WO2006130921A1

WO2006130921A1 - Investigating biological cells using enhanced raman spectroscopy

Info

Publication number: WO2006130921A1
Application number: PCT/AU2006/000796
Authority: WO
Inventors: Bayden Robert Wood; Donald Mcnaughton; Phil Heraud; John Beardall
Original assignee: Monash University
Priority date: 2005-06-08
Filing date: 2006-06-08
Publication date: 2006-12-14

Abstract

The status of one or more biological cells is measured by subjecting the cells to excitation wavelengths between 200nm and 1064 nm, and recording and analysing a wavelength band resulting from resonance enhanced Raman scattering. The method may be applied to diagnose malaria in red blood cells, where the Raman scattering is caused by hemozoin or porphyrin compounds, and also to monitor the effect of anti-malarial drugs. It may further be applied to analyse chlorophyll and carotenoids in plant and algal cells. Acoustic levitation may be used to reduce noise, by providing a container-less environment for the cell samples.

Description

INVESTIGATING BIOLOGICAL CELLS USING ENHANCED RAMAN SPECTROSCOPY

Field of the invention

The present invention provides a method of measuring the status of one or more biological cells using enhanced Raman spectroscopy. In particular the present invention provides a method of using enhanced Raman scattering for diagnosis or analysis using one or more cells. The method of the present invention is particularly useful for detection and diagnosis relating to disorders such as malaria.

Background of the invention

In this specification where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge; or known to be relevant to an attempt to solve any problem with which this specification is concerned.

While the present invention will be described with particular reference to analysis of haeme in red blood cells, it will be appreciated that the present invention is not so limited but can be applied to a range of compounds in many different types of cells. Furthermore, while particular reference is made to the use of the method of the present invention for the detection and diagnosis of malaria the present invention is not so limited but can be applied to the detection and diagnosis of other disorders and applications outside of the medical field, such as in bioprocessing and other manufacturing industries.

Spectroscopy is the branch of science devoted to discovering the chemical composition of materials by examining the interaction of electromagnetic radiation with the material. Infrared (IR) spectroscopy relates primarily to the absorption of energy by molecular vibrations having wavelengths in the infrared segment of the electromagnetic spectrum, that is energy of wave number between 200 and 4000 cm^'1. Raman spectroscopy relates to the inelastic scattering of monochromatic light giving wavelength shifts that depend on the molecular vibrations, having typically wave number shifts between 20 and 4000 cm^"1.

Vibrational spectroscopy of molecules is a relatively well known method of analysis in the chemical industry. Raman and infrared spectroscopy are two types of vibrational spectroscopy that are frequently used in laboratories. According to the theory of quantum mechanics, only certain well-defined vibrational frequencies and atomic displacements are allowed in a molecule. These are known as the normal modes of vibration of the molecule. There are several types of motion that contribute to the normal modes. Some examples are:

• stretching motion between two bonded atoms;

• bending motion between three atoms connected by two bonds;

• out-of-plan deformation modes that change an otherwise planar structure into a non- planar one.

Vibrational infrared spectroscopy allows characterisation of vibrations in molecules by measuring the absorption of light of certain energies that correspond to the vibrational excitation of the molecule from one energy state to a higher energy state. Raman spectroscopy allows characterisation of vibrations in molecules by measuring the energy shifts of monochromatic light that correspond to the difference between vibrational energy states. For a vibrational motion to be IR active, the dipole moment of the molecule must change. Therefore, the symmetric stretch in carbon dioxide is not IR active because there is no change in the dipole moment. The asymmetric stretch is IR active due to a change in dipole moment. The symmetric stretch in carbon dioxide is Raman active because the polarisability of the molecule changes. For a vibration to be Raman active, the polarisability of the molecule must change with the vibrational motion. Thus Raman spectroscopy complements IR spectroscopy. Typically micro-Raman spectroscopy is carried out by illuminating a sample with

UV/visible of near infrared energy from a laser. The laser is passed through a line filter to remove any unwanted laser lines and sidebands (such as sidebands generated from pumping light in a diode laser or plasma lines in a gas laser) then focused onto the sample via a microscope lens. Light scattered from the sample is then passed through an optical filter to remove inelastic scattered light t filter before being reflected by a series of mirrors and dispersed by a grating onto a single pint or array detector. Fourier transform techniques are also available.

Depending on the size and nature of the sample it is often convenient to direct the laser and scattered light between the spectrometer and the sample using optic fibres. The sensitivity of the process depends on the amount of Raman scattering by any analyte or other matrix in which the sample resides and on the presence of fluorescence and absorbance.

The results of Raman spectroscopy are generally recorded electronically in the form of a spectrum, that is a graph of intensity of Raman scattering (y-axis) against wavelength (x- axis). Typically a spectrum looks like a series of peaks on a rough base line and is presented as wave number shift.

The ability to detect chemical analytes and monitor nutrient status of living cells is of critical importance in a range of technologies and industries. Most analysis of living cells relies on removal of a sample set of cells from the system, then lysing or otherwise breaking open the cells (thus killing them) to obtain their contents for analysis. Typically the contents are subjected to high performance liquid chromatography (HPLC) to separate individual chemical components followed by ultraviolet/visible spectroscopy to analyse individual components. Although this type of testing is used in industry, particularly the bioprocessing industry, it is slow and expensive and does not give sufficiently rapid feedback regarding changes during bioprocessing. A number of other different sample handling procedures and spectral monitoring techniques have been proposed to deal with bioprocessing (Baena, L.R., Lendl, B. Current Opinion in Chemical Biology, 2004 (8) 534-539; Jarute, G, Kainz, A., Schroll, G., J.R. Baena, Lendl, B., Anal. Chem. 2004 (76) 6353-6358) and in this respect, Fourier Transform IR (FTIR) has shown potential (Jarute et al.). However one of the major limitations in developing FTIR for on-line bioprocessing applications is that the spectrum recorded is subject to interference from water deformation modes in the 1700-1550 cm^"1 region of the spectrum where the strong and analytically useful protein bands absorb.

By contrast Raman spectroscopy has the advantage that water does not interfere with or obscure bands in the Raman spectrum. Furthermore, Raman spectroscopy is more sensitive than FTIR for testing of small samples (micro-samples) such as one or two plant or animal cells. Most Raman measurements of micro-samples require a microscope facility to focus the Raman laser on to the sample, but due to the difficulties associated with handling such small entities, the spectrum recorded often has a very poor signal-to-noise ratio.

Effective, reliable analysis depends upon the ability to obtain clear and reproducible results, hence these prior art methods have considerable drawbacks with respect to small samples.

Acoustic levitation provides a containerless environment for monitoring and is now an established miniaturisation technique that has found myriad analytical and bioanalytical applications. For example, acoustic levitation has been used for investigating chemical reactions of micro-crystals (Musick, J., Popp, J. Phys. Chem. Chem. Phys 1, 1999 (24), 5497- 5502; Musick, J., Kiefr, W., Popp, J., App. Spectrosc. 2000 (54) 1136-1141) and organic test molecules in silver colloidal sols (Leopold, N., Haberkorn, M., Laurell, T., Nilsson, J., Baena, J.R., Frank, J., Lendl, B., Anal. Chem. 2003 (75) 2166-2171). Few, if any studies have been carried out using living cells, with the exception for example of studies by Santesson et al. (Santesson, S., Andersson, M., Degerman, E., Johansson, T., Nilsson, J., Nilsson S., Anal

Chem. 2000 (72) 3412-3418). These authors describe a system for analysing the effects of β- adrenergic agonists and insulin on lipolysis in adipocytes. The use of a levitated, adipocyte- containing droplet to simulate lipolysis was an advantage over the classic way of measuring this cellular event. It avoided the need to prevent flotation of the fat cells by extensively mixing them using magnetic stirrers.

Raman spectra may be further improved by the use of 'resonance enhanced Raman scattering'. Raman spectroscopy is usually performed using a laser operating at a wavelength below the first electronic transitions of most molecules. Resonance Raman scattering refers to the result obtained when the excitation wavelength used lies on or near an electronic transition (that is, at a 'resonance' or 'pre-resonance' wavelength). In that case the intensity of some Raman-active vibrations increases by a factor of 10²-10⁴. The vibrations whose Raman bands are resonance enhanced fall into two or three general classes. The most common case is Franck-Condon enhancement, in which a component of the normal coordinate of the vibration is in a direction in which the molecule expands during an electronic excitation. The more the molecule expands along this axis when it absorbs light, the larger the enhancement factor. The easily visualized ring breathing (in-plane expansion) modes of porphyrins fall into this class. Vibrations which couple two electronic excited states are also resonance enhanced. This mechanism is called vibronic enhancement. In both cases enhancement factors roughly follow the intensities of the absorption spectrum.

Another form of resonance Raman scattering known as 'Surface-enhanced Raman scattering (SERS) occurs when a compound (or ion ) is adsorbed on a structured metal surface, inducing Raman scattering up to 10³-10⁶ times greater than in solution. SERS is strongest on silver, but is observable on gold and copper as well. SERS arises from two mechanisms. The first is an enhanced electromagnetic field produced at the surface of the metal. When the wavelength of the incident light is close to the plasma wavelength of the metal, conduction electrons in the metal surface are excited into an extended surface electronic excited state called a surface plasmon resonance. Molecules adsorbed or in close proximity to the surface experience an exceptionally large electromagnetic field. Vibrational modes normal to the surface are most strongly enhanced.

The second mode of enhancement, often termed chemical enhancement is by the formation of a charge-transfer complex between the surface and analyte molecule. The electronic transitions of many charge transfer complexes are in the visible region of the electromagnetic spectrum, so that resonance enhancement may also occur.

It has now been found that enhanced Raman response can be obtained from a range of biological molecules and can act as useful markers to measure the status of these cells. In particular it has been found that enhanced Raman response can be used to measure the status of living cells or their contents when exposed to chemical actives such as drugs.

Summary of the invention

The present invention provides a method of measuring the status of one or more cells using enhanced Raman spectroscopy.

In particular, the method includes measurement of the status of one or more cells using enhanced Raman spectroscopy, including the steps of

(a) subjecting the one or more cells to excitation wavelengths between 200 and 1064 nm,

(b) recording at least one band resulting from enhanced Raman scattering and

(c) using the band recorded from step (b) to deduce the status of the one or more cells. It will readily be apparent to the persons skilled in the art that the excitation wavelength used will depend on the type of laser and optical configuration of the spectrometer used.

Where used herein the word 'status' indicates a quality or quantity relating to the cell. This includes for example, the health, viability or identity of the cell. The health or viability of a cell may change due to factors such as disease, environment, metabolic state and upon interaction with drugs, pollutants or other chemicals. The identity of a cell may also change. For example a stem cell or totipotent cell may change with time to being a fully differentiated cell. For fully differentiated cells, the status may include its taxonomic identification.

The status of individual cells also provides information about the status of the micro- organism, tissues or organs from which the cells were derived. Accordingly the method of the present invention could be applied to a wide range of diagnoses and analyses relating to biological systems.

The cells may be living, or alternatively the cells may have been subjected to further processing such as lysing, drying or concentration. The method of the present invention can be used for a sample consisting of a single cell.

Typically the Raman scattering will be caused by a compound, or compounds in the cell. As mentioned previously, Raman spectroscopy measures vibrational transitions associated mainly with symmetric stretching and bending in a molecule when the polarisability of the molecule changes with the vibrational motion. Typically, the present invention is used to measure Raman scattering in compounds having certain kinds of structural features called chromophores. Chromophores in the visible region include delocalised π-orbital electrons and if the resonance of the π-orbital structure matches the incident photon's wavelength then enhanced scattering of the electromagnetic energy is possible. In a particularly preferred embodiment the method of the present invention is used to measure resonance Raman scattering of a compound from cells which compound has a series of conjugated double (or triple) bonds and/or which is coordinated to a metal.

Preferably the cells tested using the method of the present invention include a compound chosen from the group including:

(1) organic species such as porphyrins, including protoporphyrins, protoporphyrinogens, coproporphyrinogens, uroporphyrinogens, coproporphyrinogens, porphobilinogens and porphyrins; quinolines; carotenes including β-carotene; fullerenes; polydiacetylenes, their precursors and degradation products, and

(2) inorganic species such as metalloporphyrins including haeme, myoglobin chlorophyll a, and chlorophyll b and their precursors and degradation products,

and combinations thereof.

Compounds having a high degree of symmetry are particularly preferred. In a particularly preferred embodiment the compound is haeme, a haemoglobin precursor such as protoporphyrin IX, protoporphyrinogen IX, coproporphyrinogen III, uroporphyrinogen III or porphobilinogen, or the product of biological breakdown or derivative of haeme such as haemozoin, porphyrin or bilirubin. In another preferred embodiment the compound is chlorophyll a or chlorophyll b or their degradation products such a pheophytin.

Other compounds that can be used with the method of the present invention include hormones, xanthophils, phycobiloproteins (including both phycocyanin and phycoerythrin) and silica in both higher plants and algae. It is noted however that the measurement of silica relies not on an enhancement mechanism but on naturally strong Raman scattering. Without wishing to be bound by theory, it is believed that using enhanced Raman spectroscopy the spectrum of the chromophoric moiety is resonance enhanced and that of the surrounding protein matrix is not. This allows the chromophoric site to be probed without spectral interference from the surrounding protein.

The method of the present invention could be used for measuring the status of:

• animal cells, based on Raman scattering by haemoglobin, myoglobin and other haem based derivatives

• plant cells, based on Raman scattering by chlorophyll, beta-carotene, xanthophils and silica in the plant cells, and

• algal cells based on Raman scattering by chlorophyll, beta-carotene, phycobiloproteins and xanthophils in the algae.

In a preferred embodiment, the method of the present invention is used to measure the status of red blood cells based on Raman scattering by haemoglobin. This could be useful for example, to measure the haematocrit, degree of oxidation, deoxygenation and metabolic state of the red blood cells. Haeme is extremely sensitive to changes in cell status such as the changes caused by sickle cell anaemia, altitude sickness and malaria (a disease caused by any one or four species of parasitic protozoa that infect human red blood cells which is transmitted to humans via the Anopheles genus of mosquito). The Raman scattering of haemoglobin will vary depending on whether iron (or other metal or chemical moiety) is coordinated to the haeme ring, whether the haemoglobin is oxygenated or deoxygenated and whether the haeme ring is disrupted or damaged.

In a particularly preferred embodiment the method of the present invention is used for diagnostics and drug screening in living cells. Malaria

In a particularly preferred embodiment the method of the present invention is used to diagnose malaria. Malaria can be suspected based on a patient's symptoms and the physical findings upon examination. However, these symptoms are similar to those of other diseases e.g. fever and for a definitive diagnosis to be made, laboratory tests must demonstrate the presence of malaria parasites or their components. Malaria must be recognized promptly in order to treat the patient in a timely manner and to prevent further spread of invention in the community. Although a range of antimalarial treatments are available it is advantageous to use a specific drug treatment for each malarial strain. Incorrect diagnoses, the unnecessary use of antimalarial drugs and the use of the incorrect drug results in additional expense and increases the risk of selecting for drug-resistant parasites. Diagnostic methods of the prior art are often prone to inaccuracy (such as microscope based examination of a patient blood droplet), prohibitively expensive (such as commercially available test kits) or have comparatively high limits of detection (above 100 parasites per μl).

The present invention may be suitable for diagnosing the species of malaria that has infected an individual. Specifically, malaria parasites consume haeme, converting it to a substance known collectively as malaria pigment (also called 'haemozoin'). Different species of malaria parasite (Plasmodium malariae, Plasmodium vivax and Plasmodium falciparum) produce haemozoin structures having slightly different chromophores and thus produce different enhanced Raman scattering. The major advantage of the present invention is that it can detect between 1 and 100 malaria trophozoites per μl, more preferably between 1 and 10 malaria trophozoites per μl, or even more preferably 1 malaria trophozoites per μl in a matter of seconds in a blood sample of 100 μl.

Furthermore, the malaria parasite catabolizes haemoglobin and converts the resulting free haem to haemozoin. Antimalarials such as chloroquine and quinoline act by binding to haem, probably through π-π stacking of the aromatic structures or alternatively forming hydrogen bonds between the propionate and vinyl groups. Other antimalarials that interact with haeme include the artemisinins, aryl-alcohols, and peroxides. The haeme-drug interactions could be monitored in the functional red blood cell using the spectroscopic technique of the present invention either directly by detecting Raman scattering from the drug in the parasite food vacuole, or indirectly by observing changes in the relative intensity of the compound haemozoin formed by the parasite when it consumes haeme. Direct evidence of drugs binding to haem in vitro are few and most rely on either measurements of crude trophozoite lysates, chemical methods under non-physiologic conditions or morphologic effects using microscopic techniques. Hitherto there has been no direct in vivo drug screening technique to monitor antimalarials accumulating in cells and binding either to the free haem or haemozoin.

Furthermore, the method of the present invention may be suitable as a diagnostic. For example, compounds such as quinolines are effective as antimalarials based on their ability to bind to haemoglobin. However in some individuals, this binding cannot occur and quinolines are thus ineffective as antimalarials in these individuals. The method of the present invention can be used to rapidly test for efficacy of quinoline in individuals.

Accordingly the present invention has enhanced detection as compared with other conventional techniques and enables malarial parasite detection at extremely low levels and allows confirmation of the presence of the disease and efficacy of treatment in patients for which existing technologies would be unable to detect. In theory, this ability to detect the disease early will enable treatment to commence prior to the parasite multiplying to levels which would cause clinical symptoms.

The method of the present invention may be useful for measuring the status of plant cells. A number of techniques have been employed in the past in order to determine factors limiting growth and production of plant cells such as algae. These include bioassays, estimation of a range of physiological parameters such as nutrient uptake rates, analysis of elemental and macromolecular composition and measurement of specific molecular markers for nutrient limitation. However most are relatively slow, complicated or expensive.

In a particularly preferred embodiment the method of the present invention can be used to monitor the status of living cells based on Raman scatter by chlorophyll, carotenoids, phycobiloproteins and xanthophils. For example the method of the present invention is used to measure the status of living plant cells based on the relative proportion of chlorophyll to beta- carotene in the cell. This could be useful for rapid testing during manufacture of bioproducts.

For example carotenoids such as beta-carotene are common vitamin supplements and food additives. Beta-carotene is produced industrially by micro-organism cultivation. The method of the present invention could be used for rapid testing of cells to monitor the level of beta carotene production in a batch of cultivated micro-organisms. Alternatively, beta carotene and other compounds are often produced in vivo by transgenic cells. The method of the present invention could be used as a method for testing the expression and level of in vivo synthesis of transgenic products.

In another embodiment the method of the present invention is used to monitor the status of cells in order to detect adverse environmental effects such as pollution. Microorganisms such as algae and diatoms often sequester pollutants, or their chlorophyll is damaged, severely inhibiting their ability to photosynthesise. The method of the present invention could be used to test micro-organisms for the presence of sequestered pollutants to monitor pollution levels. For example the invention could be used to measure the level of pollutants in algae living in waterways.

In a further embodiment relating to micro-organisms, the present invention may be used for the taxonomic differentiation of biological cells constituting micro-organisms. Different species of a micro-organism often included different chemical species, or metabolise chemical species differently. In a particularly preferred embodiment the method of the present invention is used for the taxonomic differentiation of malaria parasites. For example, malaria parasites consume haeme, converting it to a malaria pigment known as 'haemozoin'. Different species of malaria parasite {Plasmodium malariae, Plasmodium vivax and Plasmodium falciparum) produce haemozoin structures having slightly different chromopohores. The different chromophores produce different Raman scattering and thus can be used to identify different species of parasite.

In another particularly preferred embodiment the method of the present invention is used for the taxonomic differentiation of algal cells. For example, different proportions of chlorophyll a, chlorophyll b, β-carotene and the like can be used to identify different types of algae. Algal species such as Chaetoceros muelleri, Phaeodactylum tricornutum, Dunaliella tertiolecta and Porphyridium purpureum can be distinguished based on Raman scattering by chlorophyll a and β-carotene within the algae.

The method of the present invention may be carried out using any commercially available Raman spectrometer suitable for resonance Raman spectrometry. Typically the Raman spectrometer is linked by fibre optics to an acoustic levitation device. Use of a levitation device to hold the sample is particularly advantageous because it avoids the use of a container or matrix that could attenuate the Raman scattering and reduce the quality of the spectrum recorded.

The method of the present invention can be performed using a device that is sufficiently simple, inexpensive and portable that it could be used for mobile analysis and diagnosis. This would be particularly useful, for example for the diagnosis of different Plasmodium species and strains in remote areas where malaria is endemic. Examples

Various embodiments/aspects of the invention will now be described with reference to the following non-limiting examples and the graphs/spectra in which:

• Figure 1 depicts three Raman spectra, recorded on three human cells each infected with a different strain of malaria as described in Example 1;

• Figure 2 depicts Raman spectra recorded from levitated samples of Dunaliella tertiolecta as described in Example 2;

• Figures 3 and 4 depict Raman spectra recorded from levitated samples of Dunaliella tertiolecta as described in Example 3;

• Figure 5 depicts a plot of the ratio of chlα to pheophytin based on UV measurements;

• Figure 6 depicts Raman spectra recorded from levitated samples of Dunaliella tertiolecta as described in Example 4;

• Figure 7 depicts Raman spectra recorded from levitated suspensions of Chaetoceros muelleri, Phaeodactylum tricornutum, Dunaliella tertiolecta and

Porphyridium purpureum;

• Figure 8 depicts a PCl scores plot as showing individual spectra represented as points from samples of P, faciparum (P) and P.vivax (PV) from 1% parasite culture indicating the potential of the technique as a diagnostic;

• Figure 9 depicts a PCA scores plot based on the Raman spectra recorded from samples of P. falciparum (X) and P.ovale (■); • Figure 10 depicts a PCl scores plot showing individual spectra represented as points form samples of P. falciparum (Pf) clearly separated from P.vivax (PV) and P. ovale (PO) indicating the potential of the technique as a diagnostic.

• Figure 11 depicts a PCAl scores plot based on Raman spectra showing separation of P, falciparum and P.vivax from clinical samples.

• Figure 12 depicts a PCA scores plot based on Raman spectra showing separation of P. falciparum and P. ovale from spectra of single cells.

Example 1:

A portable 785 nm InPhotocs Raman spectrometer coupled to a quartz halogen light source and Raman fibre optic probe was used in the method of this example. Three 10 μL samples of parasitized red blood cells were levitated at the node of a Dantac ultrasonic acoustic levitation device. The first sample was infected with P.malariae, the second sample was infected with P.vivax and the third sample was infected with P. falciparum. Each sample was directly subjected to an excitation wave of 780 nm and a x 60 water immersion objective on a Renishaw Raman system 2000. The Raman spectra were recorded by focusing within the cell on the hemozoin deposit (ca 1-2 micron deposits). Figure 1 depicts the average of spectra taken of single red blood cells infected with the three strains of malaria mentioned above. The prior art teaches this should be carried out in containers such as Petri dishes in contradistinction to the current example using levitation.

The spectra show distinct differences in the ratios of important bands associated with heme aggregation. Different species of malaria parasites are known to have different haemozoin crystal morphologies and therefore different aggregation states. These differences are reflected by the differences in the three spectra, thus the method of the invention can be used to diagnose the type of malaria infection present in an individual, an important step to choosing appropriate treatment.

Examples 2 to 5:

In Examples 2 to 5, a portable Raman spectrometer was coupled to an acoustic levitation device to analyse living algal cells. The examples illustrate the suitability of the method of the present invention for environmental monitoring and the potential taxonomic identification of microalgae.

Cells and chemicals

Four species of microalgae, namely the green alga Dunaliella tertiolecta, two diatoms Chaetoceros muelleri and Phaeodactylum tricornutum and the red alga Porphyridium purpureum (CSIRO Marine Laboratories, Hobart, strains CS 175, CS 176, CS 29 and , CS-25 respectively) were grown in 100 mL batch cultures with artificial seawater, based on the "D" medium of Provasoli et <z/.(Provasoli, L.; McLaughlin, J.J.A., Droop, M.R. Archiv Microbiol (1957) 25 392-428) Cultures were maintained under cool white fluorescent tubes at a photon flux of 100 μmol quanta m^'V¹ at 18 ⁰C and the cells were analysed when cultures were in the mid-exponential phase. Nitrogen-limited (Nlimited) cultures of D. tertiolecta were obtained by transferring cells from a nutrient replete mid-exponentially growing culture into artificial seawater medium without nitrogen, in which they were maintained for four days before the cells were subjected to levitation. 10 mL suspensions containing ~10⁷ cells were centrifuged for 2 minutes at 5000 rpm and the supernatant removed. Aliquots of 10 μL of concentrated cells were transferred by pipette into an acoustic node of the levitation device. For Dunaliella tertiolecta approximately 10⁴ cells were transferred to the acoustic node. In a similar manner a similar number of cells were deposited in a quartz micro-cuvette for spectral acquisition. Chlorophyll a (chlά) and β-carotene were purchased from Sigma-Aldrich (Clayton, Victoria, Australia) and used as supplied. Approximately 20 mg of each compound was dissolved in 1 mL of MiIIiQ water and 10 μL was transferred by pipette in to the acoustic node.

Apparatus

An ultrasonic levitator (Dantec/Invent Measurement Technology, Erlangen, Germany) was used at a frequency of 56 kHz, the Standard wavelength is 5.9 mm, the largest drop diameter is 2.5 mm and the smallest is 15 μm. The instrument, which is based on a piezoelectric vibrator, generates a standing acoustic wave with equally spaced nodes and antinodes by multiple reflections between a solid reflector and ultrasonic transducer.(Omrane, A., Santesson, S., Alden, M., Nilsson, S. Lab Chip (2004) 4287-291).

The samples were levitated below pressure nodes, due to axial radiation pressure and radial Bernoulli stress. There are 4-5 pressure nodes, but only the inner 2-3 are used for stable levitation. Destabilization effects caused by the ultrasonic transducer and reflector influence the outer 2 nodes and hence the central or inner nodes were used for sample levitation. The cell suspension placed in the node appeared like a flattened doughnut. For long experiments (i.e. greater than 150 seconds) water was added to avoid evaporation and ultimately cell death, which may have influenced the spectra. The Raman spectra were acquired with an InPhotonicsportable InPhotote Raman Spectrometer with a 785 nm diode laser and an InPhotonics RamanProbe™

The fibre optic sampling probe was a cylindrical device, 12.7 mm in diameter. Laser light was transmitted to the probe via a 90 μm excitation fibre. The light was collimated into the probe using a collimating lens before it is transmitted through a band pass filter. Approximately 85% of the light then passed through a dichroic filter and extraneous light was reflected. Another lens focuses the light onto the sample and also collected the back scattered light from the sample and focused this light back into the probe. The dichroic filter then reflected the scattered light to the opposite side of the probe to where it is redirected parallel to the original path but in the opposite direction. The light was then transmitted to a long-pass filter assembly consisting of three filters which remove the Rayleigh and anti-Stokes scattered light. A final lens focuses the light into a 200 μm collection fibre, which transmitted the signal to the spectrometer. Power at the sample was measured at 50 mW and unless otherwise stated each spectrum was collected with 20 seconds of laser exposure. Spectra were baseline corrected using OPUS™ spectroscopic software at minima turning points (1800, 1409, 1080, 928, 763 and 600 cm-1). Spectra were also recorded of cells in the micro-cuvette using the same parameters adopted for the levitation experiment.

Example 2: - Band enhancement and spectroscopic assignments

Figure 2 depicts Raman spectra recorded from Dunaliella tertiolecta, chlα and β- carotene from levitated droplets in an acoustic node generated by the 56 kHz levitation device. The non-processed spectrum shows a large sloping baseline due to fluorescence but an excellent signal-to-noise ratio. The spectrum of Dunaliella tertiolecta is dominated by enhanced bands from chlα and β-carotene with no signals evident from other macromolecular components including proteins, thus illustrating the utility of the method of the present invention for detecting the status of living algal cells.

The strong enhancement of many β-carotene bands at 785 nra, which is well away

from the major electronic transitions, has been explained by a π-electron/phonon coupling mechanism advanced by Castilioni et α/.(Castiglioni, C, Del Zoppo, M., Zerbi, G. J.Raman Spectrosc. (1993) 24485-494) and experimentally verified by Parker et al. (Parker, S.F., Tavaender, S.M., Dixon, M., Herman, H., Williams K.P., Maddams, W.F., Appl. Spectrosc. (1999) 53 86-91). Chlα enhancement is not expected through normal pre-resonant enhancement with the Q band of Chlα centred at 660nm. It is possible that the observed enhancement in chlα results from excitonic interactions in the highly ordered environment of the chloroplasts. Band assignments for Dunaliella tertiolecta are complex because of the many overlapping vibrational modes associated with the porphyrin moiety in chlorophyll. In general CC stretching vibrations from chlα appear between 1600-1500 cm-1 along with the band at 1524 cm-1 assigned to υi from C=C double bonds in β-carotene. Bands in the 1500-1400 cm^"1 spectral window arise mainly from CC stretching, CH₃ bending and in-plane CH bending from methine groups in chlα along with CH₂ and CH₃ deformation modes from β-carotene. Bands in the 1400-1300 cm-1 result primarily from CH₃ bends along with methine in-plane CH bending contributions as well as N-C stretching of chIα.(Chen, M., Zeng, H., Larkum, A.W.D., Zheng- Li, C. Spectrochim. Acta Part A (2004) 60 527-534) Bands between 1300-1200 cm^"1 arise from combinations of CH₃ deformations, in-plane CH methine vibrations and NC stretching vibrations of chlα. Peaks observed in the 1200-1000 cm^"1 are mainly due to C-O stretching vibrations of the propionate groups of chkr. The band at 1155 cm^"1 is assigned to υ₂ from CC bonds of β-carotene, while υ₃ appears at 1006 cm^"1 .(35) Bands below 1000 cm^"1 result from NCC and CCC in-plane bending vibrations, while bands below 800 cm^"1 are associated with out-of-plane CH deformations and OCO vibrations mainly from chlα.

Example 3: - Effect of laser exposure and high white light flux

In this example the effect of constant laser exposure is illustrated firstly, by irradiating the cells with 20 and 120 sec of laser light and recording spectra during this time interval and secondly, by levitating 10 μL samples of Dunaliella tertiolecta and then exposed the cells to a high flux of white light (2000 μmol quanta mfV¹), provided via the fibre optic of a Schott KL 1500 lamp (Schott AG, Mainz, Germany), for 10 min prior to recording a 20-second exposure Raman spectrum. At 2.5 min intervals 10 μL of water was added to the suspension in the node to prevent evaporation. Consequently, the size and shape of the droplet changes over time, resulting in a change in cell density and the overall signal intensity but no change in the relative intensities of the bands. Three samples exposed to high white light flux and three controls with no light exposure were measured and representative spectra presented in Figure 3. After spectral acquisition the samples were placed in an Eppendorf tube containing 1 mL of 90% acetone and centrifuged for 7 minutes at 5000 r.p.m. to extract the chlo from the cells. UV measurements of the chlα in acetone were taken before and after removal of the Mg. Calculations based on the UV measurements and spectrophotometric equations devised by Lorenzen (Lorenzen, CJ., Limnol. Oceanogr. (1967) 12 343-346) provided a relative measure of chlα concentration versus pheophytin.

Results

Figure 3 compares spectra acquired with 20 and 120 seconds of laser exposure. Spectra recorded at the longer acquisition time show a dramatic intensity decrease in bands at 1324, 1233, 1185, and 742 cm^'1 relative to the 1155 cm^"1 band from β-carotene. The 1155 cm^'1 band was chosen as an internal standard because constant laser and white light exposure results in negligible photo-damage to β-carotene. The above mentioned bands are associated with the porphyrin skeletal vibrations and indicate photo- and/or heat damage to the porphyrin moieties at the longer exposure time, however, the cells still appeared very motile and green after the long exposure. To investigate the effects of photo-bleaching spectra were recorded before and after 10 minutes of exposure to a quartz halogen lamp. Figure 4 shows average spectra from four trials recorded after 10 minutes exposure with the quartz halogen lamp along with a control sample levitated for the same time but not exposed to the lamp. Even after 10 minutes of acoustic levitation and white light exposure the cells were still motile although they were not as visibly green as the control. The spectra of the cells exposed to the halogen light exhibit a similar profile to spectra of cells after prolonged laser exposure in Figure 3. In particular bands at 1324, 1289, 1233, 1185 and 757 cm^"1 are significantly decreased in intensity compared to the control. To corroborate these results UV measurements of the chla in acetone were taken before and after the addition 10 μL of HCl. Treatment of chlα with acid degrades it to pheophytin through removal of the Mg ion. Calculations based on the UV measurements and spectrophotometric equations devised by Lorenzen (Lorenzen, C.J., Limnol. Oceanogr. (1967) 12 343-346) enable one to determine the concentration of chlα in the cell suspension. Figure 5 shows a plot of the ratio of chlα to pheophytin based on the UV measurements, which shows a 2-fold decrease in concentration of chlα compared to the control, which is consistent with the Raman spectra. Destruction of chlα molecules in photosystem II core complexes from chlorophyte microalgae exposed to photo-inhibitory fluxes of visible light has been previously described.(Bumann, D., Oesterhelt, D., Proc. Natl Acad. Sci (1995) 92 12195-12199) Formation of singlet oxygen under high light results in damage to chlα and appears to be a primary mechanism causing the inhibition of photosynthesis under these conditions.

Example 4: - Effect of nitrogen limitation

Raman spectra of nitrogen replete {N-replete) and nitrogen limited (N-limited) levitated algal cells were compared. Samples of both N-replete and N-limited cultures of Dunaliella tertiolecta were prepared as described above. 10 mL aliquots were centrifuged at 5000 r.p.m. for 5 min and supernatant removed. 10 μL samples were levitated and spectra were acquired at full laser power with an acquisition time of 20 sec. Ten spectra for both the N- replete and N-limited cells were recorded from 10 different levitated droplets and the spectra compared.

Results

Figure 6 depicts cells under N-limited conditions show a decrease in intensity of chlorophyll bands at 1324, 1233, 1185, 993 and 757 cm^"1 relative to the 1155 cm^'1 peak of β-

carotene. It is important to note that there also may be a concomitant decrease in β-carotene in

N-limited cells as evinced by the decease in U₁ at 1524 cm^"1 . Unfortunately it was not possible to use an independent internal standard in the current experiment. Nevertheless it appears that the concentration of functional chlα is significantly less for the N-limited cells compared to N- replete cells. This is supported by other work using conventional methods(Geider, R.J., Graziano, L.M., McKay, R.M.L., Eur. J. Phycol. (1998) 33 315-332), which show that N- limitation in D. tertiolecta caused a decline in total chlα per cell as well as an increase in β- carotene to chlo ratio relative to replete cultures.

Example 5: - Preliminary taxonomic identification experiments

In the final example, spectra from three species of eukaryotic algae and one species of cyanobacteria were compared. For each species 5 spectra were recorded from 5 levitated droplets. Each spectrum was acquired with 20 seconds of laser exposure with 50 mW power.

Results

The Raman acoustic levitation technique also shows potential as a tool for the taxonomic identification of algae in remote environments. Figure 7 shows spectra recorded of levitated suspensions of Chaetoceros muelleri, Phaeodactylum tricornutum, Dunaliella tertiolecta and Porphyridium purpureum. Table 2 shows the approximate cell size, shape, along with chlorophyll a and β-carotene concentrations per cell based on previous studies.(Fukiki, T., Taguchi, S., J. Plankton Res. (2002) 24 859-874; Stramski, D., Bicaud, A., Morel, A., Λpp. Optics (2001) 40 2929-2945). It is important to note that it is virtually impossible to record a spectrum of living Porphyridium purpureum. Chaetoceros muelleri, or Phaeodactylum tricornutum, with the Raman microscope attachment due to their small cytoplasmic volume. The spectrum of Porphyridium purpureum has a poor signal-to-noise ratio compared to Dunaliella tertiolecta and Chaetoceros muelleri because the concentration of both chromophores is much lower in the former. The signal-to-noise ratio of the Porphyridium purpureum is lower than that of Phaeodactylum tricornutum because the Porphyridium purpureum has a much larger cytoplasmic volume and hence fewer cells in the droplet. The Porphyridium purpureum has a pronounced peak at 1286 cm^"1 that readily distinguishes it from the other microalgae. This peak is possibly due to phycobilins, including phycoerythrin and phycocyanin, both of which have a similar chromophoric structure and are abundant in high concentrations in red algae.(Tandeau de Marsac, N., Photosynthesis Res (2003) 76 197-205) The extraction and spectroscopic analysis of these proteins is the subject of future studies. The spectra of all four species show different ratios of β-carotene and chlorophyll and also

variation in the intensity of υi and O₂ possibly because of the different chromophoric environments of the β-carotene in each of the species. The ratio of the intensities for the 1155/1525 cm^"1 bands is much greater in the green alga Dunaliella tertiolecta than in the diatoms Chaetoceros muelleri and Phaeodactylum tricornutum. The Phaeodactyliim tricornutum lacks structural detail in the 700-600 cm^"1 region, which readily distinguishes it from Chaetoceros muelleri. The results indicate that the method of the present invention has potential for the identification of algae.

Example 6:

Figures 8 to 12 are plots illustrating the statistical accuracy of the method of the present invention and thus its suitability for use in diagnosis of malaria.

Figure 8 depicts a PCl scores plot as showing individual spectra represented as points from samples of P, faciparum (P) and P.vivax (PV) from 1% parasite culture indicating the potential of the technique as a diagnostic. The plot shows clustering of the spectra of single cells from 2 different strains. Each point represents a spectrum of an individual cell in 2D (the PC's are orthogonal) where the separation results from spectral variance. Only 1% of the cells are infected showing that even a low infection % can be diagnosed for malarial strain. This shows that each individual spectrum is diagnostic rather than just the spectral averages shown in the slide above. Figures 9 to 12 show similar statistical accuracy as depicted in Figure 8, each point in the plots representing a spectrum from a single cell.

Figure 9 depicts a PCA scores plot based on the Raman spectra recorded from samples of P, falciparum (X) and P.ovale (■). The different X's in Figure 9 represent two different incubations of cells (ie. 2 separate experiments) showing the repeatability of the method of the present invention when used for diagnosis.

Figure 10 depicts a PCl scores plot showing individual spectra represented as points form samples of P. falciparum (Pf) clearly separated from P.vivax (PV) and P.ovale (PO) indicating the potential of the technique as a diagnostic. Figure 10 is similar to Figure 9, but shows all 3 strains clustering in the PC scores plots with the Pf well separated from the other 2 and a slight overlap of PO and PV. The latter indicates that there is a high rate of differentiation between PV and PO.

Figure 11 depicts a PCAl scores plot based on Raman spectra showing separation of P.falciparum and P.vivax from clinical samples. Figure 11 is similar to Figure 9 but with cells taken from a clinic, that is, the samples were not incubated to induce malaria but isolated from real patients.

Figure 12 depicts a PCA scores plot based on Raman spectra showing separation of P.falciparum and P.ovale from spectra of single cells. This plot includes the data of Figure 9, but depicts Pf against PO.

The word 'comprising' and forms of the word 'comprising' as used in this description and in the claims does not limit the invention claimed to exclude any variants or additions.

Modifications and improvements to the invention will be readily apparent to those skilled in the art. Such modifications and improvements are intended to be within the scope of this invention.

Claims

The claims defining the invention are as follows:

1. A method of measurement using enhanced Raman spectroscopy, the method including the steps of:

(a) subjecting one or more cells to excitation wavelengths between 200 and 1064 nm,

(b) recording at least one band resulting from enhanced Raman scattering, and

(c) using the band recorded from step (b) to deduce the status of the one or more cells.

2. A method according to claim 1 wherein the status is chosen from the group comprising the health, viability or identity of the one or more cells.

3. A method according to any one of the preceding claims wherein the one or more cells are chosen from the group comprising human cells, animal cells, plant cells or algal cells.

4. A method according to any one of claims 1 to 3 wherein the Raman scattering is caused by a compound in the one or more cells.

5. A method according to claim 4 wherein the compound is chosen from the group comprising porphyrins.

6. A method according to claim 5 wherein the compound is chosen from the group comprising protoporphyrins, protoporphyrinogens, coproporphyrinogens, uroporphyrinogens, coproporphyrinogens, porphobilinogens and porphyrins; quinolines; carotenes including β-carotene; fullerenes; polydiacetylenes; metalloporphyrins including haeme, myoglobin chlorophyll a, and chlorophyll b; phycobiloproteins and xanthophils; their precursors and degradation products and combinations thereof.

7. A method according to claim 4 wherein the compound is a haeme precursor or metabolite chosen from the group comprising protoporphyrins, protoporphyrinogens, coproporphyrinogens, uroporphyrinogens, porphobilinogen, haemozoin, porphyrin or bilirubin and combinations thereof.

8. A method according to any one of the preceding claims wherein the one or more cells are blood cells and the status of the one or more cells is indicative of either the haematocrit, degree of oxidation, deoxygenation or and metabolic state of the blood cells.

9. A method according to claim 8 wherein the one or more cells are red blood cells and the status of the one or more cells is indicative of cell changes caused by sickle cell anaemia, altitude sickness or parasitic infection.

10. A method according to claim 8 wherein the status of the cell is indicative of the presence of malaria trophozoites chosen from the group comprising Plasmodium malariae, Plasmodium vivax and Plasmodium falciparum in human cells.

11. A method according to any one of the preceding claims wherein the status of a single cell is measured.

12. A method according to claim 10 wherein the change in status of the one or more cells is indicative of the presence of between 1 and 10 malaria trophozoites per μl in a blood sample of 100 μl.

13. A method according to claim 12 wherein the change in status of the one or more cells is indicative of the presence of 1 malaria trophozoite per μl in a blood sample of 100 μl.

14. A method according to any one of claims 1 to 4 wherein the status of the one or more cells is indicative of the interaction of a drug with the one or more cells.

15. A method according to any one of claims 1 to 4 wherein the status of the one or more cells is predictive of the interaction of a drug with the one or more cells.

16. A method according to any one of claims 1 to 4 wherein the one or more cells are red blood cells and their status is indicative of the interaction of a drug with haeme.

17. A method according to claim 16 wherein in the drug is chosen from the group comprising chloroquine, quinoline, artmisinins, aryl-alcohols and peroxides.

18. A method according to any one of claims 1 to 4 wherein the one or more cells are plant cells and the status of the cells is indicative of cell changes caused by environmental effects.

19. A method according to any one of the preceding claims wherein the one or more cells are plant cells and the status of the cells is indicative of the production of a bioproduct by the plant from which the one or more cells were derived.

20. A method according to any one of claims 18 or 19 wherein the one or more cells are transgenic plant cells.

21. A method according to any one of claims 1 to 4 wherein the status of the one or more cells is indicative of their taxonomy.

22. A method according to claim 18 wherein the status of the one or more cells indicates that their taxonomy as being chosen from the group comprising Chaetoceros muelleri, Phaeodactylum tricornutum, Dunaliella tertiolecta and Porphyridium purpureum.

23. A method according any one of claim 1 to 4 when used as a diagnostic.

24. A method according to any one of claims 1 to 4 when used as an assay.

25. A method according to any one of claims 1 to 4 when used for taxonomic identification of the one or more cells.

26. A method according to any one of the previous claims including the step of acoustic levitation of the one or more cells.

27. A method of measurement using enhanced Raman spectroscopy, the method including the steps of:

(a) subjecting one or more red blood cells to excitation wavelengths between 200 and 1064 nm,

(b) recording at least one band resulting from enhanced Raman scattering, and

(c) using the band recorded from step (b) to deduce the status of the one or more red blood cells as indicative of malarial infection.

28. A method of measurement using enhanced Raman spectroscopy, the method including the steps of:

(a) subjecting one or more red blood cells to excitation wavelengths between 200 and 1064 nm, (b) recording at least one band resulting from enhanced Raman scattering by haemoglobin, and

(c) using the band recorded from step (b) to deduce the status of the one or more red blood cells.

29. A method of measurement using enhanced Raman spectroscopy, the method including the steps of:

(b) recording at least one band resulting from enhanced Raman scattering by haemozoin, and

(c) using the band recorded from step (b) to deduce infection of the one or more cells by a malaria parasite chosen from the group comprising Plasmodium malariae, Plasmodium vivax and Plasmodium falciparum.