CELL SORTING

Abstract

The present invention relates to a screening chip for cell sorting, said screening chip comprising a substrate having opposing first and second surfaces, wherein at least a portion of said first surface is coated with a Raman-inactive coating material which can be vaporised by laser irradiation at a wavelength and wherein said substrate is transparent to laser radiation at wavelength In further aspects of the invention, a cell sorting method employing the screening chip and a cell sorting apparatus employing the screening chip are provided.

Claims

1. A screening chip for cell sorting, said screening chip comprising a substrate having opposing first and second surfaces, wherein at least a portion of said first surface is coated with a Raman-inactive coating material which can be vaporised by laser irradiation at a wavelength .sub.1 and wherein said substrate is transparent to laser radiation at wavelength .sub.1.

2. A screening chip according to claim 1, further comprising a cell-holding layer adjacent to the Raman-inactive coating material, wherein said cell-holding layer comprises at least one well in which at least a portion of said Raman-inactive coating material is exposed.

3. A screening chip according to claim 2, wherein said cell-holding layer comprises a plurality of wells.

4. A screening chip according to any one of claims 1 to 3, wherein .sub.1 is about 532 nm or about 1064 nm.

5. A screening chip according to any one of claims 1 to 4, wherein the Raman-inactive coating material is selected from aluminium, titanium, gold, silver, nickel, copper, platinum, palladium, and rhodium, mixtures thereof, the oxides thereof, mixtures of the oxides thereof, graphite, graphene, polyethylene naphthalate (PEN), polyethylene terephthalate, polyester, TiO.sub.2, and mixtures and composites thereof.

6. A screening chip according to any one of claims 1 to 5, wherein the Raman-inactive coating material has a thickness of 100 nm or less.

7. A screening chip according to any one of claims 1 to 6, wherein the Raman-inactive coating material is aluminium having a thickness of about 25 nm, or a composite of PEN and aluminium wherein the PEN forms a layer adjacent to the first surface of the substrate and the aluminium forms a 25 nm thick layer adjacent to the PEN layer and separated from the first surface of the substrate by the PEN layer.

8. A cell sorting method, said method comprising: (i) providing a screening chip as defined in any one of claims 1 to 7, wherein said screening chip further comprises a plurality of cells; (ii) irradiating said cells with laser radiation of wavelength .sub.2 and detecting Raman scattering from the thus irradiated cells; (iii) identifying a target region based on said Raman scattering, wherein said target region is a region of the first surface of the substrate of the screening chip coated with a Raman-inactive coating material and having at least one cell located adjacent thereto; (iv) irradiating the screening chip with laser radiation of wavelength .sub.1 such that said laser radiation of wavelength .sub.1 impinges on the second surface of the substrate of the screening chip and is transmitted through the substrate from the second surface to the first surface to selectively irradiate the coating material in the target region, thereby vaporising at least a portion of the coating material in the target region, causing ejection of the at least one cell located adjacent thereto; and (v) collecting the at least one ejected cell on a collection chip.

9. A cell sorting method according to claim 8, wherein in step (ii) said laser radiation of wavelength .sub.2 impinges on said cells without travelling through the screening chip substrate.

10. A cell sorting method according to claim 8 or claim 9, wherein the screening chip defines an xy plane, the radiation of wavelength .sub.1 impinges upon the screening chip from a location having a positive z-coordinate relative to the xy plane, and the radiation of wavelength .sub.2 impinges upon the screening chip from a location having a negative z-coordinate relative to the xy plane.

11. A cell sorting method according to claim 8 or claim 9, wherein the screening chip defines an xy plane, the radiation of wavelength .sub.1 impinges upon the screening chip from a location having a positive z-coordinate relative to the xy plane and the radiation of wavelength .sub.2 impinges upon the screening chip from a location having a positive z-coordinate relative to the xy plane.

12. A cell sorting method according to claim 11, wherein the screening chip is inverted between step (ii) and step (iv).

13. A cell sorting method according to any of claims 8 to 12, wherein the collection chip is introduced between step (ii) and step (iv).

14. A cell sorting method according to any of claims 8 to 13, wherein .sub.2 is ultraviolet radiation in the range of about 230 nm to about 390 nm, visible radiation in the range of about 400 nm to about 700 nm, or near infra-red radiation in the range of about 750 nm to about 1200 nm.

15. A cell sorting method according to any of claims 8 to 14, wherein .sub.1 is 532 nm or 1064 nm.

16. A cell sorting method according to any of claims 8 to 15, wherein the laser radiation of wavelength .sub.2 is pulsed and the laser radiation of wavelength .sub.1 is continuous-wave.

17. A cell sorting apparatus, said apparatus comprising: a screening chip as defined in any one of claims 1 to 7; an irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.2; a detection unit for detecting Raman scattering from a cell sample on the screening chip; an irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.1 such that said laser radiation of wavelength .sub.1 impinges on the second surface of the substrate of the screening chip and is transmitted through the substrate from the second surface to the first surface to selectively irradiate the Raman-inactive coating material in a target region thereof, thereby vaporising at least a portion of the coating material in the target region.

18. A cell sorting apparatus according to claim 17, wherein the irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.2 and the detection unit for detecting Raman scattering are components of a confocal Raman microscope.

19. A cell sorting apparatus according to claim 17 or claim 18, wherein the irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.1 is arranged such that the laser radiation of wavelength .sub.1 impinges upon the screening chip from a location having a negative z-coordinate relative to an xy plane defined by the screening chip and the irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.2 is arranged such that the laser radiation of wavelength .sub.2 impinges upon the screening chip from a location having a positive z-coordinate relative to an xy plane defined by the screening chip.

20. A cell sorting apparatus according to claim 17 or claim 18, wherein the irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.1 is arranged such that the laser radiation of wavelength .sub.1 impinges upon the screening chip from a location having a negative z-coordinate relative to an xy plane defined by the screening chip and the irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.2 is arranged such that the laser radiation of wavelength .sub.2 impinges upon the screening chip from a location having a negative z-coordinate relative to an xy plane defined by the screening chip.

21. A cell sorting apparatus according to any one of claims 17 to 20, further comprising a computer loaded with software for recording an xy coordinate of the sampling chip and/or an xy coordinate of the irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.1 and/or an xy coordinate of the irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.2.

22. A cell sorting apparatus according to claim 21, wherein the sampling chip, the irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.1 and/or the irradiation unit for irradiating the screening chip with laser radiation of wavelength .sub.2 are connected to driving means for moving the screening chip and/or the irradiation units relative to one another upon instruction by the computer.

Description

[0106] One or more non-limiting examples will now be described, with reference to the accompanying drawings, in which:

[0107] FIG. 1 (a) shows an example of a screening chip as herein described. The first layer (substrate) may be any material transparent to .sub.1, e.g. glass. The second layer (coating) can be vaporized by .sub.1 and is, for example, 25 nm aluminium. The coating can also contain multiple layers to provide more functionality, e.g. Al on top of a layer of (polyethylene naphthalate) PEN. The third layer, e.g. PDMS, may be present to introduce wells, e.g. a well layer/cell holding layer/sample layer. Although a single well is illustrated, the chip may contain more than one well. FIG. 1(b) shows an example of a collection chip as herein described. The substrate may be glass. The second layer, e.g. PDMS, may be present to introduce wells, e.g. a well layer/cell holding layer/sample layer. Although a single well is illustrated, the chip may contain more than one well.

[0108] FIG. 2 shows examples of alternative designs for the collection chip (A, B and C).

[0109] FIG. 3 shows an example of the screening and collection ships in combination. The right hand figures show examples of diameters and depths of the screening chip (below) and collection chip (above).

[0110] FIG. 4 shows an example of the screening step, FIG. 4(a) from above, FIG. 4(b) from below.

[0111] FIG. 5 shows an example of cell ejection. The screening chip and collection chip are shown attached via their well layers. A pulse laser of 532 nm is shown, although other wavelengths may be used. When the pulse laser is through the same objective which is used in the Raman acquisition screening step, the chip may be flipped for the ejection/collection step.

[0112] FIG. 6 shows an example of the operation principle of the RACE system (a) cell identification process, (b) cell ejection process using two objectives. The Raman spectrometer is used to identify the cell in (a). Once the cells are identified, they are ready to be ejected in (b). This configuration employs two independent objectives and therefore there is no requirement to flip over the chip during the process.

[0113] FIG. 7 shows an example of (a) The optical layout of a RACE Add-on module; (b) Chip holder design with pusher. TM1, TM2, and TM3: are turning mirrors, RL is a relay lens, BE is a beam expander. These are used to adapt an existing spectrometer for use in the present invention.

[0114] FIG. 8 shows an exemplary illustration of Raman activated single cell ejection. (A) Microscopic image of cells on RACE chip. (B) The continuous laser is used for acquiring single cell Raman spectra. (C) Spectra of one single cell (top trace) and 25 nm aluminium coating background (bottom trace). (D) The RACE chip is turned over and the target cell is ejected into the collector by a pulse laser.

[0115] FIG. 9 shows microscopy images of the RACE chip (top row) which holds seawater sample and the collector (bottom row) before and after applying the pulse laser. (A) Pulse laser focused at a position without cells. (B) Pulse laser focused on a cell.

[0116] FIG. 10 (A): Single cell Raman spectra of some typical bacteria in the Red Sea sample (traces (top to bottom): A cell with fluorescence; A cell with unknown compound; A cell with PHB; A typical bacterial cell). (B): Raman spectra of four groups of ejected cells. Each group contains 3-8 cells which had the same carotenoid spectra (traces (top to bottom): Cyanobacteria spp G610-8; Shigella spp S709-6; Pelomonas spp P610-5; Pelomonas spp P709-11). (C): Raman spectra of three groups of ejected individual cells (traces (top to bottom): Pelomonas spp P728-5; Bradyrhizobium spp B728-3; Halomonas spp H808-5). The positions of the carotenoid v1, v2 and v3 bands are labelled.

[0117] FIG. 11: Gene identified from Red Sea water in bacterial carotenoid synthesis pathway. The proteins which are present in ejected cells are underlined. IDI: isopentenyl diphosphate isomerase (or IPI, isopentenlypyrophosphate isomerase); IspA: farnesyl diphosphate synthase; CrtE: Geranylgeranyl pyrophosphate synthase; CrtB: phytoene synthase; Crtl: phytoene desaturase (dehydrogenase); CrtC: hydroxyneurosporene synthase; CrtD: methoxyneurosporene dehydrogenase; CrtF: hydroxyneurosporene-O-methyltransferase; CrtY: lycopene cyclase; CrtZ: carotene hydroxylase; PMD: phosphomevalonate decarboxylase.

[0118] In a non-limiting exemplary embodiment of the present invention, the cell of interest is identified by a commercial spectroscope with a green laser (see FIG. 6(a)). Afterwards, the target cell needs to be ejected by a pulsed green laser from the bottom using a second objective for DNA amplification (FIG. 6(b)). This corresponds to a method according to variant (A1) of method (A) as described herein. In order to achieve RACE using the existing spectroscope, a RACE add-on Module has been developed. The optical setup is shown in FIG. 7(a). A 45 degree turning mirror (TM1) is mounted on the spectrometer objective lens turret to deflect the green laser to the RACE module's input. Inside the RACS add-on, a dummy upright microscope is performed using a second 45 degree turning mirror (TM2), objective lens turret, objective (objective 1) and chip holder. For certain systems, e.g. Infinity corrected optical systems, the distance between the objective and tube lens can be varied. A relay lens (RL) can also be used between TM1 and TM2 to conjugate the objective back focal plane of the spectrometer and the objective back focal plane of the RACE add-on. The chip holder (FIG. 6(b)) may be designed with a pushing system to locate the sample slide to the specified position. This design can provide repeatable alignment for the target cells on the sample slide. This slide holder can be attached to the existing spectrometer's motorized scanning stage or mounted on a separate motorized scanning stage. A green pulsed laser is used to eject the target cell from the bottom. A beam expander (BE) is used after the pulsed laser to expand the beam size to fill the ejection objective (objective 2) back aperture to achieved full numerical aperture (NA) application. This generates the laser induced forward transfer (LIFT) to isolate the cells. A 45 degree turning mirror (TM3) is used to deflect the beam upright to the ejection objective or high NA lens. As noted above, the RACE module can also be applied to a modified common microscope (e.g. sort cells based on morphology) and fluorescence microscope (e.g. sort cells based on fluorescence labelled cells) to achieve single cell isolation and subsequent single cell genomics.

[0119] It will be understood that the description above relates to a non-limiting example and that various changes and modifications may be made from the arrangement shown without departing from the scope of this disclosure, which is set forth in the accompanying claims.

[0120] The disclosure will now be further described by way of the following non-limiting Examples:

EXAMPLE 1CHIP PRODUCTION

[0121] Slides with varying thickness of thin layer coating materials including Ti, TiO.sub.2, Al, Au and Ag were purchased from Wellsens Biotech (Beijing, China) for the test. The metal layer coating was less than 100 nm to ensure transparency.

[0122] Examples of sampling and collection RACE chips designed for single cell genomics are illustrated in FIG. 3. To make collection RACE chips, multi-layered PDMS consisting of two concentric wells were fabricated. A PDMS layer (3 mm thick) was made by pouring a mixture of PDMS base and curing agent (Sylgard 184, Dow Corning) at a ratio of 10:1 (w/w) onto a silanised wafer in a container, followed by curing at 65 C. overnight. Small wells (with 3.5 mm diameter were then punched in the PDMS layer (FIG. 3). The second PDMS layer (0.5 mm thick) was made by spinning PDMS prepolymer on a silanised wafer at 200 rpm for 30 s, followed by curing at 65 C. for 1 h (FIG. 3). The two PDMS layers were bonded together using oxygen plasma treatment, and then 2 mm diameter holes were punched in the 0.5 mm thick PDMS layer concentrically to the 3.5 mm holes (FIG. 3). The resultant PDMS well block was irreversibly bonded onto a coverslip using oxygen plasma treatment. To make sampling RACE chips, single-layered 0.5 mm thick PDMS wells with 1 mm diameter upon thin layer coating slides were manufactured in a similar manner (FIG. 3).

[0123] Various coating materials including PEN (polyethylene naphthalate), PET (polyethylene terephthalate) and POL (polyester), TiO.sub.2/Ti (75 nm), Indium-Tin Oxide (10 nm), Au (10 nm) and Al (10, 25, 50 and 100 nm) were examined to achieve RACE. The 532-nm pulsed laser (0.95 ns FWHM, Emax=4.3 J, rep rate=0.1-16.6 kHz) was found to achieve single cell ejection on a thin PEN, PET, POL, TiO.sub.2/Ti (75 nm), Au (10 nm) and Al (10 and 25 nm) coated slide. Al with 25 nm thickness resulted in good quality SCRS with minimal Raman background signal (FIG. 8C).

[0124] As discussed herein, other thicknesses of the layers and diameters of the holes are possible beyond those specifically illustrated in this Example and in FIG. 3.

EXAMPLE 2EVALUATION OF RAMAN BACKGROUND

[0125] Low Raman background is a key factor for the Raman spectra. This experiment was used to check whether the RACE add-on module (e.g. that shown in FIG. 7) induces extra Raman background. Background data with two Thorlabs 80 mm lens (AC254-080-A) as a relay lens in place was recorded. This background signal was from both reflection and lens material, as no sample is mounted, only a black sheet after the 40 objective. In order to check whether the Raman background is from the lens material, Edmund lenses were tried for the same experiment and got similar results. Once the relay lenses were removed, almost no background signal was seen (only 100 count at peak compared with 900 peak count with relay lenses). According to the Raman background signal with 532 nm excitation, the two peak signal is at 580 nm and 610 nm. These two peaks can be confirmed from the laser itself. Full power from the Raman spectrometer and 5 second acquisition time was used and thus this Raman background is still acceptable and could be improved through choice of lenses and emission filters into the spectrometer.

EXAMPLE 3EJECTION LASER POWER AND BEAM SIZE

[0126] A 532 nm pulsed laser (CryLas DPSS 20 J@1 KHz, 1.3 ns pulse width) was used to eject a target cell from the bottom of a screening chip. The minimum power using this laser to generate the laser induced forward transfer (LIFT) was 200 pJ and the maximum power using this laser to generate LIFT with an acceptable spot was 2 J.

EXAMPLE 4CELL EJECTION

[0127] 2 J@1 kHz laser power was applied to eject cell 500 m upright using the configuration shown in FIG. 7. A 40/0.4 NA objection was used to perform the ejection process. Compared with the Olympus LMPLFLN 50/0.5 NA, similar results are achieved. This experiment proves that this RACE module can be used for single cell ejection.

EXAMPLE 5SCREENINGCONFOCAL RAMAN MICRO-SPECTROSCOPY AND SPECTRAL PROCESSING

[0128] All chemicals used were purchased from Sigma-Aldrich (Dorset, UK) unless otherwise stated. Escherichia coli JM109 (Promega Co. UK) containing p18GFP was incubated at 37 C. in LB broth supplemented with 100 g/L ampicillin.

[0129] Cells in concentrated Red Sea water samples (collected from the pier of the Inter-University Institute for Marine Sciences (IUI) in Eilat, Israel) were washed with deionised water before being analysed by Raman micro-spectroscopy. Each cellular suspension (1 l) was mounted in the designed mini-wells of the RACE chip of FIG. 3 (i.e. a screening chip according to the present invention) and allowed to air dry before Raman analysis.

[0130] The cells were examined by the Raman micro-spectroscopy using a continuous 532-nm laser (FIG. 8B). SCRS were used as sorting criteria to distinguish the phenotype of cells, and the positions of targeted cells were recorded (FIG. 8C).

[0131] The Raman spectra were acquired using a confocal Raman microscope (LabRAM HR Evolution, HORIBA Scientific, London, UK) equipped with an integrated microscope (BX41, Olympus). A 50 magnifying dry objective (MPLFLN, NA 0.8, Olympus, Essex, UK) was used to observe and acquire Raman signals from single cells. The laser beam position was calibrated and marked by software Labspec6 (HORIBA Scientific, London, UK). Cells were visualised by an integrated colour camera and a motorised XYZ stage (0.1 m step). The Raman scattering was excited with a 532-nm Nd:YAG laser (Ventus, Laser Quantum, Manchester, UK). Grating 600 I/mm was used for Raman measurements, resulting in a spectral resolution of 1 cm.sup.1 with 1019 data points. The laser power on a single cell was about 0.5 mW. The detector was a 70 C. air-cooled charge coupled device detector (Andor, Belfast, UK). The system was run with a confocal pinhole diameter of 100 m, enabling a spatial resolution of 1 m to be obtained. LabSpec6 software was used to control the Raman system and acquire Raman spectra. Acquisition times for Raman spectra were 5 s for single cell measurements. The spatial location of each measured cell from the Red sea sample were recorded in Labspec6 (operating software for the Raman spectrometer) and used to identify cells for subsequent single cell ejection.

[0132] Labspec6 records the coordinates of each spot where the spectrum is obtained. A reference point on the slide was found and set to (0,0) in Labspec6, so that all cells measured would have a coordinates against that (0,0). Any other software that can record the precise movement of the sampling stage would also be appropriate. For example, any XYZ translation stage with minimum step size to fulfil the experimental requirement (0.1 m/step) can be used to locate the cell. The programmable software is used to drive the stage and record the corresponding position. Alternatively, cells may be located merely by taking an image of the cell, and then finding the cell from that image.

[0133] Thus, SCRS was used as sorting criteria to distinguish the phenotypic profile of cells (in this case the presence of carotenoids in cells), and the positions of targeted cells were recorded (FIG. 8C).

EXAMPLE 6COLLECTIONINTEGRATION OF SINGLE CELL EJECTION INTO A RAMAN MICRO-SPECTROSCOPE

[0134] Following the screening step of Example 5, the RACE sampling chip was turned over and single cells of interest were ejected using the 532-nm pulse laser and harvested in a RACE collection chip (i.e. a collection chip according to the present invention, FIGS. 8D and 3). In this study, because a continuous 532-nm laser light path was employed to guide the pulsed laser, the Al-coated slide (screening chip) was transparent to allow for locating and visualising of the cells after turning the slide over for cell ejection (FIG. 8D). However, a design such as that shown in FIG. 6 would not require this transparency by placing the pulsed laser beneath the sampling chip.

[0135] A 532-nm pulse laser (ALPHALAS GmbH, Goettingen, Germany) was integrated into a confocal Raman microscope, sharing the same continuous laser (532-nm) light path and objective lens for SCRS measurement without changing mirrors and filters (FIG. 8). The peak power was 1.1 kW, about two magnitudes lower than the traditional UV microdissection lasers. An exposure test showed that the 532-nm pulse laser did indeed cause no damage to the mirror and edge filters in this LabRAM HR Evolution Raman microscope.

[0136] All the equipment and working surfaces were wiped with 10% (v/v) domestic bleach (Domestos, UK). All the consumables were autoclaved and exposed to a UV light bulb (254 nm) inside a UV sterilised Laminar Flow cabinet for 1 hour. The reagents were carefully sterilized.

[0137] 1 l of lysis buffer (500 l of SCG-grade water and 45.5 l of 1M DTT were added to buffer DLB (single cell kit, Qiagen, UK) and aliquoted into UV sterilised Eppendorf tubes) and 1 l TE buffer (Tris-EDTA buffer and DMSO: Tris-EDTA (pH=8) buffer and DMSO were filtered through a 0.2 m filter and aliquoted into UV sterilised Eppendorf tubes) were added into each well on the collection chip which was then attached to the RACE screening chip. Alternatively, the liquid could be added after the cells are collected. The enclosed chips were moved to the stage of the Raman microscope with the RACE chip facing down. The target cells were located by their coordinates and confirmed by comparing the bright field image taken during Raman spectra acquisition. The laser spot was manually aligned to the coating layer under the target cell. Alternatively, this could be automated using software that moves the stage by input the target coordinates and then do an auto image alignment. The target cells were ejected by evaporation of the coating layer upon application of a 0.1 s exposure of 532 nm pulse laser and the cells were harvested in the collector. For a 12-well RACE chip, at least two control wells were also set up: one negative control, which remained cell-free, and one positive control, to which cell suspensions were added before whole genome amplification (WGA).

[0138] It was found that a 25-nm aluminium (Al) coating on a RACE sampling chip gave minimal Raman background (FIG. 8C). The thin coating was able to absorb the energy of the 532-nm pulse laser and eject a single cell sitting on it (FIG. 9). FIG. 9 shows that the Al (25 nm) coating material has been removed leaving a 1.5 m mark on the coating slide after laser ejection. No observable Al material reached the collection chip after ejection in the blank controls, suggesting that Al was completely vaporized. FIG. 9B indicates that the single cell was accurately isolated and collected by the RACE collection chip.

EXAMPLE 7WHOLE GENOME AMPLIFICATION ON-CHIP

[0139] The enclosed chips were carefully moved to a laminar flow chamber and the two chips were separated. Cell suspension of 0.5 l was added to the positive control well and a sterile coverslip was then placed on top of the collector chip. Cell lysis was carried out by three freeze-thaw cycles followed by heating at 65 C. for 10 min in a thermocycler (C1000, Bio-rad, UK). After adding 1 l of Stop solution to neutralise the lysis buffer, 12 l of reaction master mix was added to each well (Reaction buffer contained 1 Repliphi29 reaction buffer (Epicentre, US), 50 M random hexamers, 5% DMSO, 10 mM DTT, dNTPs (0.4 mM) and aliquoted into UV sterilised Eppendorf tubes). The collector chip was then covered by a coverslip and kept in the thermocycler at 30 C. with the hot lid activated and set at 70 C. After incubation for 8 hours, the phi29 DNA polymerase was deactivated by heating to 65 C. for 10 min. The MDA product was transferred into sterilised 200 l-PCR tubes for storage.

EXAMPLE 8SORTING CAROTENOID-CONTAINING BACTERIA FROM A RED SEA SAMPLE BASE ON THEIR RESONANT RAMAN SPECTRA

[0140] SCRS of 3,278 single cells from the surface seawater at Eilat were obtained and analysed, 31.4% of the analysed cells exhibited fluorescence and 68.6% of the cells had distinguishable SCRS for sorting. FIG. 10A shows examples of SCRS from the sample, including a typical bacterial cell, individual cells showing fluorescence, containing poly--hydroxybutyrate (PHB) and unknown compounds. Since SCRS reflect biochemical phenotypes of cells, cells can be sorted based on biomarker Raman bands of SCRS. For example, SCRS of a typical cell (FIG. 10A), the 1005 cm.sup.1 benzene ring breathing band (e.g. phenylalanine) is sharp and distinguishable and it shifts in response to .sup.13C-incorporation, which can be used to link the cells to their metabolic activity of a .sup.13C-substrate. Cells containing PHB gave distinguishable Raman biomarker at 839, 1058, 1403 and 1123 cm.sup.1 (FIG. 10A), which can be used to sort PHB containing cells.

[0141] The characteristic v1, v2 and v3 Raman bands of carotenoids were used as sorting criteria. The v1 band is the methyl rocking mode, and the v2 and v3 bands vary due to the different lengths of conjugated CC bonds and stretching modes of CC bonds. Since the Raman spectral resolution is about 1 cm.sup.1, variations in the positions of v1, v2 and v3 indicate the different structures of carotenoid presented in the cells (FIGS. 3B and 3C). According to SCRS, 744 (22.7%) cells contained carotenoids (FIGS. 10B and 10C). Based on their SCRS, four groups of carotenoid-containing cells were isolated with 3-8 cells in each group (FIG. 10B), and the position of v1, v2, v3 were exactly the same within each group. Three single cells were also isolated individually (FIG. 10C). Collectively these seven sorted samples could be resolved into five different types (FIGS. 10B and 10C). Optical images of the sorted cells showed that the cells were small with size of 0.3-1 m.

[0142] The estimated degree of putative contamination, based on multiple copies of universal marker genes with differing amino-acid sequences, was relatively low in all cases. However, sequence analyses also indicate low genome coverage of less than 20% in all cases, which might be due to UV treatment of the phi29 polymerase.

[0143] The recovered genes directly involved in carotenoid biosynthesis pathway are illustrated in FIG. 11. Specifically, isopentenyl diphosphate isomerase (IDI) and geranylgeranyl pyrophosphate synthase (ispA) are involved in making the colourless substrate farnesyl pyrophosphate (FPP) and phytoene dehydrogenase (crtl) is responsible for synthesis of red pigment lycopene (FIG. 11). Although no gene directly related to carotenoid synthesis was found in H808-5 (probably due to low genome coverage), this sample contain two types of putative she (squalene-hopene cyclase) genes whose substrates are carotenoid compounds. These results validate the RACE method since the sorted cells should contain carotenoids according to their SCRS.

[0144] The method of the invention has therefore been shown to accurately isolate individual cells from the Red Sea sample based on the characteristics SCRS of carotenoids.

[0145] The above Examples illustrate Raman activated cell sorting coupled to single cell genomics. The RACE technology has been applied to a Red Sea sample, sorting out seven groups of carotenoid-containing cells and performing subsequent genome analyses. The design of the RACE chip enabled single cell genomics to be performed under standard laboratory conditions without access to an expensive and dedicated super-clean facility.

[0146] The RACE chip of the present invention (e.g. FIG. 3) creates a sealed environment to perform single cell isolation and DNA amplification on-chip, significantly preventing cell or DNA contamination from the outside environment. Cells of interest have been directly isolated and ejected into prepared buffer in the collection well, enabling single cell DNA amplification on-chip without the need for cell transfer. By employing an advanced microfluidic device, the chip could perform on-chip cell cultivation, PCR, and even DNA sequencing. Use of a chip, method, coating or apparatus as herein described in single cell genomics, on-chip cell cultivation, PCR, and/or DNA sequencing (or in any other method as herein described) therefore forms a further aspect of this disclosure.