used to direct pulses appropriately along the different optical paths (or if multiple lasers are used in the laser system, an electronic delay generator can be used to introduce delays in the pulses of different wavelengths).

When laser pulses of different wavelengths are directed to different relative locations, the plumes generated by the pulses of the first and second wavelengths derived from a single pulse of a single laser source may not ablate the same location on the sample. By way of example, say the spot size is Ι μπι, the step of each movement of the stage is Ι μπι, and the relative distance between the location at which the first wavelength (λι) ablates the sample and the location at which the second wavelength (λ₂) ablates the sample is 3μπι. In this setup, as the sample is moved by the stage, when the pulse of the λ₁ ablates position n, λ₂ (shortly after, due to passage through a delay line) ablates position n-3 (i.e. the position which λ₁ ablated three pulses of λ₁ ago), when λ₁ ablates position n+1, λ₂ (shortly after) ablates position n-2, when λ₁ ablates position n+2, λ₂ (shortly after) ablates position n-1, when λ₁ ablates position n+3, λ₂ (shortly after) ablates position n, and so on. This produces an interleaved pattern of plumes vis-a-vis locations on the sample, but as the location of each pulse of each wavelength is known, the data from each plume can be mapped to that location and an image constructed (in the above example, the order of plumes detected would thus be λι(η), λ₂(η-3), λι(η+1), λ₂(η-2), λι(η+2), λ₂(η-1), λι(η+3), λ₂(η), λι(η+4), λ₂(η+1) and so on - i.e. six other plumes are analysed between the plume generated when a first wavelength hits a specific position and the second wavelength hitting that same position.

Laser system optics for multiple modes of operation

As discussed above, it is possible to use an arrangement of optical components to direct laser radiation of different wavelengths to different relative locations. Optical components can also be arranged in order to direct laser radiation of different wavelengths onto the sample from different directions. For example one or more wavelengths can be directed onto the sample from above, and one or more other wavelengths of laser radiation can be directed from below {i.e. through the substrate, such as a microscope slide, which carries the sample, also termed the sample carrier). This enables multiple modes of operation for the same imaging mass spectrometer, such as an imaging mass cytometer. Accordingly, in some embodiments, the laser system comprises an arrangement of optical components, arranged to direct laser radiation of different wavelengths onto the sample from different directions. In some embodiments, the arrangement directs laser radiation of different wavelengths onto the sample from opposite directions {e.g. as illustrated in figures 2 and 3). "Opposite" directions in this context is not limited to laser radiation directed perpendicularly onto the sample from above and below (which would be 180° opposite), but includes arrangements which direct laser radiation onto the sample at angles other than perpendicular to the sample. There is no requirement for the laser radiation directed onto the sample from different directions to be parallel. In some embodiments, the sample is on a sample carrier, the reflector arrangement is arranged to direct laser radiation of a first wavelength directly onto the sample and to direct laser radiation of a second wavelength to the sample through the sample carrier.

One example of such an arrangement is set out schematically in figure 2. Here, the laser radiation (110) from a laser source (100) is passed through a component which directs the laser radiation of different wavelengths along different optical paths (200; e.g. a prism). One or more wavelengths of laser radiation (111) can be directed directly onto the sample (140), for example by a reflector (210), and one or more other wavelengths can be directed by a reflector arrangement (220) to the sample (140) through the sample carrier (150).

Another example of such optics is set out schematically in figure 3. In this figure, a selectively reflective reflector (120) is used to direct laser radiation of one wavelength (111) onto the sample (140) from above. This selectively reflective reflector does not reflect the laser radiation of the second wavelength (112), which can then be directed by a reflector arrangement (121) to the sample (140) through the sample carrier (150).

Directing laser radiation through the sample carrier to the sample can be used to ablate the sample. In some embodiments, however, directing the laser radiation through the carrier can be used for "lifting" modes of operation, as discussed below. In some embodiments, the NA of the lens used to focus the first wavelength onto the sample from the first direction is different from the NA of the lens used to focus the second wavelength onto the sample from the second direction. The lifting operation (e.g. where laser radiation is directed through the sample carrier) often employs a spot size of greater diameter then when ablation is being performed.

Chamber o f the laser sampling system

The spatial resolution of the signals generated from laser ablation (i.e. when ablation is used for imaging rather than exclusively for clearing, as discussed below) in this way depends on factors including: (i) the spot size of the laser, as signal is integrated over the total area which is ablated; and the speed with which plumes are generated versus the movement of the sample relative to the laser, and (ii) the speed at which a plume can be analysed, relative to the speed at which plumes are being generated, to avoid overlap of signal from consecutive plumes as mentioned above. Accordingly, being able to analyse a plume in the shortest time possible minimises the likelihood of plume overlap (and so in turn enables plumes to be generated more frequently).

Accordingly, an ablation chamber with a short washout time (e.g. 100 ms or less) is advantageous for use with the apparatus and methods of the invention. A cell with a long washout time will either limit the speed at which an image can be generated or will lead to overlap between signals originating from consecutive sample spots (e.g. reference 6, which had signal duration of over 10 seconds). Therefore aerosol washout time is a key limiting factor for achieving high resolution without increasing total scan time. Ablation chambers with washout times of <100 ms are known in the art. For example, reference 7 discloses an ablation chamber with a washout time below 100 ms. An ablation chamber was disclosed in reference 8 (see also reference 9) which has a washout time of 30 ms or less, thereby permitting a high ablation frequency (e.g. above 20 Hz) and thus rapid analysis. Another such ablation chamber is disclosed in reference 10. The ablation chamber in reference 10 comprises a sample capture cell configured to be arranged operably proximate to the target, the sample capture cell including: a capture cavity having an opening formed in a surface of the capture cell, wherein the capture cavity is configured to receive, through the opening, target material ejected or generated from the laser ablation site and a guide wall exposed within the capture cavity and configured to direct a flow of the carrier gas within the capture cavity from an inlet to an outlet such that at least a portion of the target material received within the capture cavity is transferrable into the outlet as a sample. The volume of the capture cavity in the ablation chamber of reference 10 is less than lcm³ and can be below 0.005cm³. Sometimes the ablation chamber has a washout time of 25ms or less, such as 20ms or 10ms or less.

For completeness, sometimes the plumes from the sample can be generated more frequently than the washout time of the ablation chamber, and the resulting images will smear accordingly (e.g. if the highest possible resolution is not deemed necessary for the particular analysis being undertaken).

The ablation chamber typically comprises a translation stage which holds the sample (and sample carrier) and moves the sample relative to the laser beams. When a mode of operation is used which requires the direction of laser radiation through the sample carrier to the sample, e.g. as in the lifting methods discussed above, the stage holding the sample carrier should also be transparent to the laser radiation used.

Ionisation system

The transit time of a plume of ablated sample material or desorbed slug of sample to and through the ionisation system is easily controlled simply by minimising the length of the conduit between the ablation chamber and the ionisation system and by ensuring a sufficient gas flow to transport the aerosol at an appropriate speed directly to the ionisation system. Transport using argon and helium provides good results (other gasses and gas mixtures can be employed depending on the requirements of the ionization system [11]).

Sample material can be ionised by a variety of techniques. The use of an ICP is suited for IMS and IMC analyses. ICP is a plasma source in which the energy is supplied by electric currents produced by electromagnetic induction. Typically the plasma source is based on Argon gas. For example, the ionization system may comprise an ICP torch. FMC using ICP in the ionisation system is reported on in, for example, references 5 and 8. The ionisation system thus receives sample material from the laser sampling system and converts it into elemental ions for detection by the mass spectrometer. If the sample material is not atomised (e.g. if it is still in the form of molecules, or even an aerosol of particulate material) then the ionisation system acts to break down the material into elemental ions as part of the ionisation process.

Mass spectrometer

As noted above, the third component of the apparatus is a mass spectrometer. Many different kinds of mass spectrometer can be used.

The time taken to analyse the ionised material will depend on the type of mass analyser/mass spectrometer which is used for detection of ions. For example, instruments which use Faraday cups may be too slow for analysing rapid signals, but not all analyses will require the rapid analysis of signals, and so the skilled person will be able to select the mass spectrometer or mass analyser appropriately. Overall, the desired analysis speed (and thus the frequency with which ablation plumes can be interrogated) and degree of multiplexing (number of atoms to be monitored simultaneously/quasi-simultaneously) will dictate the type(s) of mass analyser which should be used (or, conversely, the choice of mass analyser will determine the speed and multiplexing which can be achieved).

Mass spectrometry instruments that detect ions at only one mass-to-charge ratio (m/Q, commonly referred to as m/z in MS) at a time, for example using a point ion detector, may give poor results when trying to detect multiple ions of different m/Q, whether detection is of ions from elements naturally in the sample, or in particular where ions from a range of labelling atoms have been introduced into the sample (such as a biological sample in which target molecules have been labelled using affinity reagents linked to labelling atoms, as discussed below). The time taken to switch between mass-to-charge ratios limits the speed at which multiple signals can be determined, and secondly, if ions are at low abundance then signals can be missed when the instrument is focused on other mass-to-charge ratios. Thus, although the instrument used in references 12 & 13 (Agilent/HP 4500 IPC MS) is sensitive and appropriate for certain applications, as understood by the skilled person, its quadrupole-based detector is not well suited to use with the rapid analysis of ions at multiple mass-to-charge ratios because, by design, ions of different mass-to-charge ratios pass through sequentially and so data acquisition for multiple ions is slow. The instrument used in reference 14 (Thermo Fisher ElementXR and Element2) analyses only one m/Q at a time and have a large settling time for magnet jumps when measuring multiple m/Q values over a range exceeding the range of an electrostatic field jump, and is understood to be applicable in certain applications but not others.

In some embodiments, a technique which offers substantially simultaneous detection of ions having different m/Q values is used. For instance, instead of using a point ion detector, it is possible to use an array detector (e.g. see Chapter 29 of ref. 15). These instruments can measure multiple m/Q signals substantially simultaneously. Their sensitivity can be increased by including electron multipliers in the detectors.

A particularly useful type of mass spectrometer is based on time-of-flight (TOF) detection, which can quasi-simultaneously register multiple masses in a single sample. Whereas TOF mass analyzers are normally unpopular for atomic analysis because of the compromises required to deal with the effects of space charge in the TOF accelerator and flight tube, the effectiveness of the technique can be improved by using it only to detect a subset of ranges. For example, in mass cytometry and imaging mass cytometry, a range may be chosen only such that ions from the labelling atoms used to mark target molecules in a biological samples are detected and so other atoms (e.g. those having an atomic mass below 100) can be removed. This results in a less dense ion beam, enriched in the masses in (for example) the 100-250 dalton region, which can be manipulated and focused more efficiently, thereby facilitating TOF detection and taking advantage of the high spectral scan rate of TOF. Thus rapid analyses can be achieved by combining TOF detection with choosing labelling atoms that are uncommon in the sample and ideally having masses above the masses seen in an unlabelled sample e.g. by using the higher mass transition elements. Further details on mass cytometry can be found in references 16 and 17, and on imaging mass cytometry in reference 7.

Methods of the invention

The methods of the invention are based on the principle of imaging a sample by imaging mass spectrometry, comprising performing laser ablation of the sample using laser radiation of two or more wavelengths.

For instance, the invention provides a method of analysing a sample comprising:

(ii) subjecting the plumes individually to ionisation and mass spectrometry, whereby detection of atoms in the plumes permits construction of an image of the sample. The sample analysed may be a biological sample. The method of analysis may be mass cytometry.

The invention also provides method of performing mass cytometry on a sample comprising a plurality of cells, the method comprising:

In some embodiments, a first wavelength of the two or more wavelengths preferentially ablates the proteinaceous material in the sample, and a second wavelength of the two or more wavelengths preferentially ablates nucleic acid material. In some embodiments, the two of the two or more wavelengths are emitted from the same laser source, for example by harmonics generation. For example, the first wavelength may be about 213nm and the second wavelength about 266nm. In some embodiments, pulses of the first and second wavelengths of laser radiation ablate the sample at the same known location simultaneously. In some embodiments, a first wavelength of the two or more wavelengths of laser radiation is used to ablate a known location before a second wavelength of laser radiation is used to ablate the known location.

Various other embodiments of the methods are described above, for example, ablating the sample with a spot size of controlled diameter, and ablating to a controlled depth in the "Laser system of the laser sampling system" section, and the description in that section and the other elsewhere herein applies equally as embodiments of the methods described here. For instance, where the text in the above section says: "the laser spot will have a diameter of less than 4 μπι e.g. within the range 0.1-4 μπι, 0.25-3 μπι, or 0.4-2 μπι", then the corresponding embodiment of the method is "performing laser ablation of the sample using a laser spot of diameter less than 4 μιη" etc.

Lifting methods of the invention

The technique of "lifting" is described in co-pending applications US 62/098,890 and US 62/108,911, which are hereby incorporated by reference in their entirety. In lifting, rather than ablation of the sample, the laser radiation is used to desorb a bulk mass of sample material from the sample carrier on which it is located without substantial disintegration of the sample and its conversion into small particles and/or vaporisation. Herein, the term slug is used to refer to this desorbed material.

In certain embodiments, sample material may be desorbed by thermal mechanisms. In such embodiments, the surface of the sample carrier at the site irradiated by laser radiation becomes sufficiently hot to desorb the biological material. As discussed in more detail below, in some embodiments, the plurality of discrete sites comprises a thermally desorbable material configured to release the biological sample. Likewise, discussed in more detail below, the sample can be desorbed from the sample by thermal energy, mechanical energy, kinetic energy, and a combination of any of the foregoing. One example is the use of laser radiation energy, in a technique called lifting (laser induced forward transfer; see e.g. Doraiswamy et al., 2006, Applied Surface Science, 52: 4743-4747; Fernandez-Pradas, 2004, Thin Solid Films 453-454: 27-30; Kyrkis et al., in Recent Advances in Laser Processing of Materials, Eds. Perriere et al., 2006, Elsivier). Accordingly, in some embodiments, the sample carrier comprises a desorption film layer. In other words, the sample carrier has a film layer on the surface of the carrier which is for receiving the sample, and which assists the separation of the sample from the sample carrier by absorbing laser radiation. The desorption film can absorb the radiation to cause release of the desorption film and/or the biological sample (e.g. in some instances the sample film desorbs from the sample carrier together with the biological sample, in other instances, the film remains attached to the sample carrier, and the biological sample desorbs from the desorption film). The sample carrier may be coated to facilitate the bulk desorption process, for example with polyethylene naphthalate (PEN) polymer or PMMA polymer film.

The energy applied to the surface should be sufficient to desorb the biological material, preferably without altering the biological sample. Any suitable radiation wavelength can be used, which can depend in part on the absorptive properties of the sample carrier. In certain embodiments, a surface or layer of the sample carrier may be coated with or include an absorber that absorbs laser radiation for conversion to heat. The radiation may be delivered to a surface of the carrier other than the surface on which the sample is located, or it may be delivered to the surface carrying the sample, such as through the thickness of the carrier. The heated surface may be a surface layer or may be an inner layer of a multilayer structure of the sample carrier.

Desorption by heating can take place on a nanosecond, picosecond or femtosecond time scale, depending on the laser used for desorption. In some embodiments, the sample may be attached to the sample carrier by a cleavable photoreactive moiety. Upon irradiating the cleavable photoreactive moiety with radiation (e.g. from a laser in the laser system of the laser sampling system), the photoreactive moiety can cleave to release sample material. In some embodiments, the sample carrier may comprise (i) a cleavable photoreactive moiety that couples the sample to the sample carrier and (ii) a desorption film as discussed above. In this situation, a first laser radiation pulse may be used to cause cleavage of the photoreactive moiety and a second laser radiation pulse may be used to target the desorption film to cause separation of the sample from the sample carrier by lifting (or a thermal energy pulse introduced by other means may be used to heat the desorption film and so cause separation of sample material from the sample carrier). The first and second pulses may be of different wavelengths. Thus in some embodiments of the methods discussed in more detail below (e.g. comprising both ablation and desorption), separation of the sample from the sample carrier may involve multiple laser pulses of different wavelengths. In some instances, cleavage of the photoreactive moiety and lifting may be accomplished by the same laser pulse.

In some embodiments, the sample carrier may include a coating or layer of a chemically reactive species that imparts kinetic energy to the biological sample to release the sample from the surface. For example, a chemically reactive species may release a gas such as, for example, H₂, C0₂, N₂ or hydrochlorofluorocarbons. Examples of such compounds include blowing and foaming agents, which release gas upon heating. Generation of gas can be used to impart kinetic energy to desorbing sample material that can improve the reproducibility and direction of release of sample material.

In some embodiments, the same carrier may be coated with photoinitiated chemical reactants that undergo an exothermic reaction to generate heat for desorbing sample material.

The coating of the carrier, or indeed particular chemical linkages in that carrier, discussed in the above paragraphs (that is irradiated by the laser to release the slug of sample material from the carrier) is an example of a material that can be targeted by a wavelength of laser radiation, as discussed herein in relation to the methods and apparatus of the invention.

Each, or a combination, of these techniques permits ordered detachment of sample material from the sample carrier. However, often, the location on the sample that is of interest does not represent a discrete entity, such as a lone cell, at a discrete site which can be easily lifted in isolation. Instead, the cell of interest may be surrounded by other cells or material, of which analysis is not required or desired. Trying to perform lifting of the location of interest may therefore desorb both the cell of interest and surrounding material together. Atoms, such as labelling atoms (see discussion below), from the surrounding area of the sample (e.g. from other cells which have been labelled) that are carried in a slug of material in addition to the specific location (e.g. cell) of interest could therefore contaminate the reading for the location of interest.

A solution to this problem is provided by the invention whereby the techniques of ablation and lifting can be combined in a single method. For example, to perform precise lifting of a location (e.g. cell) of interest in a sample, e.g. a tissue section sample or cell suspension dispersion, on the sample carrier, laser ablation can be used to ablate the area around the cell of interest to clear it of other material. After clearing the surrounding area by ablation, the location of interest can then be desorbed from the sample carrier by using the desorption laser wavelength, and then ionised and analysed by mass spectrometry in line with standard mass cytometry procedures. This is shown in figures 4 and 5.

Figure 4 is a schematic of the steps of the combined ablation and lifting method. In figure 4A, first laser radiation of a first wavelength (410) is directed on the sample (140), which ablates that part of the sample (as indicated by the gap between sample fragments (431) and 432, the slug of material to be analysed, in figures 4B and 4C). The sample (140) is on a sample carrier (150) and in between the sample and the carrier is a functionalised layer (400) of the type discussed in the preceding paragraphs, which assists the desorption of sample material from the surface of carrier. Figures 4B and 4C illustrate the same step - the irradiation of the functionalised layer (400), with laser radiation of a different wavelength (420) from that used to clear the area surrounding the portion of the sample to be analysed (432), or if multiple wavelengths were used to clear the surrounding area, then just one of those wavelengths might be used to desorb the sample. Figure 4B and 4C are alternative modes of irradiation. 4B shows irradiation of the functionalised layer (400) through the sample carrier (150), whereas 4C shows irradiation of the functionalised layer (400) through the sample material to be desorbed (432). Figure 4D illustrates the production of a gas (480) by the functionalised layer (400) following irradiation which ejects the slug of sample material (432) into the gas phase, wherein it is carried by the flow of gas (490) into the conduit (495) leading to the ionisation system. In some instances, there is no production of gas by the functionalized layer to eject the slug of sample material, and instead another kind of laser- induced desorption occurs when the functionalized layer, such as a desorption film, absorbs laser radiation.

In some embodiments, the wavelength of laser radiation used for lifting is a harmonic of the same laser for ablation. For example, the third harmonic on an Nd-YAG laser at 355 nm can be used for lifting. This means that a single Nd-YAG laser supporting three wavelengths can be employed in a system combining the laser ablation and lifting. The 3^rd harmonic at 355 nm will support lifting, the 4^th harmonic at 266 nm will support the laser ablation of DNA material and the 5^th harmonic at 213 nm will support the laser ablation of the protein material.

Accordingly, the invention provides a method of analysing a sample comprising:

(i) performing laser ablation of a sample using laser radiation of a first wavelength;

(ii) desorbing a slug of sample material using a second wavelength of laser radiation; and

(iii) ionising the slug of sample material and detecting atoms in the slug by mass spectrometry.

In some embodiments, the ablation of the sample generates one or more plumes of sample material, and wherein the plumes are individually ionised and the atoms in the plume detected by mass spectrometry. In some embodiments, the sample is on a sample carrier comprising a functionalised layer, and the second wavelength of laser radiation targets the functional layer, of the kind discussed above (such as a desorption film), in order to cause the desorption of the slug of sample material that separates it from the sample (i.e. lifting).

In some embodiments of the invention, the parts of the sample that are removed by desorption and by ablation may be different. For example, where there is a cluster of cells, ablation with (such as with subcellular resolution) may be performed, to enable the imaging of all cells in the cluster (e.g. where desorbing the sample material could remove multiple cells at once, which may not be desired where cell-by-cell analysis is required). On the same sample, however, there may be cells which are spaced apart from the other cells, and so can be lifted. In some embodiments, step (ii) is performed before step (i).

In some embodiments, the sample is a biological sample, such as a tissue section, or a cell solution dispersed on the slide (and optionally fixed). In some embodiments, prior to step (i), the method comprises the additional step labelling a plurality of different target molecules in the sample with one or more different labelling atoms, to provide a labelled sample. The labelling step thereby enables imaging mass cytometry, in addition to imaging mass spectrometry.

In some embodiments, the laser ablation is used to ablate the material around a location of interest to clear the surrounding area before the sample material at the location of interest is desorbed as a slug of material.

The location of interest (both in methods using lifting and those reliant only on ablation using multiple wavelengths of laser radiation as discussed elsewhere herein) can be identified by another technique before the laser ablation and lifting is performed (see also co-pending applications US 62/097406 and US 62/1 17542, which are hereby incorporated by reference in their entirety). The inclusion of a camera (such as a charged coupled device image sensor based (CCD) camera or a CMOS camera or an active pixel sensor based camera), or any other light detecting means in an imaging mass spectrometer as described in the preceding sections is one way of enabling these techniques. The camera can be used to scan the sample to identify cells of particular interest or locations of particular interest (for example cells of a particular morphology). Once such locations have been identified, the locations can be lifted after laser pulses have been directed at the area around the location of interest to clear other material by ablation before the location (e.g. cell) is lifted. This process may be automated (where the system both identifies, ablates and lifts the location(s) of interest) or semi-automated process (where the user of the system, for example a clinical pathologist, identifies the location(s) of interest, following which the system then performs ablation and lifting in an automated fashion). This enables a significant increase in the speed at which analyses can be conducted, because instead of needing to ablate the entire sample to analyse particular cells, the cells of interest can be specifically ablated.

The camera can record the image from a confocal microscope. The identification may be by light microscopy, for example by examining cell morphology or cell size. Sometimes, the sample can be specifically labelled to identify the location(s) (e.g. cell(s)) of interest. In the cells in the images in figure 5, a dye that stains that nucleic acids in the nucleus has been used. The majority of cells are red blood cells which can be distinguished from white blood cells by both size and morphology (the red blood cells are anuclear). The image contains two white blood cells, which are larger than the red blood cells and comprise a nucleus. The two white blood cells can be distinguished from one another on the basis of the morphology of their nuclei. The cell of interest (surrounded by black dotted border in figure 5B) can be classified as a band neutrophil on the basis of its nucleus, and the other white blood cell is a segmented neutrophil. On recognition of the characteristic nucleus, the steps of ablation and lifting can be performed to clear the area surrounding the cell and then the cell lifted for analysis, as illustrated in figure 5C and 5D.

Often, fluorescent markers are used to specifically stain the cells of interest (such as by using labelled antibodies or labelled nucleic acids). These fluorescent makers can be used to stain specific cell populations (e.g. expressing certain genes and/or proteins) or specific morphological features on cells (such as the nucleus, or mitochondria) and when illuminated with an appropriate wavelength of light, these regions of the sample are specifically identifiable. In some instances, the absence of a particular kind fluorescence from a particular area may be characteristic. For instance, a first fluorescent label targeted to a cell membrane protein may be used to broadly identify cells, but then a second fluorescent label targeted to the ki67 antigen (encoded by the MKI67 gene) can discriminate between proliferating cells and non-proliferating cells. Thus by targeting cells which lack fluorescence from the second label fluorescent, non-replicating cells can be specifically targeted for analysis. In some embodiments, the systems described herein therefore can comprise a laser for exciting fluorophores in the labels used to label the sample. Alternatively, an LED light source can be used for exciting the fluorophores. Non-confocal (e.g. wide field) fluorescent microscopy can also be used to identify certain regions of the biological sample, but with lower resolution than confocal microscopy.

When a laser is used to excite fluorophores for fluorescence microscopy, in some embodiments this laser is the same laser that generates the laser radiation used to ablate material from the biological sample and for lifting (desorption), but used at a fluence that is not sufficient to cause ablation or desorption of material from the sample. In some embodiments, the fluorophores are excited by a wavelength of laser radiation that is used for sample desorption. In others, a different wavelength may be used, for example by exploiting different harmonics of the laser to obtain laser radiation of different wavelengths. The laser radiation that excites the fluorophores may be provided by a different laser source from the ablation and/or lifting laser source(s).

By using an image sensor (such as a CCD detector or an active pixel sensor, e.g. a CMOS sensor), it is possible to entirely automate the process of identifying regions of interest and then ablating them, by using a control module (such as a computer or a programmed chip) which correlates the location of the fluorescence with the x,y coordinates of the sample and then directs the ablation laser radiation to the area surrounding that location before the cell at the location is lifted. As part of this process, in some embodiments, the first image taken by the image sensor has a low objective lens magnification (low numerical aperture), which permits a large area of the sample to be surveyed. Following this, a switch to an objective with a higher magnification can be used to home in on the particular features of interest that have been determined to fluoresce by higher magnification optical imaging. These features recorded to fluoresce may then be lifted. Using a lower numerical aperture lens first has the further advantage that the depth of field is increased, thus meaning features buried within the sample may be detected with greater sensitivity than screening with a higher numerical aperture lens from the outset. In methods and systems in which fluorescent imaging is used, the emission path of fluorescent light from the sample to the camera may include one or more lenses and/or one or more optical filters. By including an optical filter adapted to pass a selected spectral bandwidth from one or more of the fluorescent labels, the system is adapted to handle chromatic aberrations associated with emissions from the fluorescent labels. Chromatic aberrations are the result of the failure of lenses to focus light of different wavelengths to the same focal point. Accordingly, by including an optical filter, the background in the optical system is reduced, and the resulting optical image is of higher resolution. A further way to minimise the amount of emitted light of undesired wavelengths that reaches the camera is to exploit chromatic aberration of lenses deliberately by using a series of lenses designed for the transmission and focus of light at the wavelength transmitted by the optical filter, akin to the system explained in reference 18.

A higher resolution optical image is advantageous in this coupling of optical techniques and lifting, because the accuracy of the optical image then determines the precision with which the ablating laser source can be directed to ablate the area surrounding the cell of interest.

Accordingly, the invention provides a method of performing mass cytometry on a sample comprising a plurality of cells, the method comprising steps of: (i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms, to provide a labelled sample; (ii) illuminating the sample with light (e.g. laser radiation) to identify one or more locations of interest; (iii) recording locational information of the one or more locations of interest on the sample; (iv) using the locational information of the locations of interest to desorb a slug of sample material from a location of interest, comprises first performing laser ablation to remove sample material surrounding the location of interest using laser radiation of a first wavelength, before the slug of sample material is desorbed from the location using laser radiation of a second wavelength; (v) ionising the desorbed slug of sample material; and (vi) subjecting the ionised sample material to mass spectrometry, for detection of labelling atoms in the sample material.

The invention also provides a method of performing mass cytometry on a sample comprising a plurality of cells, the method comprising steps of: (i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms and one or more fluorescent labels, to provide a labelled sample; (ii) illuminating the sample with light (e.g. laser radiation) to excite the one or more fluorescent labels; (iii) recording locational information of one or more locations of the sample based on the pattern of fluorescence; (iv) using the locational information based on the pattern of fluorescence to desorb a slug of sample material from a location of interest, comprising first performing laser ablation to remove sample material surrounding the location of interest using laser radiation of a first wavelength, before the slug of sample material is desorbed from the location using laser radiation of a second wavelength; (v) ionising the desorbed slug of sample material; and (vi) subjecting the ionised sample material to mass spectrometry, for detection of labelling atoms in the sample material.

In some embodiments of the methods described above, the sample is on a sample carrier (e.g. microscope slide or other appropriate solid platform from which the sample can be ablated or desorbed, typically without significant removal of atoms of the carrier). In some embodiments, the wavelength of laser radiation used to desorb sample material is directed to the sample material through the sample carrier. In some embodiments, the slug of sample material is desorbed by lifting. In some embodiments, the sample is on a sample carrier which comprises a layer which absorbs laser radiation to assist lifting of the slug of sample material. Examples of suitable layers, which may be a desorption film coated on the surface of the sample carrier, include triazene polymer, such as shown in Figure 1 of Doraiswamy et al., 2006, Applied Surface Science, 52: 4743-4747, or other polymers which evaporate on upon laser radiation.

In some embodiments, no data are recorded from the ablation performed to clear the area around the location to be desorbed (e.g. the cell of interest). In some embodiments, data is recorded from the ablation of the surrounding area. Useful information that can be obtained from the surrounding area includes what target molecules, such as proteins and RNA transcripts, are present in the surrounding cells and intercellular milieu. This may be of particular interest when imaging solid tissue samples, where direct cell-cell interactions are common, and what proteins etc. are expressed in the surrounding cells may be informative on the state of the cell of interest.

The invention also provides an imaging mass spectrometer, such as an imaging mass cytometer, comprising a control module programmed to perform the methods set out in this section.

Constructing an image

IMS and IMC can provide signals for multiple labelling atoms in plumes. Detection of a label in a plume reveals the presence of its cognate target at the position of ablation (or, correspondingly, the position of desorption of the slug of material). By generating a series of plumes at known spatial locations on the sample's surface the MS signals reveal the location of the labels on the sample, and so the signals can be used to construct an image of the sample. By labelling multiple targets with distinguishable labels it is possible to associate the location of labelling atoms with the location of cognate targets, so the invention can build complex images, reaching levels of multiplexing which far exceed those achievable using existing techniques. The inventors have shown that images generated by the methods of the invention can reproduce the staining patterns and the proportion of cells expressing a given marker as determined by IFM, thereby confirming the invention's suitability for imaging.

Assembly of signals into an image will use a computer and can be achieved using known techniques and software packages. For instance, reference 3 used the GRAPHIS package from Kylebank Software, but other packages such as TERAPLOT, ImageJ and CellProfiler can also be used. Imaging using MS data from techniques such as MALDI-MSI is known in the art e.g. reference 19 discloses the 'MSiReader' interface to view and analyse MS imaging files on a Matlab platform, and reference 20 discloses two software instruments for rapid data exploration and visualisation of both 2D and 3D MSI data sets in full spatial and spectral resolution e.g. the 'Datacube Explorer' program.

Samples

The invention provides a method of imaging a sample. All kinds of samples can be analysed by the methods, including alloys, geological samples and archaeological samples. Biological samples can also be analysed. Such samples comprise a plurality of cells, a plurality of these cells can be subjected to IMC in order to provide an image of these cells in the sample. In general, the invention can be used to analyse tissue samples which are now studied by IHC techniques, but with the use of labels which are suitable for detection by FMC.

Any suitable tissue sample can be used in the methods described herein. For example, the tissue can be epithelium tissue, muscle tissue, nerve tissue, etc., and combinations thereof. For diagnostic or prognostic purposes the tissue can be from a tumor. In some embodiments a sample may be from a known tissue, but it might be unknown whether the sample contains tumor cells. Imaging can reveal the presence of targets which indicate the presence of a tumor, thus facilitating diagnosis. The tissue sample may comprise breast cancer tissue, for example human breast cancer tissue or human mammary epithelial cells (HMLE). The tissue sample may comprise formalin-fixed, paraffin-embedded (FFPE) tissue. The tissues can be obtained from any living multicellular organism, but will usually be human.

The tissue sample will usually be a section e.g. having a thickness within the range of 2-10 μπι, such as between 4-6 μπι. Techniques for preparing such sections are well known from the field of IHC e.g. using microtomes, including dehydration steps, including embedding, etc. Thus a tissue may be chemically fixed and then sections can be prepared in the desired plane. Cryosectioning or laser capture microdissection can also be used for preparing tissue samples. Samples may be permeabilised e.g. to permit of reagents for labelling of intracellular targets (see above).

The size of a tissue sample to be analysed will be similar to current IHC methods, although the maximum size will be dictated by the laser ablation apparatus, and in particular by the size of sample which can fit into its ablation chamber. A size of up to 5 mm x 5 mm is typical, but smaller samples (e.g. 1 mm x 1 mm) are also useful (these dimensions refer to the size of the section, not its thickness).

In addition to being useful for imaging tissue samples, the invention can instead be used for imaging of cellular samples such as monolayers of adherent cells or of cells which are immobilised on a solid surface (as in conventional immunocytochemistry). These embodiments are particularly useful for the analysis of adherent cells that cannot be easily solubilized for cell- suspension mass cytometry. Thus, as well as being useful for enhancing current immunohistochemical analysis, the invention can be used to enhance immunocytochemistry.

After being prepared, the sample will be placed into the chamber of the laser sampling system or laser desorption ionisation system, and then subjected to analysis according to the invention.

Labelling of the tissue sample

In some embodiments, as described above, the apparatus and methods of the invention detect atoms that have been added to a sample (i.e. which are not normally present). Such atoms are called labelling atoms. The sample is typically a biological sample comprising cells, and the labelling atoms are used to label target molecules in the cells/on the cell surface. In some embodiments, simultaneous detection of many more than one labelling atom, permitting multiplex label detection e.g. at least 3, 4, 5, 10, 20, 30, 32, 40, 50 or even 100 different labelling atoms is enabled. Labelling atoms can also be used in a combinatorial manner to even further increase the number of distinguishable labels. By labelling different targets with different labelling atoms it is possible to determine the presence of multiple targets on a single cell.

Labelling atoms that can be used with the invention include any species that are detectable by MS and that are substantially absent from the unlabelled sample. Thus, for instance, ¹²C atoms would be unsuitable as labelling atoms because they are naturally abundant, whereas ^UC could in theory be used because it is an artificial isotope which does not occur naturally. In preferred embodiments, however, the labelling atoms are transition metals, such as the rare earth metals (the 15 lanthanides, plus scandium and yttrium). These 17 elements provide many different isotopes which can be easily distinguished by MS. A wide variety of these elements are available in the form of enriched isotopes e.g. samarium has 6 stable isotopes, and neodymium has 7 stable isotopes, all of which are available in enriched form. The 15 lanthanide elements provide at least 37 isotopes that have non-redundantly unique masses. Examples of elements that are suitable for use as labelling atoms include Lanthanum (La), Cerium (Ce), Praseodymium (Pr), Neodymium (Nd), Promethium (Pm), Samarium (Sm), Europium (Eu), Gadolinium, (Gd), Terbium (Tb), Dysprosium (Dy), Holmium (Ho), Erbium (Er), Thulium (Tm), Ytterbium (Yb), Lutetium (Lu), Scandium (Sc), and Yttrium (Y). In addition to rare earth metals, other metal atoms are suitable for detection by MS e.g. gold (Au), platinum (Pt), iridium (Ir), rhodium (Rh), bismuth (Bi), etc. The use of radioactive isotopes is not preferred as they are less convenient to handle and are unstable e.g. Pm is not a preferred labelling atom among the lanthanides.

In order to facilitate TOF analysis (see above) it is helpful to use labelling atoms with an atomic mass within the range 80-250 e.g. within the range 80-210, or within the range 100-200. This range includes all of the lanthanides, but excludes Sc and Y. The range of 100-200 permits a theoretical 101-plex analysis by using different labelling atoms, while permitting the invention to take advantage of the high spectral scan rate of TOF MS. As mentioned above, by choosing labelling atoms whose masses lie in a window above those seen in an unlabelled sample {e.g. within the range of 100-200), TOF detection can be used to provide rapid analyses at biologically significant levels.

Labelling the particles generally requires that the labelling atoms are attached to one member of a specific binding pair (sbp). This labelled sbp is contacted with a sample such that it can interact with the other member of the sbp (the target sbp member) if it is present, thereby localising the labelling atom to a target molecule in the sample. The method of the invention then detects the presence of the labelling atom on a particle as it is analysed by the mass cytometer. Rare earth metals and other labelling atoms can be conjugated to sbp members by known techniques e.g. reference 21 describes the attachment of lanthanide atoms to oligonucleotide probes for MS detection, reference 22 describes the use of ruthenium to label oligonucleotides, and Fluidigm Canada sells the MaxPar™ metal labelling kits which can be used to conjugate over 30 different labelling atoms to proteins (including antibodies).

Various numbers of labelling atoms can be attached to a single sbp member, and greater sensitivity can be achieved when more labelling atoms are attached to any sbp member. For example greater than 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 labelling atoms can be attached to a sbp member. For example, monodisperse polymers containing multiple monomer units may be used, each containing a chelator such as DTPA. DTP A, for example, binds 3+ lanthanide ions with a dissociation constant of about 10^"6 M [1]. These polymers can terminate in a thiol-reactive group (e.g. maleimide) which can be used for attaching to a sbp member. For example the thiol- reactive group may bind to the Fc region of an antibody. Other functional groups can also be used for conjugation of these polymers e.g. amine-reactive groups such as N-hydroxy succinimide esters, or groups reactive against carboxyls or against an antibody's glycosylation. Any number of polymers may bind to each sbp member. Specific examples of polymers that may be used include straight-chain ("X8") polymers or third-generation dendritic ("DN3") polymers, both available as MaxPar™ reagents. Use of metal nanoparticles can also be used to increase the number of atoms in a label.

As mentioned above, labelling atoms are attached to a sbp member, and this labelled sbp member is contacted with the sample where it can find the target sbp member (if present), thereby forming a labelled sbp. The labelled sbp member can comprise any chemical structure that is suitable for attaching to a labelling atom and then for detection according to the invention.

In general terms, methods of the invention can be based on any sbp which is already known for use in determining the presence of target molecules in samples (e.g. as used in IHC or fluorescence in situ hybridisation, FISH) or fluorescence-based flow cytometry, but the sbp member which is contacted with the sample will carry a labelling atom which is detectable by MS. Thus the invention can readily be implemented by using available flow cytometry reagents, merely by modifying the labels which have previously been used e.g. to modify a FISH probe to carry a label which can be detected by MS.

The sbp may comprise any of the following: a nucleic acid duplex; an antibody/antigen complex; a receptor/ligand pair; or an aptamer/target pair. Thus a labelling atom can be attached to a nucleic acid probe which is then contacted with a sample so that the probe can hybridise to complementary nucleic acid(s) therein e.g. to form a DNA/DNA duplex, a DNA/RNA duplex, or a RNA/RNA duplex. Similarly, a labelling atom can be attached to an antibody which is then contacted with a sample so that it can bind to its antigen. A labelling atom can be attached to a ligand which is then contacted with a sample so that it can bind to its receptor. A labelling atom can be attached to an aptamer ligand which is then contacted with a sample so that it can bind to its target. Thus labelled sbp members can be used to detect a variety of target molecules in a sample, including DNA sequences, RNA sequences, proteins, sugars, lipids, or metabolites.

In a typical embodiment of the invention the labelled sbp member is an antibody. Labelling of the antibody can be achieved through conjugation of one or more labelling atom binding molecules to the antibody, for example using the MaxPar™ conjugation kit as described above. The target molecule of an antibody is called its antigen, and may be a protein, carbohydrate, nucleic acid etc. Antibodies which recognise cellular proteins that are useful for mass cytometry are already widely available for IHC usage, and by using labelling atoms instead of current labelling techniques {e.g. fluorescence) these known antibodies can be readily adapted for use in methods of the invention, but with the benefit of increasing multiplexing capability. Antibodies used with the invention can recognise targets on the cell surface or targets within a cell. Antibodies can recognise a variety of targets e.g. they can specifically recognise individual proteins, or can recognise multiple related proteins which share common epitopes, or can recognise specific post-translational modifications on proteins {e.g. to distinguish between tyrosine and phospho-tyrosine on a protein of interest, to distinguish between lysine and acetyl- lysine, to detect ubiquitination, etc.). After binding to its target, labelling atom(s) conjugated to an antibody can be detected to reveal the presence of that target in a sample.

The labelled sbp member will usually interact directly with a target sbp member in the sample. In some embodiments, however, it is possible for the labelled sbp member to interact with a target sbp member indirectly e.g. a primary antibody may bind to the target sbp member, and a labelled secondary antibody can then bind to the primary antibody, in the manner of a sandwich assay. Usually, however, the invention relies on direct interactions, as this can be achieved more easily and permits higher multiplexing. In both cases, however, a sample is contacted with a sbp member which can bind to a target sbp member in the sample, and at a later stage label attached to the target sbp member is detected.

One feature of the invention is its ability to detect multiple {e.g. 10 or more, and even up to 100 or more) different target sbp members in a sample e.g. to detect multiple different proteins and/or multiple different nucleic acid sequences on particles such as cells or beads. To permit differential detection of these target sbp members their respective sbp members should carry different labelling atoms such that their signals can be distinguished by MS. For instance, where ten different proteins are being detected, ten different antibodies (each specific for a different target protein) can be used, each of which carries a unique label, such that signals from the different antibodies can be distinguished. In some embodiments, it is desirable to use multiple different antibodies against a single target e.g. which recognise different epitopes on the same protein. Thus a method may use more antibodies than targets due to redundancy of this type. In general, however, the invention will use a plurality of different labelling atoms to detect a plurality of different targets. If more than one labelled antibody is used with the invention, it is preferable that the antibodies should have similar affinities for their respective antigens, as this helps to ensure that the relationship between the quantity of labelling atoms detected by MS and the abundance of the target antigen will be more consistent across different sbps (particularly at high scanning frequencies).

If a target sbp member is located intracellularly, it will typically be necessary to permeabilise cell membranes before or during contacting of the sample with the labels. For example when the target is a DNA sequence but the labelled sbp member cannot penetrate the membranes of live cells, the cells of the sample can be fixed and permeabilised. The labelled sbp member can then enter the cell and form a sbp with the target sbp member.

Usually, a method of the invention will detect at least one intracellular target and at least one cell surface target. In some embodiments, however, the invention can be used to detect a plurality of cell surface targets while ignoring intracellular targets. Overall, the choice of targets will be determined by the information which is desired from the method.

Accordingly, in some embodiments, the methods of analysis described above comprise the step of labelling a sample with at least one labelling atom. This atom can then be detected using the methods described above.

Single cell analysis

Methods of the invention include laser ablation of multiple cells in a sample, and thus plumes from multiple cells are analysed and their contents are mapped to specific locations in the sample to provide an image. In most cases a user of the method will need to localise the signals to specific cells within the sample, rather than to the sample as a whole. To achieve this, the boundaries of cells {e.g. the plasma membrane, or in some cases the cell wall) in the sample can be demarcated.

Demarcation of cellular boundaries can be achieved in various ways. For instance, a sample can be studied using conventional techniques which can demarcate cellular boundaries, such as microscopy as discussed above. When performing these methods, therefore, an IMC system comprising a camera is particularly useful for general FMC systems beyond the particular discussion above of systems adapted for the desorption of a slug of sample. An image of this sample can then be prepared using a method of the invention, and this image can be superimposed on the earlier results, thereby permitting the MS signals to be localised to specific cells. Indeed, as discussed above, in some cases the laser ablation may be directed only to a subset of cells in the sample as determined to be of interest by the use of microscopy based techniques.

To avoid the need to use multiple techniques, however, it is possible to demarcate cellular boundaries as part of the imaging method of the invention. Such boundary demarcation strategies are familiar from IHC and immunocytochemistry, and these approaches can be adapted by using labels which can be detected by MS. For instance, the method can involve labelling of target molecule(s) which are known to be located at cellular boundaries, and signal from these labels can then be used for boundary demarcation. Suitable target molecules include abundant or universal markers of cell boundaries, such as members of adhesion complexes (e.g. β-catenin or E-cadherin). Some embodiments can label more than one membrane protein in order to enhance demarcation.

In addition to demarcating cell boundaries by including suitable labels, it is also possible to demarcate specific organelles in this way. For instance, antigens such as histones (e.g. H3) can be used to identify the nucleus, and it is also possible to label mitochondrial-specific antigens, cytoskeleton-specific antigens, Golgi-specific antigens, ribosome-specific antigens, etc., thereby permitting cellular ultrastructure to be analysed by methods of the invention.

Signals which demarcate the boundary of a cell (or an organelle) can be assessed by eye, or can be analysed by computer using image processing. Such techniques are known in the art for other imaging techniques e.g. reference 23 describes a segmentation scheme that uses spatial filtering to determine cell boundaries from fluorescence images, reference 24 discloses an algorithm which determines boundaries from brightfield microscopy images, reference 25 discloses the CellSeT method to extract cell geometry from confocal microscope images, and reference 26 discloses the CellSegm MATLAB toolbox for fluorescence microscope images. A method which is useful with the invention uses watershed transformation and Gaussian blurring. These image processing techniques can be used on their own, or they can be used and then checked by eye.

Once cellular boundaries have been demarcated it is possible to allocate signal from specific target molecules to individual cells. It can also be possible to quantify the amount of a target analyte(s) in an individual cell e.g. by calibrating the methods against quantitative standards. Methods of the invention are highly quantitative. In comparison with known methods they do not suffer from sample autofluorescence, there are minimal or no matrix effects as compared to those common in MALDI and SFMS imaging, there is no need for an amplification step such as is often applied in IHC, the tissue can be completely sampled, and the method has a wide dynamic range of ~10⁵. General

The term "comprising" encompasses "including" as well as "consisting" e.g. a composition "comprising" X may consist exclusively of X or may include something additional e.g. X + Y.

The term "about" in relation to a numerical value x is optional and means, for example, x+10%.

The word "substantially" does not exclude "completely" e.g. a composition which is "substantially free" from Y may be completely free from Y. Where necessary, the word "substantially" may be omitted from the definition of the invention.

DESCRIPTION OF THE FIGURES

FIGURE 1. Schematic showing arrangement of optical components to direct laser radiation of different wavelengths to the same location (1 A) and to different locations (IB).

FIGURE 2. Schematic showing arrangement of optical components to direct laser radiation of different wavelengths to opposite surfaces of a sample, using selectively reflective reflectors.

FIGURE 3. Schematic showing arrangement of optical components to direct laser radiation of different wavelengths to opposite surfaces of a sample, using a prism to separate the laser beam in laser radiation of different wavelengths.

FIGURE 4. Schematic showing arrangement showing the cell lifting mode of operation, where the area around a location {e.g. a cell) of interest is first ablated by a first wavelength of laser radiation directed onto the sample (figure 4A), before a second wavelength of laser radiation is directed to the same through the sample carrier to interaction with a layer between the sample and the sample carrier (figure 4B) or through the sample itself (figure 4C), which causes the release of a slug of sample material from the location of interest as a solid block of matter, which is then carried away for ionisation and detection of labelling atoms by MS by the gas flow passing over the sample (figure 4D).

FIGURE 5. Series of images representing the process steps conducted on a sample (figure 5A), comprising identifying a cell of interest on a sample (figure 5B), ablating the area that surrounds the cell of interest (figure 5C) and indicating the area to which laser radiation would be directed through the sample carrier to lift the cell of interest in a slug of material (figure 5D), for ionisation and analysis by MS.

FIGURE 6. Graph showing absorption (log scale) at different wavelengths of 100% DNA, 100% protein and a selection of mixtures. REFERENCES

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Claims

1. An apparatus comprising:

(i) a laser sampling system, wherein the laser system is adapted to emit pulses of laser radiation at two or more wavelengths, and wherein a first wavelength targets a first material, and a second wavelength targets a second material;

2. The apparatus according to claim 1, where the laser system comprises one or more sources of laser radiation, and wherein two or more wavelengths of laser radiation are emitted by a single source.

3. The apparatus according to claim 2, in which the single source comprises a nonlinear frequency conversion component configured to generate radiation at at least one of said wavelengths by way of a nonlinear frequency conversion process.

4. The apparatus according to claim 3, wherein the nonlinear frequency conversion component comprises a harmonic generation component configured to generate at least one said wavelengths by harmonic generation.

5. The apparatus according to claim 1, in which the laser system comprises two or more laser sources, and a first wavelength of the two or more wavelengths of laser radiation emitted by the laser system is emitted by a first laser source, and a second wavelength of the two or more wavelengths of laser radiation is emitted by a second laser source.

6. The apparatus according to any preceding claim, wherein the laser system comprises one or more filters, arranged to control a wavelength of laser radiation emitted by the laser system.

7. The apparatus of any preceding claim in which the first and second wavelengths are discrete.

8. The apparatus of any preceding claim, wherein one of the two or more wavelengths of laser radiation emitted by the laser system is selected to preferentially ablate nucleic acid over protein.

9. The apparatus of claim 8, wherein the selected wavelength is between about 250nm to about 270nm, such as about 255nm to about 266nm, optionally about 260nm or about 266nm.

10. The apparatus of any preceding claim wherein one of the two or more wavelengths of laser radiation emitted by the laser system is selected to preferentially ablate protein over nucleic acid.

11. The apparatus of claim 10, wherein the selected wavelength is between about 185nm to about 235nm, such as about 195nm to about 225nm, optionally about 193nm or about 213nm.

12. The apparatus of claim 11, wherein the laser system is adapted to emit laser radiation at 213nm and 266nm.

13. The apparatus of claim 12, wherein the laser system is adapted to emit laser radiation at 213nm, 266nm and 355nm.

14. The apparatus of any preceding claim, wherein the laser system comprises a translation device to move the sample, and an optical arrangement configured to direct pulses at said first and second wavelengths to different locations relative to the optical arrangement.

15. The apparatus of claim 14, wherein the optical arrangement is configured so that pulses of different wavelengths are received at the same location on the moving sample, optionally further comprising a delay component arranged so that said pulses of different wavelengths are received asynchronously at said same location on the moving sample.

16. The apparatus of any preceding claim, wherein the laser system comprises a reflector arrangement, arranged to direct laser radiation of different wavelengths onto a sample in the mass spectrometer from different directions.

17. The apparatus of claim 16, wherein the sample is on a sample carrier, and wherein the reflector arrangement is arranged to direct laser radiation of a first wavelength directly onto the sample and to direct laser radiation of a second wavelength to the sample through the sample carrier.

18. The apparatus of any preceding claim, comprising a laser pulse power controller, programmed to control the fluence of laser pulses of differing wavelengths.

19. The apparatus of claim 18, in which the fluence is controlled by optical attenuation, for example where each wavelength of laser radiation is controlled by a separate channel of optical attenuation.

20. The apparatus of any preceding claim, comprising an optical delay line, positioned in the optical path of a wavelength of laser radiation.

21. The apparatus of claim 5, wherein the laser system further comprises a laser pulse controller, adapted to generate asynchronous pulses from the two or more lasers or adapted to synchronise pulses from the two or more lasers.

22. The apparatus of any preceding claim, which is an imaging mass cytometer.

23. A method of imaging a sample by imaging mass spectrometry, comprising performing laser ablation of the sample using laser radiation of two or more wavelengths.

24. A method of analysing a sample comprising:

(ii) subjecting the plumes individually to ionisation and mass spectrometry, whereby detection of atoms in the plumes permits construction of an image of the sample.

25. A method of performing mass cytometry on a sample comprising a plurality of cells, the method comprising:

26. The method of any one of claims 23-25, wherein a first wavelength of the two or more wavelengths, preferentially ablates the proteinaceous material in the sample, and a second wavelength of the two or more wavelengths preferentially ablates nucleic acid material.

27. The method of claim 26, wherein the first wavelength may be about 213nm and the second wavelength about 266nm.

28. The method of any one of claims 23-27, wherein at least two wavelengths of the two or more wavelengths of laser radiation ablate a known location on the sample simultaneously.

29. The method of any one of claims 23-27, wherein a first wavelength of the two or more wavelengths of laser radiation is used to ablate a known location before a second wavelength of laser radiation is used to ablate the known location.

30. A method of analysing a sample comprising:

31. The method of claim 30, wherein the ablation of the sample generates one or more plumes of sample material, and wherein the plumes are individually ionised and the atoms in the plume detected by mass spectrometry.

32. The method of claim 30 or 31, in which the sample is a biological sample, and the method further comprises, prior to step (i), the additional step labelling a plurality of different target molecules in the sample with one or more different labelling atoms, to provide a labelled sample.

33. The method of any one of claims 30-32, in which laser ablation is used to ablate the material around a location of interest to clear the surrounding area before the sample material at the location of interest is desorbed from the sample carrier as a slug of material.

34. A method of performing mass cytometry on a sample comprising a plurality of cells, the method comprising steps of:

(ii) illuminating the sample with light to identify one or more locations of interest;

(iii) recording locational information of the one or more locations of interest on the sample;

(iv) using the locational information of the locations of interest to desorb a slug of sample material from a location of interest, comprising first performing laser ablation to remove sample material surrounding the location of interest using laser radiation of a first wavelength, before the slug of sample material is desorbed from the location using laser radiation of a second wavelength;

(v) ionising the desorbed slug of sample material; and

(vi) subjecting the ionised sample material to mass spectrometry, for detection of labelling atoms in the sample material.

35. A method of performing mass cytometry on a sample comprising a plurality of cells according to claim 34, comprising steps of: (i) labelling a plurality of different target molecules in the sample with one or more different labelling atoms and one or more fluorescent labels, to provide a labelled sample;

(ii) illuminating the sample with light to excite the one or more fluorescent labels;

(iii) recording locational information of one or more locations of the sample based on the pattern of fluorescence;

(iv) using the locational information of based on the pattern of fluorescence to desorb a slug of sample material from a location of interest, comprising first performing laser ablation to remove sample material surrounding the location of interest using laser radiation of a first wavelength, before the slug of sample material is desorbed from the location using laser radiation of a second wavelength;

(v) ionising the desorbed slug of sample material; and

36. The method of any one of claims 30-35, wherein the sample is on a sample carrier.

37. The method of claim 36, wherein the second wavelength of laser radiation is directed through the sample carrier to desorb the slug of sample material from the sample carrier.

38. A method of analysing a sample, comprising using the apparatus of any one of claims 1-22, optionally in which the method is a method according to one of claims 23-37.