METHOD FOR CALIBRATING SPECTROSCOPY APPARATUS AND EQUIPMENT FOR USE IN THE METHOD

Abstract

This invention concerns a method of calibrating spectroscopy apparatus including illuminating a reference sample, identifying spectrum from light emitted from the sample and calibrating the spectroscopy apparatus based upon the spectrum, wherein the reference sample has been dried. The invention also concerns a reference sample for use in this method and a kit including such a reference sample. The reference sample may be lyophilised dye labelled oligonucleotides.

Description

FIELD OF INVENTION

This invention concerns a method for calibrating spectroscopy apparatus and equipment for use in the method. The invention has particular, but not exclusive, application to the calibration of Raman spectroscopy apparatus to be used in the identification of dye labelled nucleic acid sequences in a sample.

INTRODUCTION

It is known to use Raman spectroscopy to identify a molecule in situ in a sample. However, Raman spectroscopy used in its basic form often lacks the sensitivity to identify molecules, particularly when attempting to detect multiple analytes simultaneously in a single interrogation. To enhance the Raman signal, surface enhanced resonance Raman scattering (SERRS) may be used. SERRS uses the principal that the molecule to be identified is absorbed on an active surface and comprises a chromophore having an electronic transition in the region of the laser wavelength used to excite the Plasmon on the enhancing surface.

For a biological sample, to provide a sufficiently distinct chromophore for each type of molecule to be identified, the sample may be treated to attach different dyes to each type of molecule to be identified (eg different types of oligonucleotides). Examples of such techniques are described in WO09/022,125 and US2006246460, which are incorporated herein by reference. In order to accurately detect the Raman signal produced by the dye labelled oligonucleotide it may be necessary to calibrate the Raman spectroscopy apparatus to take into account factors specific to that apparatus.

One method for calibrating the system is for the user to apply the dyes to plates, independent from the sample, and carry out Raman spectroscopy of these plates to identify the Raman spectra that occur for those dyes. Knowledge of the spectra can then be used when analysing the processed sample to determine if any of the dyes are present. A problem with this technique is that the user may make errors when applying the dyes to the plates and the technique is time consuming. This is particularly the case for SERRS where the exact method of applying the dye to the surface is crucial.

US2010/0291599 discloses a technique for automatically calibrating Raman spectroscopy apparatus using reference samples built into the apparatus. A problem with such a technique is that it assumes the response of the reference sample remains unchanged over time. This problem is acknowledged in U.S. Pat. No. 5,850,623, which attempts to overcome this problem through the use of a complex correction algorithm established through the irradiation of a plurality of reference samples.

WO2006/134376 is directed towards a similar problem. This document discloses a method for identifying the presence of probe/target complex molecules using Raman spectroscopy without the use of labels. A problem with the technique is that exposure of the SERS chip to sample solution may result in a change in an efficiency of the chip. To calibrate for this change, the chip comprises probe regions for receiving the sample interspersed with calibration regions, which contain calibration molecules. These calibration regions can be used for calculating a normalization factor to calibrate the instrument for changes in overall SERS efficiency during application of the sample.

SUMMARY OF INVENTION

According to a first aspect of the invention there is provided a method of calibrating spectroscopy apparatus comprising illuminating a reference sample, identifying a spectrum from light emitted from the sample and calibrating the spectroscopy apparatus based upon the spectrum, characterised in that the reference sample has been dried.

It is believed that drying of a reference sample, and, optionally, subsequent rehydration, does not significantly change the characteristic spectra obtained from the reference sample. Accordingly, the spectrum obtained from such a reference sample can be used for calibrating the spectroscopy apparatus for identifying substances corresponding to that of the reference sample. Drying of the reference sample lengthens the time over which characteristic spectrum can be obtained from the reference sample allowing storage of the reference sample before use in calibrating spectroscopy apparatus. Furthermore, the reference sample can be prepared in a controlled environment and then delivered to the location of the spectroscopy apparatus for calibration of the apparatus. This may ensure consistency and reduce the likelihood of human error.

In a preferred arrangement, the dried reference sample has been lyophilised on a substrate. A reference sample that has been dried in this way may retain the critical structures that produce the characteristic spectrum on which a calibration can be based. Alternatively, the reference sample may have been dried by another method, for example, air dried or, for samples that can withstand high temperatures and/or pressures, supercritical drying.

The reference sample may be an organic reference sample. In one embodiment, the reference sample comprises dye labelled oligonucleotides. The dried reference sample may be treated with one or more reagents before spectra are obtained. The reagents may be the same reagents used to treat samples of unknown elements. The reagents may be used in substantially the same relative quantities as that used to treat the samples of unknown elements. In this way, the conditions under which spectroscopy is carried out is consistent for both the reference sample and the sample(s) of unknown elements. Alternatively, additional reagents may be used or there may be a difference in the relative quantities of the reagents from that used to treat the samples of unknown elements, for example, to compensate for the fact that the reference sample is or has been dried. For example, a greater proportion of water may be used with the reference sample to take account of the relatively high water retention of the reference sample. The reagents used may be one or more selected from water, spermine and silver or gold colloid.

The light emitted by the reference sample may be scattered light, for example the method may comprise identifying from the scattered light a Raman spectrum for calibrating Raman spectroscopy apparatus. The invention has particular application to Surface Enhanced Resonance Raman spectroscopy (SERRS). However, the method may be used for other forms of spectroscopy, such as fluorescence spectroscopy.

The method may be carried out automatically by the spectroscopy apparatus. For example, the dried reference sample may be stored within the apparatus, the apparatus arranged to direct illumination laser light at the sample periodically for calibrating the apparatus. The apparatus may comprise multiple reference samples, each sample comprising a different label, such as a dye. For example, different dyes may be arranged to attach to different target molecules such that the presence of a particular dye corresponds to the presence of a particular target molecule in the sample. The apparatus may have to be calibrated for each dye that is used.

Calibrating the spectroscopy apparatus may comprise updating a library of component reference spectra with the spectrum of the reference sample. Such a library of component reference spectra may be used in direct classical least squares (DCLS) analysis of a spectrum from an unknown sample, such as the modified DCLS method described in European patent application 11250530.0. Accordingly, it will be understood that “calibrating the spectroscopy apparatus” as used herein is intended to include updating/adjusting reference spectra used to analyse a spectrum in order to take into account factors that may have changed since the previous reference spectra fewer obtained.

Accordingly, a second aspect of the invention provides a method of calibrating spectroscopy apparatus comprising illuminating a reference sample, determining a spectrum characteristic of the reference sample from light emitted from the sample and updating a library of component reference spectra with the spectrum characteristic of the reference sample.

Such a method may be carried out periodically and/or immediately before using the spectroscopy apparatus to determine components present in an unknown sample.

According to a third aspect of the invention there is provided a reference sample for use in calibrating spectroscopy apparatus, the reference sample comprising lyophilised dye labelled oligonucleotides.

Lyophilising the dye labelled oligonucleotide preserves the dye labelled oligonucleotides such that properties of the material remain substantially unchanged between formation of the reference sample and calibration of spectroscopy apparatus.

The reference sample may comprise a substrate on to which the dye labelled oligonucleotides are lyophilised. The reference sample may comprise a plurality of different dye types lyophilised to the substrate. The substrate may comprise an array of wells, each well comprising a different dye. The reference sample may or may not require processing before use in calibrating spectroscopy apparatus, for example reagents may be applied to the reference sample.

According to a fourth aspect of the invention there is provided a kit for calibrating spectroscopy apparatus comprising labels for attaching to target molecules that are potentially present in an organic sample and a dried reference sample comprising the labels and organic matter.

According to a fifth aspect of the invention there is provided a method of calibrating Raman spectroscopy apparatus comprising illuminating a reference sample and identifying a Raman spectrum from light scattered from the sample that is characteristic of the reference sample, characterised in that the reference sample has been stabilized without significantly altering the Raman spectrum that is obtained from that which would have been obtained from the reference sample before the reference sample was stabilized.

It will be understood that the term “the reference sample has been stabilized” means that the reference sample has been placed in a state from which it does not significantly change for a longer period, such as weeks, months or years longer, than would have been the case if the reference sample had not been stabilized.

The Raman spectrum identified as characteristic of the reference sample may be used as a reference for use in a method for determining components/elements present in a sample. For example, the identified Raman spectrum may be used in a Direct Least Squares method (DCLS) for identifying components present in a sample, such as the modified DCLS method described in European patent application 11250530.0.

BRIEF DESCRIPTION OF DRAWINGS

Embodiments of the invention will now be described, by example only, with reference to the accompanying drawings, in which:—

FIG. 1 is a flowchart showing a method according to one embodiment of the invention;

FIG. 2 shows Raman spectroscopy apparatus according to the invention;

FIG. 3 is a cross-section of a reference plate according to one embodiment of the invention;

FIG. 4 is a graph showing the Raman spectrum of a TET control plate and TET lyophilised plate according to the invention that has been stored for 1 week;

FIG. 5 is a graph showing average SERRS intensity for specified Raman peaks of different dyes of air dried reference plates stored for 1, 2, 3, 4 and 5 weeks at 4° C.;

FIG. 6 is a graph showing average SERRS intensity for specified Raman peaks of different dyes of air dried reference plates stored for 1, 2, 3, 4 and 5 weeks at −20 C;

FIG. 7 is a graph showing average SERRS intensity for specified Raman peaks of different dyes of lyophilised reference plates stored for 1, 2, 3, 4 and 5 weeks at 4° C.; and

FIG. 8 is a graph showing average SERRS intensity for specified Raman peaks of different dyes of lyophilised reference plates stored for 1, 2, 3, 4 and 5 weeks at −20° C.;

DESCRIPTION OF EMBODIMENTS OF THE INVENTION

Referring to the drawings, a method of calibrating spectroscopy apparatus comprises forming a reference sample by lyophilising 101 dye labelled oligonucleotides 301 on to a substrate, in this case a micro plate 302. In this embodiment, the plate comprises an array of wells 303, with each well containing a different dye corresponding to the set of dyes to be used in analysing a sample. The set of dyes may be in accordance with those set out in European patent application 12163369.7. The dye labelled oligonucleotides may be lyophilised to the substrate in a controlled environment to reduce the risk of contamination of the reference sample.

Once the reference sample has been prepared, the reference sample is delivered 102 to a site of the spectroscopy apparatus 201 to be calibrated. The reference sample may be delivered as part of a kit of parts for carrying out medical diagnostics. For example, the kit may comprise the reference plate 302 and a set of dyes corresponding to the plurality of reference samples for labelling oligonucleotides in a sample to be analysed.

To calibrate a spectroscopy apparatus 201, the reference plate 302 is treated with one or more reagents corresponding to those to be used to treat unknown samples to be analysed. In this embodiment, the reagents are water, spermine and silver colloid. The treated reference plate 302 is then placed on a table (not shown) of the spectroscopy apparatus and illuminated with a laser 202. Light scattered by the reference sample is detected.

A Raman spectroscopy apparatus will typically comprise a dichroic filter 212 placed at 45 degrees to the optical path to reflect the laser beam 202 towards the sample 206. The laser beam 201 then passes though an objective lens 204, which focuses it to a spot or line at a focal point on the sample and, optionally, a mirror 208 for redirecting the beam towards the sample. Light is scattered by the sample, collected by objective lens 204 and collimated into a parallel beam, which passes back through dichroic filter 212. The filter 212 rejects Raleigh scattered light having the same wavelength as the laser beam and transmits Raman scattered light of a different wavelength. The Raman scattered light then passes to Raman analyser 220.

The Raman analyser 220 comprises a dispersive element, such as a diffraction grating, that disperses the scattered light into a spectrum, which is focussed by lens 222 onto a suitable photo-detector. In this embodiment, the photo-detector is a charge-coupled device (CCD) 224. The photo-detector is connected to a computer 225, which acquires data from the photo-detector 224 and analyses the data as required.

When calibrating the apparatus, computer 225 may identify from light scattered from the reference sample a Raman spectrum characteristic of the dye used to label the oligonucleotides. The spectrum may be specific to that spectroscopy apparatus as a result of shifts in the spectrum resulting from changes in specific aspects of the apparatus and the local environment at that time, such as the ambient temperature. The identified Raman spectrum is stored in data storage 229, for example as part of a library, for later use as reference component spectra in a DCLS analysis of an unknown sample such as described in European patent application 11250530.0.

Calibration of the spectroscopy apparatus may be carried out periodically or each time an unknown sample is to be analysed.

Example 1

A dye phosphoramidite (trade name TET) was lyophilised on to a plate and stored in a fridge at 4° C. for 1 week. The plate was then treated with reagents, water, spermine and a colloid, and a Raman spectrum obtained. A Raman spectrum of a control plate of TET was also obtained. The two Raman spectra are illustrated in FIG. 4. The Raman spectrum of the control plate is shown as a solid black line and the Raman spectrum of the lyophilised plate is shown as a dotted line. As can be seen, the Raman spectrum of the lyophilised plate substantially matches that of the control plate.

Example 2

Method

Plates were prepared with 5 repetitions of each dye and blanks. The dyes were added to each plate in accordance with the plate set-up shown in the table below:

1

2

3

4

5

6

7

8

9

10

11

12

A

TET

TAM

R.G.

Cy3

HEX

MAX

TYE

488

565

Blank

B

TET

TAM

R.G.

Cy3.5

HEX

MAX

TYE

488

549

Blank

C

TET

JOE

R.G.

Cy3.5

HEX

MAX

520

488

549

Blank

D

TET

JOE

R.G.

Cy3.5

FAM

MAX

520

488

549

E

TET

JOE

Cy3

Cy3.5

FAM

MAX

520

565

549

F

TAM

JOE

Cy3

Cy3.5

FAM

TYE

520

565

549

G

TAM

JOE

Cy3

HEX

FAM

TYE

520

565

Blank

H

TAM

R.G.

Cy3

HEX

FAM

TYE

488

565

Blank

The dye labelled oligonucleotides were diluted in concentrations shown in the table below from a 1×10⁻⁶ M stock solution in 0.15% polysorbate 20 (Tween-20). A 2.5 μL aliquot of dye labelled oligonucleotides was added to each well of the microplate and allowed to dry (either through air drying or using a Lyophiliser).

Dye List Detailing Concentration Used

Dye       Concentration (M)
TET       2 × 10−8
TAMRA     2 × 10−8
JOE       2 × 10−8
Rho Green 2 × 10−8
Cy3       1 × 10−8
Cy3.5     2 × 10−8
HEX       2 × 10−8
FAM       4 × 10−8
BHQ2      5 × 10−7
MAX       2 × 10−8
TYE       2 × 10−8
TEX       1.5 × 10−7
ATTO520   4 × 10−8
ATTO488   1 × 10−8
ATT0565   2 × 10−8
DY549     1.5 × 10−8

A Lyophiliser was switched on one hour prior to use to allow the temperature and pressure to equilibrate. Once the oligonucleotides were added the plate, the plate was frozen for 30 minutes and then transferred to the Lyophiliser. The plate remained in the Lyophiliser for 3 hours and then removed and stored in one of three storage conditions, namely room temperature, 4° C. and −20° C.

Analysis of the plate with the Raman spectroscopy apparatus at 2.5% laser power for 1 second with a single accumulation.

Part 1

The plates were stored at room temperature, 4° C. and −20° C., sealed using a sealing plate, chillout wax or no seal and stored in upright or inverted positions. One plate was prepared according to each condition (therefore, 18 for each drying process) in addition to a control plate. The control plate was prepared immediately prior to analysis. Therefore, in total 37 plates were analysed.

Part 2

The plates were stored at 4° C. and −20° C., sealed using a sealing plate and stored in an upright position. Three plates were prepared according to each condition plus three control plates. Therefore, in total 15 plates were analysed.

Part 3

The conditions set out in Part 2 were repeated but using only 2 plates for each condition and 2 control plates. The plates were prepared for weekly analysis at 1, 2, 3 and 4 weeks.

SERRS Analysis

The following peaks were chosen for each dye, their SERRS intensity recorded and relative standard deviations calculated:

TET       1636 cm−1
TAMRA     1653 cm−1
JOE       1503 cm−1
Rho Green 1368 cm−1
Cy3       1589 cm−1
Cy3.5     1520 cm−1
HEX       1503 cm−1
FAM       1542 cm−1
MAX       1512 cm−1
TYE       1589 cm−1
520       1359 cm−1
488       1645 cm−1
565       1653 cm−1
549       1394 cm−1

Results

Part 1

Before the peaks where chosen, the spectra from each plate were examined and general observations recorded. The details of the air dried and Lyophiliser plates of part 1 are shown in the two tables below.

Observations from Air Dried Plates

Plate         Dyes giving poor signal
RT P U        JOE, Cy3, Cy3.5, MAX, TYE, 549
RT P I        Cy3, Cy3.5, TYE
RT C U        Cy3, Cy3.5, MAX, TYE, 549
RT C I        Cy3, Cy3.5, TYE
RT N U        Cy3, Cy3.5, TYE
RT N I        Cy3, Cy3.5, TYE
4 P U         Cy3, Cy3.5, TYE
4 P I         Good all round performing
4 C U         Cy3, Cy3.5, MAX, TYE
4 C I         Cy3, Cy3.5, TYE
4 N U         Cy3, Cy3.5, TYE
4 N I         Cy3, Cy3.5, TYE
20 P U        Good all round performing
20 P I        Cy3, Cy3.5, TYE
20 C U        Good all round performing
20 C I        Cy3, Cy3.5, TYE
20 N U        Good all round performing
20 N I        Cy3, Cy3.5, TYE
Control Plate Cy3, Cy3.5, TYE (signals a bit low)

Observations of Lyophiliser Plates

Plate Dyes giving poor signal
RTPU  Similar to control plate
RTPI  Similar to control plate
RTCU  MAX
RTCI  Similar to control plate
RTNU  Similar to control plate
RTNI  MAX
4PU   Best so far
4PI   Best so far
4CU   Similar to control plate
4CI   Similar to control plate
4NU   Similar to control plate
4NI   Similar to control plate
20PU  Similar to control plate
20PI  Similar to control plate
20CU  MAX
20CI  TYE
20NU  Similar to control plate
20NI  Similar to control plate

The abbreviations for the plates refer to the conditions in which the plates were stored and are as follow:

RT=Room temperatureP=Plate seal

U=Upright

I=Inverted

C=Chillout wax

N=No seal

4=4° C.

20=−20° C.

Observations indicate that the lyopholiser plates gave better quality spectra in that the spectra were most similar to the spectra from the control plate.

For part 1 plates, an approximate peak height was estimated using the highest value on the axis of SERRS intensity for each spectrum, which roughly corresponded to the peak intensity of the most intense peak of the spectrum. Results for both the air dried and lyophiliser plates are summarised in the tables below:

Summary of peak intensity data form air dried plates
	TET	TAMRA	JOE	Rho Green	Cy3	Cy3.5	HEX	FAM	MAX	TYE	520	488	565	549
RT P U	12500	15000	4800	5000	3500	no peaks	7000	5000	2500	2000	11000	6000	7500	8000
RT P I	13000	17500	6500	7500	4000	3000	8500	5500	3500	2300	21000	12500	7500	9000
RT C U	8000	12500	4500	5000	3500	no peaks	7000	5500	3000	2500	12000	10000	7500	6000
RT C I	7500	15000	6500	6000	3500	3000	9000	4500	2500	2000	15000	10000	8000	11000
RT N U	11000	16000	5500	7000	4000	3000	8000	6500	4000	3000	14000	10000	8000	9000
RT N I	9500	18000	4500	6000	3500	no peaks	9000	6000	3000	2500	16000	12000	8000	9000
4 P U	12000	17000	5500	8000	4500	3500	10000	6500	2500	2500	13000	13000	8000	9000
4 P I	14000	18000	7000	8000	4500	3500	10000	6000	2500	2000	13000	14000	8000	9000
4 C U	8000	13000	5000	6500	3500	no peaks	9000	5500	1800	1500	12000	9000	7000	9000
4 C I	9000	13000	5500	7000	3500	3000	10000	6000	2500	2500	13000	13000	7500	8000
4 N U	11000	13000	5000	7000	3500	3000	8000	5500	2500	2000	12000	11000	8000	8500
4 N I	10000	13000	5500	8000	3500	3000	9000	5500	2000	2000	11000	14000	7500	10000
20 P U	10000	14000	5500	7500	4000	3500	8000	6000	2500	2000	12000	12000	8000	10000
20 P I	14000	16000	7000	9000	4500	3500	11000	6000	2500	2000	14000	15000	9000	11000
20 C U	8000	14000	6000	6000	4000	3000	10000	5500	2000	2000	14000	13000	8000	10000
20 C I	7000	13000	5500	7000	3500	3000	9000	4000	3000	2500	13000	12000	7000	10000
20 N U	14000	15000	6000	9000	4500	4000	11000	6000	2500	2000	13000	15000	9000	11000
20 N I	15000	15000	7000	10000	4000	3500	11000	6000	2500	2000	13000	17000	9000	10000
Control	16000	16000	6000	8000	6000	3500	12000	7000	3000	3500	14000	13000	10000	11000

Summary of peak intensity data form lyophilised plates
	TET	TAMRA	JOE	Rho Green	Cy3	Cy3.5	HEX	FAM	MAX	TYE	520	488	565	549
RT P U	16000	16500	9000	8500	6500	3500	10000	8500	3000	3000	13500	15000	9500	11500
RT P I	13000	14500	8000	9500	6500	3500	9500	6500	2500	2500	12000	11500	8000	10000
RT C U	9000	13500	5000	6500	4500	3000	8000	5500	3000	2000	12500	11500	8000	9500
RT C I	10500	12000	5500	6000	4000	3000	7500	5000	2500	2500	10500	11000	7500	10500
RT N U	15000	16500	7500	7000	6500	4000	10000	7500	3500	3000	11500	13000	8000	9500
RT N I	9500	14500	6000	8500	5000	3000	8500	6500	2000	2500	12500	11000	8500	8500
4 P U	12500	17500	9500	9500	5500	3500	9000	6500	3000	3500	10000	13000	8500	10000
4 P I	15000	15500	8500	10500	7000	4500	12000	7500	2500	3000	12000	14500	8500	9000
4 C U	11500	13000	6000	6500	4500	3000	8500	5500	2500	2500	12000	12000	8500	9500
4 C I	12500	14500	6500	7500	4500	3500	10000	6000	2000	2500	11500	12500	7500	9000
4 N U	14500	15000	8500	9500	5500	4000	10000	7000	2500	3000	11000	13000	9000	10000
4 N I	15000	17000	9500	10000	6000	4500	8500	7000	2500	2500	11500	15000	9000	11000
20 P U	14500	15000	9500	10500	7000	4000	10500	7000	2500	2500	10500	14000	9000	10000
20 P I	16500	16000	8500	10500	7000	4500	10500	7500	2500	2500	12000	16000	10000	11000
20 C U	12000	13500	7000	7000	4000	3000	8000	5500	2000	2000	13000	10000	8000	9000
20 C I	9500	12000	5500	8000	5000	3500	10000	5000	2000	1500	10000	10500	7000	88500
20 N U	13500	13500	9000	9000	6000	4000	9500	6500	3000	2500	12500	12500	8500	9500
20 N I	14500	15500	9500	10500	5500	4000	10500	7000	2500	2500	10000	14000	8500	7500

From these results it would appear that:

• • 1) Chillout wax causes a reduction in signal intensity
  • 2) Storage of plates in an inverted position reduces the SERRS signal
  • 3) Plates dried in the lyophiliser produced better quality, more reproducible spectra than air dried plates
  • 4) Storing the plates at room temperature generally results in lower peak intensity than storing the plates at 4° C. or −20° C.

Part 2

Characteristic SERRS peaks (as detailed above) were chosen for each dye and the average peak intensity determined

Summary of Part 2 results with average peak intensity (within the reps
of each plate) and average between plates also detailed (in bold).
	Average	TET	TAMRA	JOE	Rho Green	Cy3	Cy3.5	HEX	FAM
Air dried	4PU-1	7602.204	13656.4	5367.28	5689.13	4069.686	3532.672	8972.968	5647.212
	4PU-2	8269.198	12910.87	5519.446	6072.132	4335.976	3668.158	10327.35	5972.175
	4PU-3	7935.701	13283.63	5443.363	5880.631	4202.831	3600.415	9650.16	5809.694
Average		7935.701	13283.63	5443.363	5880.631	4202.831	3600.415	9650.16	5809.694
Lyophiliser	4PU-1	10280.06	11622.45	8513.096	11100.598	5170.28	4117.07	10677.44	7895.872
	4PU-2	10021.37	12313.34	7608.642	11043.038	4979.256	3947.116	10023.45	8154.688
	4PU-3	10042.87	13441.49	8902.106	11487.752	5208.724	3986.654	11330.02	8288.95
Average		10114.77	12459.09	8341.281	11210.463	5119.42	4016.947	10676.97	8113.17
Air dried	20PU-1	6743.778	10768.64	5381.69	6336.36	3725.472	3606.952	8982.48	6696.884
	20PU-2	9010.607	12453.18	7090.506	8991.3532	4658.402	3839.367	10141.53	7252.707
	20PU-3	7451.09	11038.19	6298.536	7465.148	4042.498	3747.566	10181.96	6794.104
Average		7735.158	11420	6256.911	7597.6204	4142.124	3731.295	9768.654	6914.565
Lyophiliser	20PU-1	8897.415	12078.99	7270.45	9422.3523	4622.295	3857.434	10222.73	7550.084
	20PU-2	10893.08	14701.84	8218.378	11766.692	5284.984	4212.834	10564.45	8006.378
	20PU-3	10793.05	12626.34	7625.76	11694.65	5431.642	4228.006	12630.65	8655.612
Average		10194.52	13135.72	7704.868	10961.231	5112.974	4099.425	11139.28	8070.691
Control	Plate 1	7351.395	8572.52	5339.605	3218.96	1819.262	1819.408	5729.816	6593.11
	Plate 2	6521.778	6062.102	2633.506	3262.4975	2120.094	2000.392	5769.102	7848.636
	Plate 3	6493.584	6478.616	4045.146	4121.466	2039.988	2202.92	6853.936	8819.316
Average		6788.919	7071.079	4006.086	3534.3078	1993.115	2007.573	6117.618	7753.687
		Average	MAX	TYE	520	488	565	549
	Air dried	4PU-1	3615.484	4264.922	9570.596	8846.684	6780.468	9296.826
		4PU-2	2573.31	3684.112	9410.023	9880.72	6452.566	8517.548
		4PU-3	3094.397	3974.517	6500.206	6405.135	4599.345	6121.125
	Average		3094.397	3974.517	8493.608	8377.513	5944.125	7978.5
	Lyophiliser	4PU-1	2809.43	3429.162	9983.91	12686.6	6387.426	8005.095
		4PU-2	1826.06	2210.076	12898.29	10127.77	6188.326	7839.582
		4PU-3	1760.768	2 4.888	12951.97	12298.45	6593.75	8367.076
	Average		2132.083	2768.042	11944.72	11704.27	6389.834	8070.584
	Air dried	20PU-1	2974.01	3670.04	9368.178	8206.902	857.158	8595.326
		20PU-2	2527.305	3241.606	10305.84	9972.375	5994.281	7853.898
		20PU-3	2896.11	2990.765	11248.39	9679.35	6478.794	9239.298
	Average		2799.142	3300.804	10307.47	9286.209	6110.078	8562.841
	Lyophiliser	20PU-1	2352.721	2924.236	11452.9	10331.52	6250.357	8327.627
		20PU-2	1337.215	2414.022	12069.29	11268.51	6834.14	8103.678
		20PU-3	1481.98	1955.992	12274.71	12003.37	6821.59	8451.478
	Average		1723.972	2431.417	11932.3	11201.13	6335.362	8294.261
	Control	Plate 1	1215.173	1665.618	12569.31	3596.788	3161.942	5681.596
		Plate 2	#DIV/O1	1220.174	9524.656	2886.7	2825.132	4516.338
		Plate 3	#DIV/O1	1502.814	11015.11	3636.12	2995.592	4337.126
	Average		#DIV/O1	1462.869	11036.36	3373.203	2994.222	4845.02
indicates data missing or illegible when filed

From these results it would appear that storage of the plates at the different temperatures has no significant effect on the SERRS intensity. The drying process, however does appear to affect SERRS intensity. For both drying techniques, the Raman peaks are present but lyophiliser drying appears to increase the SERRS intensity (with the exception of MAX and TYE) compared to air drying. For TAMRA and DY549 the SERRS counts were approximately the same, whilst for the remaining dyes the counts were significantly higher.

Part 3

All dyes with the exception of MAX survived the full 4 weeks. MAX signals disappeared after 1 week and TYE also produced poor quality spectra.

For all dyes, the average SERRS intensity was calculated and the results are shown in FIGS. 5 to 8 from week to week. FIGS. 5 and 6 are for air dried plates and FIGS. 7 and 8 are for lyophilised plates. In general, there is a fall in SERRS intensity between week 0 and week 4 but peaks are still sufficiently distinct in week 4.

Accordingly, from the above, it can be determined that the Raman peaks were reproducible after drying of the plates and after storage for 4 weeks. Therefore, the air dried or lyophilised dye labelled oligonucleotides can be used to create component reference spectra for calibrating Raman spectroscopy apparatus.

It will be understood that the invention is not limited to the above described embodiments and modifications and alterations can be made without departing from the scope of the invention as defined in the claims. For example, it is anticipated that other dyes may be used. Furthermore, SERRS may be carried out on naturally occurring biological matter comprising a chromophore and therefore, lyophilised or air dried reference plates may be formed from such matter without the need to attach a dye.

Claims

1. A method of calibrating spectroscopy apparatus comprising illuminating a reference sample, identifying a spectrum from light emitted from the sample and calibrating the spectroscopy apparatus based upon the spectrum, characterised in that the reference sample has been dried.

2. A method according to claim 1, comprising rehydrating the dried reference sample before illuminating the reference sample.

3. A method according claim 1, wherein the dried reference sample has been lyophilised on a substrate.

4. A method according to claim 1, wherein the reference sample is an organic reference sample.

5. A method according to claim 4, wherein the reference sample comprises dye labelled oligonucleotides.

6. A method according to claim 1, wherein the dried reference sample is treated with one or more reagents before illuminating the reference sample.

7. A method according to claim 6, wherein the reagents are the same reagents used to treat samples of unknown elements.

8. A method according to claim 6, wherein the reagents are used in substantially the same relative quantities as that used to treat the samples of unknown elements.

9. A method according to claim 6, wherein the reagents used are one or more selected from water, spermine and silver colloid.

10. A method according to claim 1, comprising identifying from scattered light a Raman spectrum for calibrating Raman spectroscopy apparatus.

11. A method according to claim 1, wherein the method steps are carried out automatically by the spectroscopy apparatus.

12. A method of calibrating spectroscopy apparatus comprising illuminating a reference sample, identifying a spectrum characteristic of the reference sample from light emitted from the sample and updating a library of component reference spectra with the spectrum characteristic of the reference sample.

13. A reference sample for use in calibrating spectroscopy apparatus, the reference sample comprising lyophilised dye labelled oligonucleotides and, optionally, comprises a substrate on to which the dye labelled oligonucleotides are lyophilised.

14. A kit for calibrating spectroscopy apparatus comprising labels for attaching to target molecules that are potentially present in an organic sample and a dried reference sample comprising the labels and organic matter.

15. A method of calibrating Raman spectroscopy apparatus comprising illuminating a reference sample, determining a Raman spectrum from light scattered from the sample and calibrating the Raman spectroscopy apparatus based upon the Raman spectrum, characterised in that the reference sample has been stabilized without significantly altering the Raman spectrum that is obtained from that which would have been obtained from the reference sample before the reference sample was stabilized.