Title of Invention	APPARATUS AND METHODS FOR IMAGING AND MODIFICATION OF BIOLOGICAL SAMPLES
Abstract	This invention relates to enrichment of a biological sample comprising a plurality of cells to assist further analysis thereof. It provides a technique comprising the steps of: (a) providing a sample comprising a plurality of cells which include a photosensitive compound that can be induced by light irradiation to inactivate or kill at least part of the respective cell (b) acquiring an image of at least a portion of the sample; (c) identifying cells of interest in the sample image; (d) selecting cells other than the cells identified in step (d) and (e) irradiating only those cells selected in step (d) with a light beam so as induce the photosensitive compound therein to inactivate or kill at least part of these cells, and thereby enrich the sample with respect to the cells of interest for further analysis.

Title of Invention

APPARATUS AND METHODS FOR IMAGING AND MODIFICATION OF BIOLOGICAL SAMPLES

Abstract

This invention relates to enrichment of a biological sample comprising a plurality of cells to assist further analysis thereof. It provides a technique comprising the steps of: (a) providing a sample comprising a plurality of cells which include a photosensitive compound that can be induced by light irradiation to inactivate or kill at least part of the respective cell (b) acquiring an image of at least a portion of the sample; (c) identifying cells of interest in the sample image; (d) selecting cells other than the cells identified in step (d) and (e) irradiating only those cells selected in step (d) with a light beam so as induce the photosensitive compound therein to inactivate or kill at least part of these cells, and thereby enrich the sample with respect to the cells of interest for further analysis.

Full Text	Title: Apparatus and methods for immaging and modification bilogical samples Field of the invention The present invention relates to modification of biological samples which comprise a plurality of cells. More particularly, it concerns enrichment of a sample with respect to cells of interest to assist further analysis thereof. Background to the invention During the study of biological samples, it is common to have samples that contain cells of particular interest within a mixture of cells. It may be useful to enrich the sample with respect to the cells of interest in the population for further processing and analysis. This capability is of interest within the research area to provide an understanding of mechanisms of a cellular process, where the technique has applications for example in the study of cell differentiation, cell lineage and fate, of system development, in the area of regulation, in the response to any external stimulus, to provide new knowledge and understanding. This capability is also of interest for the diagnosis of a disease or disorder (detecting presence of a condition, evaluating the extent of a condition) by comparing the results with normals; and for evaluating the effectiveness of a treatment by looking at changes over time. Typically, a particular cell type exists within a heterogeneous population of multiple cell types. Examples include differentiated cells within a tissue sample and stem cells [So 2004]; cancer cells and blood vessels within a tissue sample; neurons growing in a substrate of supporting cells; fetal cells in maternal blood; white blood ceils of various types (natural killer cells. etc) in blood samples; metastatic cancer cells in tissues samples, etc. In addition a particular cell type may itself consist of a heterogeneous population with respect to some particular characteristic. For example, the cells may consist of subgroups of cells at different positions in the cell cycle, including being quiescent (non-dividing). In addition, the cells may be responding to the environment in different ways or be activated in different ways, and thus expressing different proteins or molecules of interest or entire processes or pathways. One current approach to sort cells is to use a flow cytometer or a Fluorescence Activated Cell Sorter (FACS) machine. The practice is to label the cells with one or more fluorescent labels, to disperse the cells in a suitable carrier medium, and to pass that carrier medium containing the cells through the FACS machine, in which the carrier is formed into droplets by passing through a nozzle. The resulting droplet is exposed to a beam of excitation light, causing the fluorescent label in an individual cell to fluoresce and emit light which is detected in the form of a signal, which is used to form the basis of a classification decision to accept or reject the cell, and divert it into a receptacle for further processing. Multiple labels can be used to determine various states of the cell and thus make more complex classification decisions. Such an approach is able to sort cells at modest rates, with good discriminating ability when the fluorescent label has the necessary sensitivity and selectivity. However this approach suffers from a number of drawbacks. Sample preparation and rough handling may perturb the sample and limits it to a single passage. This will be a problem if the cells exist in a supporting matrix and/or the interaction between cells is to be maintained so that only non-adherent cells are used. In addition, since the FACS process involves some delay, this means that some fine-grained temporal and kinetic information is lost making some studies very difficult eg synchronization of cell cycle. In addition, the data collected from each cell is of a fairly coarse nature, making it difficult to distinguish between distinct spatial organizations of the label, due e.g. to translocation processes. In an alternative approach, the sample may be analysed manually under a microscope with a micro-dissecting stage. Markers (stains) may be added to allow different cell types, components and structures to be visualized, distinguished and manipulated. This is slow, laborious and error-prone, and it is difficult to prevent cross-contamination between sample components or the use of live objects. It is not feasible if the cell type is rare. In a further technique, a laser may be used for micro-dissection and to cut around the cells of interest [Wiltke 2005; Schuetze 1997], This helps to prevent contamination problems. Alternatively, optical tweezers may be used to displace small scale, biological objects, but cannot effectively perform certain types of operation such as the cutting or removal of undesired attached material. Laser ablation can also be used to remove unwanted material. In an another approach, the sample may be lysed and probed for particular molecules or combinations of molecules such as DNA, UNA or proteins using a microarray. This approach can be very sensitive and selective. However it can be quite slow, preventing fine temporal information from being accessed, and it is generally destructive of the cell under study, thus preventing the collection and further use of cells of a particular type. Summary and detailed description of the invention. The present invention provides a method of modifying a biological sample, comprising the steps of: (a) providing a sample comprising a plurality of cells which include a photosensitive compound that can be induced by light irradiation to inactivate or kill at least part of the respective cell; (b) acquiring an image of at least a portion of the sample; (c) identifying cells of interest in the sample image; (d) selecting cells other than the cells identified in step (c); and (e) irradiating only those cells selected in step (d) with a light beam so as induce the photosensitive compound therein to inactivate or kill at least part of those cells, and thereby enrich the sample with respect to the cells of interest for further analysis. The technique proposed herein involves locating and identifying the cells of interest from a population of live cells, preferably using one or more suitable markers, with suitable image acquisition instrumentation capable of targeting an irradiating light beam onto a specific cell or part, of an unwanted cell. With the introduction of one or more suitable photosensitive comoouncs, the irradiating light beam acts to modify the photosensitive compound. Based on the appearance of a cell, a decision may be made to direct and activate the irradiating light beam, and thus result in the release of a species in such a way as to kill it. Benefits of the present approach over the prior art are that it allows examination of greater numbers of samples than would be possible using a manually graded approach, and facilitates enrichment based, on: rarer events (such as cancer metastasis); faster events or events with fine grained temporal information (such as phase synchrony); multiple events, coincident events or events separated in time or space; difficult to see events or fine spatial detail; events occurring over a longer time scale, and enrichment of greater quality (more true positives, less false positive, less contamination), and with less interference with the sample under study. Preferred techniques have been developed by the present inventors for acquiring and analyzing images with marker probes and photo-sensitive compounds, which allows individual cells to be selectively irradiated and thus deactivated or killed. Enrichment of a sample by eliminating only a proportion of the cells of interest may facilitate some long term studies (tracking changes over time), and/or modulation or manipulation of the interaction between cells types. The use of a photosensitive compound enables enrichment in a well controlled manner, using a lower power light source (such as a low power laser) than is needed to destroy a cell using light alone. The following steps are envisaged in one implementation: 1) Present sample consisting of mixed population of cells; 2) Probe with marker molecule; 3) Introduce photosensitive compound (optional); 4) Stimulate sample (optional); 5) View sample with viewing optics (various imaging modes); 6) Refine viewing parameters including focus (optional); 7) Acquire digital image set; 8) Process the image(s) to obtain an image measure ; 9) Classify cell of interest based on coincidence of one or more image measures; 10) Direct an irradiating light beam to a particular location in the sample at a particular moment in time in order to carry out an action on the sample; 11) Repeat from step 3 (introduce) step 4 (stimulate) or step 5 (view); or 12) Exit and further processing of the sample. The sample is typically a particular cell type within a heterogeneous population of multiple cell types. Examples include differentiating tissues with stem cells, progenitor, precursor and differentiated cells within a tissue sample. Furthermore a particular cell type may itself consist of a heterogeneous population with respect to some particular characteristic. For example, the cells .may consist of subgroups of cells at different positions in the cell cycle, including being quiescent (non-dividing). Alternatively the population of cells may be a heterogeneous population of cells of different cell types, or with some genetic variation, mutation or phenotype, or with different degrees of transfection of an agent. The cells may be responding to the environment in different ways or be activated in different ways, and thus expressing different proteins or molecules of interest or entire processes. The sample may be part of a cell, an organdie and/or cell region in a cell. Alternatively the population of cells may be an assembly of cells in 2D or 3D; a tissue sample; a biological fluid such as blood; a biopsy sample; an organ; an embryo; a part of an animal or a plant: or an entire animal or a plant. The marker molecule and photosensitive compounds may comprise one or more of the following: a fluorescence molecule; a luminescent molecule; an uncaging agent with an active part board to a chrornophore or fluorophore by a photo-oleavable link [Gnrney 1999]; a lisand conjugated with a dye; a CALI reagent (a non-blocking autibody conjugated with a dye [llag 2000]); a photosensitizer such, as KillerRed (marketed by Evrogen); a geneticallv encoded moleculs such as GFP; a photo-activatable molecule such as genetically encoded PA-GFP or caged molecule [Sawin 1999]; a photo-switching molecule such as KFP; a timer protein [Terskikh 2000][Verkhusha 2004]; a cell cycle marker; a voltage or potential sensitive marker; a biosensor; a multi-functional marker probe (multi-functional marker probes radicate the presence of multiple species by changes in the excitation emission profile with the presence of metal ions [Komatsu 2005] or kinases/phospholipases [Schultz 2005]); an exogenous molecule that binds to a genetically encoded tag; a conjugated quantum dot; a conjugated metallic nanop article; a conjugated encapsulated fluorescent nanoparticle; or it may rely on the fluorescence of an intrinsic molecule. Exogenous (non-genetically encoded) markers may be cell permeable. Genetically and non-genetically encoded markers may be introduced or may have been previously introduced into cells by a number of known techniques. Stimulation of the sample may include any external stimulus (biochemical, chemical, physical, thermal, optical, electrical, magnetic, electromagnetic, etc). The imaging modes may include one or more of the following: fluorescence at one or more excitation wavebands; luminescence; widefield; confocal; phase contrast; DIC; structured illumination; fluorescence lifetime; polarization; or multi-photon processes. The viewing parameters which may be modified may include the focus,plane of focus, field of view, excitation power level, wavelength and waveband, polarization, pulse regime, as well as detector parameters such as wavelength sensitivity, exposure timing and duration, location within a multi-sample holder, and the like. The digital image set may comprise one or more of the following: bright-field images; fluorescence images; confocal images at particular focal planes; fluorescence lifetime images in a range; images at particular wavebands from the infrared to the ultraviolet; a time sequence; an image stack, or any combination of the above. Suitable image measures may include cell location, boundary, extent; cell morphology; cell division or position in the cell cycle; cell viability; image features such as intensity, relative intensity, change in intensity, texture measures; lifetime, relative lifetime, change in lifetime; spectral shift; movement, migration or motility; outgrowth; a cellular or sub-cellular event such as translocaiion from cytoplasm to nucleus, membrane to nucleus, organeile to nucleus, membrane to cytoplasm, organeile to cytoplasm, nucleus to cytoplasm, cytoplasm to membrane, nucleus to membrane, organelle to membrane, cytoplasm to organeile, membrane to organeile, nucleus to organeile, endocytosis, exocytosis, invasion by a particle or virus; vesicle movement; membrane ruffling; ceil blobbing; growth cone extension; particular pH value or range of pH values, particular ion concentration or range of ion concentration; DNA content; DNA fragmentation; membrane potential; number and size of DNA loci; binding event; signaling; adhesion; or other measurement such as current, voltage, impedance, or transconductance. A classification decision may be made based on one or more measures falling above or below or within limits, comparison with a model, or statistical distance from, a set of examples; or some linear or non-linear mapping of the measures; or a classifier based on nearest neighbour; artificial neural network; support vector machine; pattern recognition; or genetic algorithm. The action carried out may inactivate or kill at least part of a cell. This may be achieved by flash photolysis, un-caging or releasing (a luminescence substrate such as luciferin [Yang 1993] or coelenterazme; free radicals) a toxic substance, perforation of a cell wall, transfection, ablation or other modification of specific structures such as micro tubules; or activation of a pathway such as apoptosis. Further processing or analysis can include examination, harvesting, sorting, culture or treatment of the sample by dispensing of reagents. The invention further provides apparatus for carrying out the techniques described herein, which comprises: a detector for acquiring an image of at least a portion of the sample; a light source for generating a light bearn to irradiate a selected region of the sampale and a controller arranged to manipulate the light source to only irradiate at least part of each of a plurality of selected cells. Instrumentation which may be configured to provide steps 5 (view) to 10 (action) is described in an earlier patent application [Courtney 2005]. The classification step may be based on data obtained from previous acquisitions; acquired from ocher imaging modalities (X-ray. CT, MRI, PET); from databases, or other external sources. The location, region and time point for the irradiation to take place may be based on information about the sample and the desired experimental protocol. The apparatus may be configured to study a single sample or a larger sample requiring motion of the viewing location between locations. Additionally multiple samples may be presented on a larger carrier, whereby the samples differ in some way (different cells, different reagents, different environment). Further applications of the techniques disclosed herein may include: screening for activity of drug'candidates by observing changes; - screening for safety of drug candidates or therapeutic agents; detecting the presence of a disease or disorder (diagnosis); evaluating the extent of a disease or disorder (stratification); - evaluating the effectiveness of a treatment; and discovery of biomatker to indicate any of the above. References P. Courtney et al, 2005, A method of analysing a sample and apparatus therefor, UK patent publication no. 2418018, in the name of PerkinElmer Singapore PTE Ltd.. W. Wittke and C. May, 2005, EP1537401: Carrier device for a biological preparation which can be cut by means of laser micro-dissection, Leica Microsystems. K. Schuetze and R. Schuetze, 1997, EP0879408 Bl: Method and Device for the contactless laser-assisted microinjection, sorting and production of biological objects generated in a planer manner, PALM. P-L So and E. H. Epstein, Adult stem cells: capturing youth from a bulge? Trends in Biotechnology, Volume 22, Issue 10, October 2004, pp. 493-496. J. Yang and D.B. Thomason, 1993, An easily synthesized, photolyzable luciferase substrate for in vivo luciferase activity measurement, Biotechniques. Nov, 15(5), pp.848-50. L.L. Hag, J.H. Ng and D.G. Jay, 2000 Cbromoph.ore-Assi.sted Laser Inactivation (CALI) to Validate Drug Targets and Pharmacogenomic Markers. Drug Development Research 49, pp. 65-73. A. Terskikh, A. Fradkov, G. Ermakova, A. Zaraisky, P. Tan, A.V. Kajava, X. Zhao, S. Lukyanov, M. Matz, S. Kim, I. Weissman and P. Siebert, 2000, Fluorescent Timer: Protein That Changes Color with Time, Science, 290 (5496), 24 November, pp.1585-1588. V.V. Verkhusha, D.M. Chudalcov, N.G. Gurskaya, S. Lukyanov and K.A. Lukyanov, 2004, Common pathway for the red chromophore formation in fluorescent proteins and chromoproteins, Chem Biol. 2004 Jun;11(6) pp. 845-54, C. Schultz, A. Schleifenbaum, J. Goedhart and T.W. Gadella, 2005, Multiparameter imaging for the analysis of intraceilular signalling, Chembiochem, Aug, 6(8), pp. 1323-30. H. Komatsu, T. Milki, D. Citterio, T. Kubota, Y. Shindo, Y. Kitamura, K. Oka and K. Suzuki, 2005, Single Molecular Multi-analyte (Ca2+, Mg2-) Fluorescent Probe and Applications to Bioimaging, Am. Chem. Soc, 127 (31), pp. 10798 -10799. K.E." Sawin, J.A. Theriot and T.J. Mitchison, Photoactivation of Fluorescence as a Probe for Cytoskeletal Dynamics m Mitosis and Cell Motility. 1999, In Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide to Technology for Quantitative Real-Time Analysis, Edited By W. Mason, Academic Press. A.M. Gurney, Photolabile Caged Compounds. 1999, In Fluorescent and Luminescent Probes for Biological Activity: A Practical Guide to Technology for Quantitative Real-Time Analysis, Edited By W. Mason, Academic Press. Claims 1. A method of modifying a biological sample, comprising the steps of: (a) providing a sample comprising a plurality of cells which include a photosensitive compound that can be induced by light irradiation to inactivate or kill at least part of the respective cell; (b) acquiring an image of at least a portion of the sample; (c) identifying cells of interest in' the sample image; (d) selecting cells other than the cells identified in step (c); and (e) irradiating only those cells selected in step (d) with a light beam so as induce the photosensitive compound therein to inactivate or kill at least part of those cells, and thereby enrich the sample with respect to the cells of interest for further analysis. 2. A method of claim 1 wherein the sample comprises cells of different cell types, and in step (c) cells of a particular type are identified, 3. A method of claim 1 wherein the plurality of cells consists of cells of the same type, and in. step (c) a subgroup of the plurality of cells which have a common characteristic is identified. 4. A method of any preceding claim wherein the image acquired in step (b) is displayed to a user, and the cells of interest are identified in step (c) by user input signals. 5. A method of any of claims 1 to 3 wherein the cells of interest are identified in step (c) by analysis-of the image acquired in step (b) by image processing means. 5. A method of any preceding claim wherein cells are identified as of interest in step (c) bv: comparing a measured parameter of a cell with a predetermined threshold; by linear or non-linear mapping of a measured parameter; by means of a classifier based on nearest neighbour; using an artificial neural network; using a support vector machine; using pattern recognition; or using a genetic algorithm. 7. A method of any preceding claim wherein cells are identified as of interest in step (c) by reference to: data obtained from other samples; or images or data relating to the sample and acquired using a different imaging modality to that employed in step (b). 8. A method of any preceding claim wherein the photosensitive compound is one of the following: a CALI reagent; an uncaging agent; a photosensitizer. 9. A method of any preceding claim including a step of introducing the photosensitive compound into each of the plurality of cells. 10. A method of any of claims 1 to 8 wherein the photosensitive compound is genetically encoded in each of the plurality of cells. 11. A method of any preceding claim wherein at least part; of the selected cells is killed in step (e) by activation of apoptosis, or by the release of a toxic substance. 12. A method of any preceding claim wherein the sample comprises cells which include a marker compound that is able to respond to light irradiation to assist analysis of the sample. 13. A method of any preceding claim wherein the sample comprises one of: an assembly of cells in 2D or 3D; a tissue sample; a biological fluid such as blood; a biopsy sample; an organ; an embryo; a part of an animal or a plant; or an entire animal or a plant. 14. A method of any preceding claim including a step of stimulating the sample prior to step (b). 15. A method of any preceding claim wherein an image is acquired in step (b) using one of the following imaging techniques: fluorescence at one or more excitation wavebands: luminescence; widefield; confocal; phase contrast; DIG; structured illumination; fluorescence lifetime; polarization; or a multi-photon process. 16. A method, of any preceding claim wherein a sequence of images is acquired in step (b), and identification step (c) is carried out with reference to the sequence, 17. A method of claim 16 wherein the sequence of acquired images comprises: bright- field images; fluorescence images; confocal images at particular focal planes; fluorescence lifetime images in a range; images at particular wavebands from the infrared to the ultraviolet; a time sequence; an image stack; or any combination thereof, 18. A method of any preceding claim wherein said further analysis comprises one of: examination; harvesting; sorting; culture; or treatment of the sample by dispensing of reagents. 19. Apparatus for modifying a biological sample in accordance with a method of any preceding claim, comprising: a detector for acquiring an image of at least a portion of the sample; a light source for generating a. light beam to irradiate a selected region of the sample; and a controller arranged to manipulate the light source to only irradiate at least part of each of a plurality of selected cells. This invention relates to enrichment of a biological sample comprising a plurality of cells to assist further analysis thereof. It provides a technique comprising the steps of: (a) providing a sample comprising a plurality of cells which include a photosensitive compound that can be induced by light irradiation to inactivate or kill at least part of the respective cell (b) acquiring an image of at least a portion of the sample; (c) identifying cells of interest in the sample image; (d) selecting cells other than the cells identified in step (d) and (e) irradiating only those cells selected in step (d) with a light beam so as induce the photosensitive compound therein to inactivate or kill at least part of these cells, and thereby enrich the sample with respect to the cells of interest for further analysis.

Full Text

Title: Apparatus and methods for immaging and modification bilogical samples
Field of the invention
The present invention relates to modification of biological samples which comprise a
plurality of cells. More particularly, it concerns enrichment of a sample with respect to cells
of interest to assist further analysis thereof.
Background to the invention
During the study of biological samples, it is common to have samples that contain cells of
particular interest within a mixture of cells. It may be useful to enrich the sample with
respect to the cells of interest in the population for further processing and analysis.
This capability is of interest within the research area to provide an understanding of
mechanisms of a cellular process, where the technique has applications for example in the
study of cell differentiation, cell lineage and fate, of system development, in the area of
regulation, in the response to any external stimulus, to provide new knowledge and
understanding. This capability is also of interest for the diagnosis of a disease or disorder
(detecting presence of a condition, evaluating the extent of a condition) by comparing the
results with normals; and for evaluating the effectiveness of a treatment by looking at
changes over time.
Typically, a particular cell type exists within a heterogeneous population of multiple cell
types. Examples include differentiated cells within a tissue sample and stem cells [So 2004];
cancer cells and blood vessels within a tissue sample; neurons growing in a substrate of
supporting cells; fetal cells in maternal blood; white blood ceils of various types (natural
killer cells. etc) in blood samples; metastatic cancer cells in tissues samples, etc. In addition
a particular cell type may itself consist of a heterogeneous population with respect to some
particular characteristic. For example, the cells may consist of subgroups of cells at different

positions in the cell cycle, including being quiescent (non-dividing). In addition, the cells
may be responding to the environment in different ways or be activated in different ways,
and thus expressing different proteins or molecules of interest or entire processes or
pathways.
One current approach to sort cells is to use a flow cytometer or a Fluorescence Activated
Cell Sorter (FACS) machine. The practice is to label the cells with one or more fluorescent
labels, to disperse the cells in a suitable carrier medium, and to pass that carrier medium
containing the cells through the FACS machine, in which the carrier is formed into droplets
by passing through a nozzle. The resulting droplet is exposed to a beam of excitation light,
causing the fluorescent label in an individual cell to fluoresce and emit light which is
detected in the form of a signal, which is used to form the basis of a classification decision to
accept or reject the cell, and divert it into a receptacle for further processing. Multiple labels
can be used to determine various states of the cell and thus make more complex
classification decisions. Such an approach is able to sort cells at modest rates, with good
discriminating ability when the fluorescent label has the necessary sensitivity and selectivity.
However this approach suffers from a number of drawbacks. Sample preparation and rough
handling may perturb the sample and limits it to a single passage. This will be a problem if
the cells exist in a supporting matrix and/or the interaction between cells is to be maintained
so that only non-adherent cells are used. In addition, since the FACS process involves some
delay, this means that some fine-grained temporal and kinetic information is lost making
some studies very difficult eg synchronization of cell cycle. In addition, the data collected
from each cell is of a fairly coarse nature, making it difficult to distinguish between distinct
spatial organizations of the label, due e.g. to translocation processes.
In an alternative approach, the sample may be analysed manually under a microscope with a
micro-dissecting stage. Markers (stains) may be added to allow different cell types,
components and structures to be visualized, distinguished and manipulated. This is slow,
laborious and error-prone, and it is difficult to prevent cross-contamination between sample
components or the use of live objects. It is not feasible if the cell type is rare.

In a further technique, a laser may be used for micro-dissection and to cut around the cells of
interest [Wiltke 2005; Schuetze 1997], This helps to prevent contamination problems.
Alternatively, optical tweezers may be used to displace small scale, biological objects, but
cannot effectively perform certain types of operation such as the cutting or removal of
undesired attached material. Laser ablation can also be used to remove unwanted material.
In an another approach, the sample may be lysed and probed for particular molecules or
combinations of molecules such as DNA, UNA or proteins using a microarray. This
approach can be very sensitive and selective. However it can be quite slow, preventing fine
temporal information from being accessed, and it is generally destructive of the cell under
study, thus preventing the collection and further use of cells of a particular type.
Summary and detailed description of the invention.
The present invention provides a method of modifying a biological sample, comprising the
steps of:
(a) providing a sample comprising a plurality of cells which include a photosensitive
compound that can be induced by light irradiation to inactivate or kill at least part of the
respective cell;
(b) acquiring an image of at least a portion of the sample;
(c) identifying cells of interest in the sample image;
(d) selecting cells other than the cells identified in step (c); and
(e) irradiating only those cells selected in step (d) with a light beam so as induce the
photosensitive compound therein to inactivate or kill at least part of those cells, and thereby
enrich the sample with respect to the cells of interest for further analysis.
The technique proposed herein involves locating and identifying the cells of interest from a
population of live cells, preferably using one or more suitable markers, with suitable image
acquisition instrumentation capable of targeting an irradiating light beam onto a specific cell
or part, of an unwanted cell. With the introduction of one or more suitable photosensitive
comoouncs, the irradiating light beam acts to modify the photosensitive compound. Based

on the appearance of a cell, a decision may be made to direct and activate the irradiating
light beam, and thus result in the release of a species in such a way as to kill it.
Benefits of the present approach over the prior art are that it allows examination of greater
numbers of samples than would be possible using a manually graded approach, and
facilitates enrichment based, on: rarer events (such as cancer metastasis); faster events or
events with fine grained temporal information (such as phase synchrony); multiple events,
coincident events or events separated in time or space; difficult to see events or fine spatial
detail; events occurring over a longer time scale, and enrichment of greater quality (more
true positives, less false positive, less contamination), and with less interference with the
sample under study.
Preferred techniques have been developed by the present inventors for acquiring and
analyzing images with marker probes and photo-sensitive compounds, which allows
individual cells to be selectively irradiated and thus deactivated or killed.
Enrichment of a sample by eliminating only a proportion of the cells of interest may
facilitate some long term studies (tracking changes over time), and/or modulation or
manipulation of the interaction between cells types.
The use of a photosensitive compound enables enrichment in a well controlled manner,
using a lower power light source (such as a low power laser) than is needed to destroy a cell
using light alone.
The following steps are envisaged in one implementation:
1) Present sample consisting of mixed population of cells;
2) Probe with marker molecule;
3) Introduce photosensitive compound (optional);
4) Stimulate sample (optional);
5) View sample with viewing optics (various imaging modes);
6) Refine viewing parameters including focus (optional);

7) Acquire digital image set;
8) Process the image(s) to obtain an image measure ;
9) Classify cell of interest based on coincidence of one or more image measures;
10) Direct an irradiating light beam to a particular location in the sample at a particular
moment in time in order to carry out an action on the sample;
11) Repeat from step 3 (introduce) step 4 (stimulate) or step 5 (view); or
12) Exit and further processing of the sample.
The sample is typically a particular cell type within a heterogeneous population of multiple
cell types. Examples include differentiating tissues with stem cells, progenitor, precursor and
differentiated cells within a tissue sample.
Furthermore a particular cell type may itself consist of a heterogeneous population with
respect to some particular characteristic. For example, the cells .may consist of subgroups of
cells at different positions in the cell cycle, including being quiescent (non-dividing).
Alternatively the population of cells may be a heterogeneous population of cells of different
cell types, or with some genetic variation, mutation or phenotype, or with different degrees
of transfection of an agent.
The cells may be responding to the environment in different ways or be activated in different
ways, and thus expressing different proteins or molecules of interest or entire processes.
The sample may be part of a cell, an organdie and/or cell region in a cell. Alternatively the
population of cells may be an assembly of cells in 2D or 3D; a tissue sample; a biological
fluid such as blood; a biopsy sample; an organ; an embryo; a part of an animal or a plant: or
an entire animal or a plant.
The marker molecule and photosensitive compounds may comprise one or more of the
following: a fluorescence molecule; a luminescent molecule; an uncaging agent with an
active part board to a chrornophore or fluorophore by a photo-oleavable link [Gnrney 1999];
a lisand conjugated with a dye; a CALI reagent (a non-blocking autibody conjugated with a

dye [llag 2000]); a photosensitizer such, as KillerRed (marketed by Evrogen); a geneticallv
encoded moleculs such as GFP; a photo-activatable molecule such as genetically encoded
PA-GFP or caged molecule [Sawin 1999]; a photo-switching molecule such as KFP; a timer
protein [Terskikh 2000][Verkhusha 2004]; a cell cycle marker; a voltage or potential
sensitive marker; a biosensor; a multi-functional marker probe (multi-functional marker
probes radicate the presence of multiple species by changes in the excitation emission profile
with the presence of metal ions [Komatsu 2005] or kinases/phospholipases [Schultz 2005]);
an exogenous molecule that binds to a genetically encoded tag; a conjugated quantum dot; a
conjugated metallic nanop article; a conjugated encapsulated fluorescent nanoparticle; or it
may rely on the fluorescence of an intrinsic molecule. Exogenous (non-genetically encoded)
markers may be cell permeable. Genetically and non-genetically encoded markers may be
introduced or may have been previously introduced into cells by a number of known
techniques.
Stimulation of the sample may include any external stimulus (biochemical, chemical,
physical, thermal, optical, electrical, magnetic, electromagnetic, etc).
The imaging modes may include one or more of the following: fluorescence at one or more
excitation wavebands; luminescence; widefield; confocal; phase contrast; DIC; structured
illumination; fluorescence lifetime; polarization; or multi-photon processes.
The viewing parameters which may be modified may include the focus,plane of focus, field
of view, excitation power level, wavelength and waveband, polarization, pulse regime, as
well as detector parameters such as wavelength sensitivity, exposure timing and duration,
location within a multi-sample holder, and the like.
The digital image set may comprise one or more of the following: bright-field images;
fluorescence images; confocal images at particular focal planes; fluorescence lifetime images
in a range; images at particular wavebands from the infrared to the ultraviolet; a time
sequence; an image stack, or any combination of the above.

Suitable image measures may include cell location, boundary, extent; cell morphology; cell
division or position in the cell cycle; cell viability; image features such as intensity, relative
intensity, change in intensity, texture measures; lifetime, relative lifetime, change in lifetime;
spectral shift; movement, migration or motility; outgrowth; a cellular or sub-cellular event
such as translocaiion from cytoplasm to nucleus, membrane to nucleus, organeile to
nucleus, membrane to cytoplasm, organeile to cytoplasm, nucleus to cytoplasm, cytoplasm
to membrane, nucleus to membrane, organelle to membrane, cytoplasm to organeile,
membrane to organeile, nucleus to organeile, endocytosis, exocytosis, invasion by a particle
or virus; vesicle movement; membrane ruffling; ceil blobbing; growth cone extension;
particular pH value or range of pH values, particular ion concentration or range of ion
concentration; DNA content; DNA fragmentation; membrane potential; number and size of
DNA loci; binding event; signaling; adhesion; or other measurement such as current,
voltage, impedance, or transconductance.
A classification decision may be made based on one or more measures falling above or
below or within limits, comparison with a model, or statistical distance from, a set of
examples; or some linear or non-linear mapping of the measures; or a classifier based on
nearest neighbour; artificial neural network; support vector machine; pattern recognition; or
genetic algorithm.
The action carried out may inactivate or kill at least part of a cell. This may be achieved by
flash photolysis, un-caging or releasing (a luminescence substrate such as luciferin [Yang
1993] or coelenterazme; free radicals) a toxic substance, perforation of a cell wall,
transfection, ablation or other modification of specific structures such as micro tubules; or
activation of a pathway such as apoptosis.
Further processing or analysis can include examination, harvesting, sorting, culture or
treatment of the sample by dispensing of reagents.
The invention further provides apparatus for carrying out the techniques described herein,
which comprises: a detector for acquiring an image of at least a portion of the sample; a light
source for generating a light bearn to irradiate a selected region of the sampale and a

controller arranged to manipulate the light source to only irradiate at least part of each of a
plurality of selected cells.
Instrumentation which may be configured to provide steps 5 (view) to 10 (action) is
described in an earlier patent application [Courtney 2005].
The classification step may be based on data obtained from previous acquisitions; acquired
from ocher imaging modalities (X-ray. CT, MRI, PET); from databases, or other external
sources.
The location, region and time point for the irradiation to take place may be based on
information about the sample and the desired experimental protocol.
The apparatus may be configured to study a single sample or a larger sample requiring
motion of the viewing location between locations. Additionally multiple samples may be
presented on a larger carrier, whereby the samples differ in some way (different cells,
different reagents, different environment).
Further applications of the techniques disclosed herein may include:
screening for activity of drug'candidates by observing changes;
- screening for safety of drug candidates or therapeutic agents;
detecting the presence of a disease or disorder (diagnosis);
evaluating the extent of a disease or disorder (stratification);
- evaluating the effectiveness of a treatment; and
discovery of biomatker to indicate any of the above.
References
P. Courtney et al, 2005, A method of analysing a sample and apparatus therefor, UK patent
publication no. 2418018, in the name of PerkinElmer Singapore PTE Ltd..
W. Wittke and C. May, 2005, EP1537401: Carrier device for a biological preparation which
can be cut by means of laser micro-dissection, Leica Microsystems.

K. Schuetze and R. Schuetze, 1997, EP0879408 Bl: Method and Device for the contactless
laser-assisted microinjection, sorting and production of biological objects generated in a
planer manner, PALM.
P-L So and E. H. Epstein, Adult stem cells: capturing youth from a bulge? Trends in
Biotechnology, Volume 22, Issue 10, October 2004, pp. 493-496.
J. Yang and D.B. Thomason, 1993, An easily synthesized, photolyzable luciferase substrate
for in vivo luciferase activity measurement, Biotechniques. Nov, 15(5), pp.848-50.
L.L. Hag, J.H. Ng and D.G. Jay, 2000 Cbromoph.ore-Assi.sted Laser Inactivation (CALI) to
Validate Drug Targets and Pharmacogenomic Markers. Drug Development Research 49, pp.
65-73.
A. Terskikh, A. Fradkov, G. Ermakova, A. Zaraisky, P. Tan, A.V. Kajava, X. Zhao, S.
Lukyanov, M. Matz, S. Kim, I. Weissman and P. Siebert, 2000, Fluorescent Timer: Protein
That Changes Color with Time, Science, 290 (5496), 24 November, pp.1585-1588.
V.V. Verkhusha, D.M. Chudalcov, N.G. Gurskaya, S. Lukyanov and K.A. Lukyanov, 2004,
Common pathway for the red chromophore formation in fluorescent proteins and
chromoproteins, Chem Biol. 2004 Jun;11(6) pp. 845-54,
C. Schultz, A. Schleifenbaum, J. Goedhart and T.W. Gadella, 2005, Multiparameter imaging
for the analysis of intraceilular signalling, Chembiochem, Aug, 6(8), pp. 1323-30.
H. Komatsu, T. Milki, D. Citterio, T. Kubota, Y. Shindo, Y. Kitamura, K. Oka and K.
Suzuki, 2005, Single Molecular Multi-analyte (Ca2+, Mg2-) Fluorescent Probe and
Applications to Bioimaging, Am. Chem. Soc, 127 (31), pp. 10798 -10799.
K.E." Sawin, J.A. Theriot and T.J. Mitchison, Photoactivation of Fluorescence as a Probe for
Cytoskeletal Dynamics m Mitosis and Cell Motility. 1999, In Fluorescent and Luminescent

Probes for Biological Activity: A Practical Guide to Technology for Quantitative Real-Time
Analysis, Edited By W. Mason, Academic Press.
A.M. Gurney, Photolabile Caged Compounds. 1999, In Fluorescent and Luminescent Probes
for Biological Activity: A Practical Guide to Technology for Quantitative Real-Time
Analysis, Edited By W. Mason, Academic Press.

Claims
1. A method of modifying a biological sample, comprising the steps of:
(a) providing a sample comprising a plurality of cells which include a photosensitive
compound that can be induced by light irradiation to inactivate or kill at least part of the
respective cell;
(b) acquiring an image of at least a portion of the sample;
(c) identifying cells of interest in' the sample image;
(d) selecting cells other than the cells identified in step (c); and
(e) irradiating only those cells selected in step (d) with a light beam so as induce the
photosensitive compound therein to inactivate or kill at least part of those cells, and thereby
enrich the sample with respect to the cells of interest for further analysis.

2. A method of claim 1 wherein the sample comprises cells of different cell types, and
in step (c) cells of a particular type are identified,
3. A method of claim 1 wherein the plurality of cells consists of cells of the same type,
and in. step (c) a subgroup of the plurality of cells which have a common characteristic is
identified.
4. A method of any preceding claim wherein the image acquired in step (b) is displayed
to a user, and the cells of interest are identified in step (c) by user input signals.
5. A method of any of claims 1 to 3 wherein the cells of interest are identified in step
(c) by analysis-of the image acquired in step (b) by image processing means.
5. A method of any preceding claim wherein cells are identified as of interest in step (c)
bv: comparing a measured parameter of a cell with a predetermined threshold; by linear or
non-linear mapping of a measured parameter; by means of a classifier based on nearest

neighbour; using an artificial neural network; using a support vector machine; using pattern
recognition; or using a genetic algorithm.
7. A method of any preceding claim wherein cells are identified as of interest in step (c)
by reference to: data obtained from other samples; or images or data relating to the sample
and acquired using a different imaging modality to that employed in step (b).
8. A method of any preceding claim wherein the photosensitive compound is one of the
following: a CALI reagent; an uncaging agent; a photosensitizer.
9. A method of any preceding claim including a step of introducing the photosensitive
compound into each of the plurality of cells.
10. A method of any of claims 1 to 8 wherein the photosensitive compound is genetically
encoded in each of the plurality of cells.
11. A method of any preceding claim wherein at least part; of the selected cells is killed
in step (e) by activation of apoptosis, or by the release of a toxic substance.
12. A method of any preceding claim wherein the sample comprises cells which include
a marker compound that is able to respond to light irradiation to assist analysis of the
sample.

13. A method of any preceding claim wherein the sample comprises one of: an assembly
of cells in 2D or 3D; a tissue sample; a biological fluid such as blood; a biopsy sample; an
organ; an embryo; a part of an animal or a plant; or an entire animal or a plant.
14. A method of any preceding claim including a step of stimulating the sample prior to
step (b).
15. A method of any preceding claim wherein an image is acquired in step (b) using one
of the following imaging techniques: fluorescence at one or more excitation wavebands:

luminescence; widefield; confocal; phase contrast; DIG; structured illumination;
fluorescence lifetime; polarization; or a multi-photon process.
16. A method, of any preceding claim wherein a sequence of images is acquired in step
(b), and identification step (c) is carried out with reference to the sequence,
17. A method of claim 16 wherein the sequence of acquired images comprises: bright-
field images; fluorescence images; confocal images at particular focal planes; fluorescence
lifetime images in a range; images at particular wavebands from the infrared to the
ultraviolet; a time sequence; an image stack; or any combination thereof,
18. A method of any preceding claim wherein said further analysis comprises one of:
examination; harvesting; sorting; culture; or treatment of the sample by dispensing of
reagents.
19. Apparatus for modifying a biological sample in accordance with a method of any
preceding claim, comprising:
a detector for acquiring an image of at least a portion of the sample;
a light source for generating a. light beam to irradiate a selected region of the sample;
and
a controller arranged to manipulate the light source to only irradiate at least part of
each of a plurality of selected cells.

This invention relates to enrichment of a biological sample comprising a plurality of cells to assist further analysis
thereof. It provides a technique comprising the steps of: (a) providing a sample comprising a plurality of cells which include a
photosensitive compound that can be induced by light irradiation to inactivate or kill at least part of the respective cell (b) acquiring
an image of at least a portion of the sample; (c) identifying cells of interest in the sample image; (d) selecting cells other than the
cells identified in step (d) and (e) irradiating only those cells selected in step (d) with a light beam so as induce the photosensitive
compound therein to inactivate or kill at least part of these cells, and thereby enrich the sample with respect to the cells of interest
for further analysis.

Documents:

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Patent Number

271977

Indian Patent Application Number

3215/KOLNP/2008

PG Journal Number

12/2016

Publication Date

18-Mar-2016

Grant Date

11-Mar-2016

Date of Filing

06-Aug-2008

Name of Patentee

PERKINELMER SINGAPORE PTE LTD.

Applicant Address

47, AYER RAJAH CRESCENT, #06-12

Inventors:

#	Inventor's Name	Inventor's Address
1	COURTNEY, PATRICK	PERKINELMER LIFE SCIENCES, CHALFONT ROAD, SEER GREEN, BEACONSFIELD, BUCKINGHAMSHIRE HP9 2FS
2	FITCH, ALISTAIR	PERKINELMER LIFE SCIENCE, CHALFONT ROAD, SEER GREEN, BEACONSFIELD, BUCKINGHAMSHIRE HP9 2FX

PCT International Classification Number

G01N 33/487

PCT International Application Number

PCT/GB2007/000698

PCT International Filing date

2007-02-28

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	0603923.4	2006-02-28	U.K.