WO2015114379A1

WO2015114379A1 - A low background raman probe for optical biopsy of brain tissue

Info

Publication number: WO2015114379A1
Application number: PCT/GB2015/050268
Authority: WO
Inventors: John Charles Clifford Day; Oliver Andrew Charles Stevens
Original assignee: The University Of Bristol
Priority date: 2014-01-31
Filing date: 2015-02-02
Publication date: 2015-08-06
Also published as: GB201401727D0

Abstract

A probe for providing medical guidance information comprises a hollow needle (1) defining an optical path within the needle for a collimated laser beam to illuminate a sample (2), the optical path further providing a return path for light scattered from the sample (2) due to inelastic scattering such as Raman scattering or fluorescence. The probe further comprises an optical input (3) for providing light of an input wavelength from a laser source to the optical path, an optical output (6) for outputting light from the return path for spectral analysis, and a wavelength selective beam splitter (8) which directs light at the input wavelength from the optical input (3) to the optical path and directs light from the return path at wavelengths other than the input wavelengths to the optical output (6).

Description

A LOW BACKGROUND RAMAN PROBE FOR OPTICAL BIOPSY OF BRAIN TISSUE

[0001] This invention relates to a probe for providing medical guidance information.

BACKGROUND

[0002] Removal of intrinsic brain tumours is a delicate process, where a high degree of specificity is required to remove all of the tumour tissue without damaging healthy brain. The accuracy of this process can be greatly enhanced by intraoperative guidance. Optical biopsies using Raman spectroscopy are a minimally invasive and lower cost alternative to current guidance methods.

[0003] Removal of intrinsic brain tumours, especially gliomas, is a challenging process, and requires careful consideration to selectively and completely remove tumour tissue, thus improving outcome, without damage to healthy brain [Reference 1].

[0004] Surgical guidance is currently assisted by a variety of imaging techniques, such as pre- or intra-operative magnetic resonance imaging (MR), computerised tomography (CT), intra-operative ultrasound, 5-ALA fluorescent imaging [Reference 2], and reflectance spectroscopy [Reference 3] many of which have been shown to improve the patient prognosis [References 4 & 5] by ensuring maximal resection of radiologically-identified tumour. Optical biopsies using Raman spectroscopy have been demonstrated to be a less invasive and potentially more effective alternative for traditional biopsy for a number of different applications [References 6 & 7], and potentially offer a reliable and lower cost method of identifying residual tumour intra-operatively, as well as defining apparent tumour edges. This is especially relevant to glial tumours whose edges are invasive and indistinct. In addition, an alternative to traditional biopsy is particularly important for pontine tumours due to their location in the brain stem, where any removal of tissue carries a risk of irreversible damage to the patient.

[0005] To perform optical biopsies in-vivo, small Raman spectroscopy probes are typically required. A variety of such probes for different applications have been

constructed [References 8-10].

[0006] Optical fibres are an efficient method of guiding light where physical dimensions are constrained, for this reason the majority of compact Raman probes employ fibres to carry the laser light to the sample and the Raman scattered photons back to the

spectrometer. However, these fibres impart a background Raman spectrum of silica onto the spectrum of the sample, and hence Raman background caused by silica is an issue that affects most Raman probes [Reference 11] The silica background is present at all Raman shifts, but is particularly prevalent at low wavenumbers. Whilst background subtraction can be performed after collection of the spectra, care must be used in its application to prevent artefacts that confound statistical analysis occurring on the spectra. Additionally, as the total number of background counts is increased for a given amount of Raman signal, the Shot noise is also increased, leading to a reduced signal to noise ratio. This problem can be avoided by restricting analysis to higher wavenumber regions (above 800 cm^-1) [Reference 12 & 13] away from the silica fluorescence, but this risks losing some valuable chemical information at lower wavenumbers. Brain tissue in particular contains peaks potentially useful for classification at Raman shifts of 400 cm^-1 [Reference 14], and possibly lower.

[0007] There are a number of techniques that can be used to reduce the background in a probe. Some groups have used hollow-cored waveguides to reduce the silica signal

[References 15 & 16] but as these use a metal coating to guide the light rather than total internal refection there is an associated attenuation of the signal, resulting in long (>100s) acquisition times.

[0008] Hollow-cored photonic crystal fibres have been used [Reference 17] to replace the excitation fibre, but these are expensive and difficult to work with due to small core diameters and limitations of the range of wavelengths they will accept.

BRIEF SUM MARY OF THE DISCLOSURE

[0009] In accordance with the present invention there is provided a probe for providing medical guidance information. The probe comprises a hollow needle defining an optical path within the needle for a collimated laser beam to illuminate a sample, the optical path further providing a return path for light scattered from the sample due to inelastic scattering such as Raman scattering or fluorescence. The probe further comprises an optical input for providing light of an input wavelength from a laser source to the optical path, an optical output for outputting light from the return path for spectral analysis, and a wavelength selective beam splitter which directs light at the input wavelength from the optical input to the optical path and directs light from the return path at wavelengths other than the input wavelengths to the optical output.

[0010] Embodiments of the invention provide a miniature Raman probe design that reduces the background by avoiding the use of fibres in the main section of the probe altogether.

[0011] The optical input may comprise an input optical fibre transmitting light from the laser source to the optical input. The input optical fibre may be a single mode optical fibre. The input optical fibre may have a core diameter of less than 10 microns. [0012] The probe may comprise a collimating lens provided between the optical input and the beam splitter. The collimating lens may be an aspheric lens or a gradient index (GRIN) lens, for example.

[0013] The probe may comprise a filter provided between the optical input and the beam splitter. The probe may comprise a filter, in particular a dichroic filter, provided between the beam splitter and the optical output. The beam splitter may comprise a dichroic filter. The beam splitter may allow only light of wavelengths longer than the input wavelength to pass from the return path to the optical output. The optical output may comprise an output optical fibre. The probe may comprise a focussing lens provided between the beam splitter and the optical output.

[0014] The needle may have an internal diameter of less than 5 mm, preferably less than 3mm, more preferably less than 2 mm. The needle may have a length of at least 100 mm. The needle may be formed of stainless steel.

[0015] The input wavelength may be greater than 700 nm, preferably greater than 800 nm. The input wavelength may be less than 900 nm.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 shows an assembled probe according to an embodiment of the invention pointed at an IR viewing card;

Figure 2 is a schematic diagram of the optical layout of the probe of Figure 1 ;

Figure 3 shows Raman spectra of the miniature standoff probe of Figure 1 and a comparable needle probe, demonstrating that the miniature standoff probe has

significantly lower background at low wavenumbers;

Figure 4 shows Raman spectra of porcine brain tissue (60s, 20mw, background subtracted), with the spectra offset for illustrative purposes;

Figure 5 shows principal component analysis (PCA) scores plot of porcine brain tissue, demonstrating that good visual separation is obtained between white matter and grey matter; and

Figure 6 shows PCA loadings plots for PC1 and PC2, where differences in PC scores between white and grey matter are mostly caused by fatty acid/cholesterol peaks. DETAILED DESCRIPTION

[0017] A miniature Raman probe for performing optical biopsies of human brain tissue is presented. The probe allows sampling inside a conventional stereotactic brain biopsy system: a needle of length 200mm and inner diameter of 1.8mm. The probe achieves a very low fluorescent background whilst maintaining good collection of Raman signal by employing a miniature stand-off Raman design. To illustrate this, the probe is compared with a Raman probe that uses a pair of optical fibres for collection. The miniature stand-off Raman probe is shown to collect a comparable number of Raman scattered photons, but the fluorescence caused by silica fibres in a Raman needle probe is reduced by a factor of two for Raman shifts under 500 cm^"1 , and by 30% at 600-700 cm^"1 . In addition, this design contains only medically approved materials at the distal end. The probe's suitability for use on tissue is demonstrated by discriminating between different types of porcine brain tissue.

[0018] The probe is designed to fit inside a standard stereotactic brain biopsy needle (available from AD-TECH Medical Instrument Corporation of Racine Wl, USA). A picture of the assembled probe is shown in Figure 1. The needle tip of the probe is 200mm long and 2mm in diameter.

[0019] The optical configuration of the probe is shown in Figure 2. As shown in Figure 2, the probe comprises a biopsy needle 1 directed at a sample 2. Light from an excitation fibre 3 is directed through the biopsy needle 1 by a collimating lens 4, a laser clean-up filter 5 and a first dichroic filter (beam splitter) 8. Light returning from the biopsy needle 1 passes through the first dichroic filter 8, a second dichroic filter 9 and a focussing lens 7 to a collection fibre 6. The probe employs a miniaturised version of 'standoff Raman probe, and employs no optics at the distal end; the only component at the tip is the steel biopsy needle 1 , which is already approved for medical use.

[0020] The probe operates at an excitation wavelength of 830nm to minimise sample fluorescence. It uses a single mode fibre 3 connected to an aspheric lens 4 for collimation of the excitation laser. This produces the desired 2mm beam profile at the tip of the probe. This results in a low numerical aperture of 0.005, but a total light throughput comparable to a 40μηι fibre of Numerical Aperture (NA) 0.2, due to the larger collection area of 1.8 mm. A dichroic beam splitter 8 is used to reflect the laser light down the needle 1 , but allow the longer Raman wavelengths to return towards the spectrometer. An additional long wavelength pass filter 9 further removes unwanted elastically scattered light from the Raman signal before the collected light is passed into another lens 7, which focusses light into a 100μηι diameter fibre 6. [0021] The probe is attached to a Raman Explorer spectrometer (available from Headwall Photonics, Inc. of Fitchburg MA, USA), and iDus 420BR-DD (available from Andor Technology Limted of Belfast, UK) camera controlled with Solis software (available from Andor Technology Limted of Belfast, UK).

[0022] The probe was tested using a PTFE (Polytetrafluoroethylene/Teflon) reference sample, and compared to a fibre-based needle probe. The needle probe is constructed from low-OH silica fibres of numerical aperture 0.22 and 200μηι diameter. Full details are described in Day et al [Reference 18]. Both probes were connected to the same Raman Explorer spectrometer and iDus 420BR-DD camera. Laser power at the sample was identical between probes (20mw, 830nm at the sample), and acquisition time was set to 30 seconds for both probes. Figure 3 contains a PTFE spectrum taken with the miniature standoff Raman probe, a fibre-based needle probe of the same length (20cm), and a reference spectrum acquired using a Renishaw desktop Raman system ( 5mw, 633nm, 10s integration time, 5x objective). The principle peak at 530 cm^-1 is of similar size between the two spectra, indicating similar Raman collection performance between the probes. However the background from the silica of the fibre causes significant additional background at low wavenumbers in the needle probe (double at 200 cm^-1). By contrast the miniature standoff Raman probe has a relatively flat background. The signal to noise ratio, calculated from the ratio of the height of the 530 cm^-1 peak to the standard deviation of the baseline at 1400 cm^-1 , is similar between the two probes.

[0023] To demonstrate the effectiveness of the probe on biological samples, it was used to identify different types of brain tissue. Spectra were acquired from porcine brain tissue (obtained from pigs destined for human consumption). 120 spectra were acquired of white matter, grey matter and blood vessels (identified by eye) from a pig brain. A laser excitation of 20mw at the sample was used, and an acquisition time of 60s. Figure 5 shows the result of an exploratory principal component analysis (PCA) on the dataset. Good visual separation is obtained between white and grey matter, so simple classification algorithms would easily discriminate between white and grey matter. Inspection of the loadings of the first two components (Figure 6) shows the discrimination is mostly due to the peaks associated with the presence of fatty acids, for example bands at 1298 cm^-1 and 1441 cm^-1. Of particular interest are the useful discriminatory peaks at 438 cm^-1 , 310 cm^-1 and 194 cm^-1 which are in a region where the silica background of the needle probe is very prevalent.

[0024] In summary, a probe for providing medical guidance information comprises a hollow needle 1 defining an optical path within the needle for a collimated laser beam to illuminate a sample 2, the optical path further providing a return path for light scattered from the sample 2 due to inelastic scattering such as Raman scattering or fluorescence. The probe further comprises an optical input 3 for providing light of an input wavelength from a laser source to the optical path, an optical output 6 for outputting light from the return path for spectral analysis, and a wavelength selective beam splitter 8 which directs light at the input wavelength from the optical input 3 to the optical path and directs light from the return path at wavelengths other than the input wavelengths to the optical output 6.

[0025] A miniature Raman probe without fibres in the distal portion of the probe has been demonstrated. The probe was compared to a silica fibre needle probe, and shown to have a much lower background at low wavenumbers, which allows features below 600 cm^-1 to be more readily investigated. The suitability of the probe for classification of brain tissue was also demonstrated using porcine tissue. It is anticipated that the probe will be used in future for optical biopsy of human brain tissue for identification of brain tumour tissue.

[0026] In addition to medical applications, in industrial applications the probe may be used, for example, inside remote containers or structures where contamination or corrosion are to be measured or where a chemical analysis of material, such as in chemical or pharmaceutical manufacture or nuclear waste analysis. The probe is particularly useful where the Raman measurement at low Stokes shift is necessary.

[0027] Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0028] Features, integers, characteristics, or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

REFERENCES

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[2] Stummer, W., Pichlmeier, U., Meinel, T., Wiestler, O. D., Zanella, F., and Reulen, H.-J., "Fluorescence- guided surgery with 5-aminolevulinic acid for resection of malignant glioma: a randomised controlled mul ticentre phase III trial.," The lancet oncology 7, 392-401 (May 2006).

[3] Toms, S. a., Lin, W.-C, Weil, R. J., Johnson, M. D., Jansen, E. D., and Mahadevan- Jansen, A., "Intraoperative Optical Spectroscopy Identifies Infiltrating Glioma Margins with High Sensitivity," Neurosurgery 57,382-391 (Oct. 2005).

[4] Haberland, N., Ebmeier, K., Hliscs, R., Grnewald, J. P., Silbermann, J., Steenbeck, J., Nowak, H., and Kalff, R., "Neuronavigation in surgery of intracranial and spinal tumors.," Journal of cancer research and clinical oncology 126, 529-41 (Sept. 2000).

[5] Litofsky, N. S., Bauer, A. M., Kasper, R. S., Sullivan, C. M., and Dabbous, O. H., "Image-guided resection of high-grade glioma: patient selection factors and outcome.," Neurosurgical focus 20, E16 (Jan. 2006).

[6] Ellis, D. I., Cowcher, D. P., Ashton, L, O'Hagan, S., and Goodacre, R., "Illuminating disease and enlightening biomedicine: Raman spectroscopy as a diagnostic tool.," The Analyst 138, 3871-84 (July 2013).

[7] Nijssen, A., Koljenovi^'c, S., Bakker Schut, T. C, Caspers, P. J., and Puppels, G. J., "Towards oncological application of Raman spectroscopy.," Journal of biophotonics 2, 29- 36 (Mar. 2009).

[8] Esmonde-White, K. a., Esmonde-White, F. W. L, Morris, M. D., and Roessler, B. J., "Fiber-optic Raman spectroscopy of joint tissues.," The Analyst 136, 1675-85 (Apr. 201 1).

[9] Day, J. C. C, Bennett, R., Smith, B., Kendall, C, Hutchings, J., Meaden, G. M., Born, C, Yu, S., and Stone, N., "A miniature confocal Raman probe for endoscopic use.," Phys Med Biol 54, 7077-7087 (Dec.2009). [10] Kirsch, M., Schackert, G., Salzer, R., and Krafft, C, "Raman spectroscopic imaging for in vivo detection of cerebral brain metastases.," Analytical and bioanalytical chemistry 398, 1707-13 (Oct. 2010).

[1 1] Ma, J. and Li, Y. S., "Fiber Raman background study and its application in setting up optical fiber Raman probes.," Applied optics 35, 2527-33 (May 1996).

[12] Koljenovi^'c, S., Schut, T. C. B., Wolthuis, R., Vincent, a. J. P. E., Hendriks-Hagevi, G., Santos, L, Kros, J. M., and Puppels, G. J., "Raman spectroscopic characterization of porcine brain tissue using a single fiber-optic probe.," Analytical chemistry 79, 557-64 (Jan. 2007).

[13] Santos, L. F., Wolthuis, R., Koljenovi^'c, S., Almeida, R. M., and Puppels, G. J., "Fiberoptic probes for in vivo Raman spectroscopy in the high-wavenumber region.," Analytical chemistry 77, 6747-52 (Oct. 2005).

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spectroscopy 61 , 1529-35 (May 2005).

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Claims

1. A probe for providing medical guidance information, the probe comprising:

a hollow needle defining an optical path within the needle for a collimated laser beam to illuminate a sample, the optical path further providing a return path for light scattered from the sample due to inelastic scattering such as Raman scattering or fluorescence;

an optical input for providing light of an input wavelength from a laser source to the optical path;

an optical output for outputting light from the return path for spectral analysis; a wavelength selective beam splitter which directs light at the input wavelength from the optical input to the optical path and directs light from the return path at wavelengths other than the input wavelengths to the optical output.

2. A probe as claimed in claim 1 , wherein the optical input comprises an input optical fibre transmitting light from the laser source to the optical input.

3. A probe as claimed in claim 2, wherein the input optical fibre is a single mode optical fibre.

4. A probe as claimed in claim 2 or 3, wherein the input optical fibre has a core diameter of less than 10 microns.

5. A probe as claimed in any preceding claim comprising a collimating lens provided between the optical input and the beam splitter.

6. A probe as claimed in claim 5, wherein the collimating lens is an aspheric lens or a gradient index (GRIN) lens.

7. A probe as claimed in any preceding claim comprising a filter provided between the optical input and the beam splitter.

8. A probe as claimed in any preceding claim comprising a filter, in particular a dichroic filter, provided between the beam splitter and the optical output.

9. A probe as claimed in any preceding claim, wherein the beam splitter comprises a dichroic filter.

10. A probe as claimed in any preceding claim, wherein the beam splitter allows only light of wavelengths longer than the input wavelength to pass from the return path to the optical output.

11. A probe as claimed in any preceding claim, wherein the optical output comprises an output optical fibre.

12. A probe as claimed in any preceding claim comprising a focussing lens provided between the beam splitter and the optical output.

13. A probe as claimed in any preceding claim, wherein the needle has an internal diameter of less than 5 mm, preferably less than 3mm, more preferably less than 2 mm.

14. A probe as claimed in any preceding claim, wherein the needle has a length of at least 100 mm.

15. A probe as claimed in any preceding claim, wherein the needle is formed of stainless steel.

16. A probe as claimed in any preceding claim, wherein the input wavelength is greater than 700 nm, preferably greater than 800 nm.

17. A probe as claimed in any preceding claim, wherein the input wavelength is less than 900 nm.