NIR Raman spectroscopy in medicine and biology: results and aspects

Journal of Molecular Structure 480–481 (1999) 21–32

NIR Raman spectroscopy in medicine and biology: results and aspects B. Schrader a,*, B. Dippel a, I. Erb a, S. Keller a, T. Lo¨chte a, H. Schulz b, E. Tatsch a, S. Wessel a a

Institut fu¨r Physikalische und Theoretische Chemie, Universita¨t Essen, D-45117 Essen, Germany b Bundesanstalt fu¨r Zu¨chtungsforschung an Kulturpflanzen, D-06484 Quedlinburg, Germany Received 25 August 1998; accepted 30 September 1998

Abstract Analyses of biomaterial by ‘classical’ Raman spectroscopy with excitation in the visible range has not been possible since the fluorescence of many essential constituents of all animal and plant cells and tissues overlays the Raman spectra completely. Fluorescence, however, is virtually avoided, when Raman spectra are excited with the Nd : YAG laser line at 1064 nm. Within seven dissertations we explored different fields of potential applications to medical diagnostics. Identification and qualification of tissues and cells is possible. Tumors show small but significant differences to normal tissues; in order to develop a reliable tool for tumor diagnostics more research is necessary, especially a collection of reference spectra in a data bank is needed. Raman spectra of biomineralization structures in teeth and bones show pathological tissues as well as the development of new mineralized structures. NIR Raman spectra of flowers, leaves, and fruit show, without special preparation, their constituents: alkaloids, the essential oils, natural dyes, flavors, spices and drugs. They allow application to taxonomy, optimizing plant breeding and control of food. 䉷 1999 Elsevier Science B.V. All rights reserved. Keywords: Fluorescence-free Raman spectroscopy; Raman diagnostics; Identification of microorganisms; Natural products of plants; Control of food

1. Introduction All cells and tissues are composed of proteins, nucleic acids, polysaccharides, lipids, coenzymes, vitamins and many other essential components, constituents of supramolecular complexes with highly complicated structures. All disease states, without exception, are caused by fundamental changes in cellular and/or tissue biochemistry [1]. Therefore it

* Corresponding author. Soniusweg 20, D-45259 Essen, Germany. Tel.: ⫹ 49 201 460638; fax: ⫹ 49 201 466650. E-mail address: [email protected] (B. Schrader)

is a challenge to investigate these by non-destructive methods [2]. Non-destructive analysis means analysis with: • • • •

no no no no

mechanical decomposition; chemical decomposition; photochemical decomposition; thermal decomposition.

Only few analytical methods fulfill these conditions and are sensitive enough to reveal the composition and its structural details: • NMR spectroscopy; • Infrared spectroscopy;

0022-2860/99/$ - see front matter 䉷 1999 Elsevier Science B.V. All rights reserved. PII: S0022-286 0(98)00650-4

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Fig. 1. Linear decadic absorption coefficient of H2O, D2O, ethanol and cyclohexane in the near infrared region. Insertion: Range of the Raman spectrum, excited by the Nd : YAG laser with radiation of l ˆ 1064 nm. The left ordinate scale shows the penetration depth: the thickness of a sample of water in cm, for which the transmission is 10%.

• Raman spectroscopy. Raman spectroscopy is in principle the most powerful method of these. As Paul Carey [3] has put it: Raman in Biochemistry, the sleeping Giant awakes. However, when excited with visible radiation the fluorescence of the natural constituents conceals the Raman spectra completely. This is because of the fact, that the quantum yield of fluorescence is of the order of 1, the quantum yield of Raman spectroscopy is however, about 6–10 orders of magnitude smaller. Since fluorescence is a consequence of the absorption spectrum, it is completely avoided when exciting radiation is used, the wavelength of which is long, beyond the range of electron absorption spectra, namely at the wavelength of the Nd : YAG laser, at 1064 nm [4]. It became evident that the conditions of NIR Raman spectroscopy with excitation at 1064 nm define a global optimum [5–8]. This method is a very potent tool for the non-destructive analysis of biomaterials. We therefore investigated systematically Raman spectra of tissues and cells in order to learn if NIR Raman spectroscopy may help in medical diagnostics. Two years ago, it was reported at the EUCMOS XXIII in Balatonfu¨red, regarding the state of the art of six dissertations, employing 1064 nm excitation, at the Universita¨tsklinikum Essen [9–11]. Now, after

completion of these theses [12–17] it is time to give a critical overview of the achievements and limitations of NIR FT Raman spectroscopy in medical diagnostics and to give an outlook to other fields of NIR FT Raman spectroscopy of biosamples, especially of plants. 2. Experimental Since a Raman spectrum excited at 1064 nm covers a spectral range, where NIR absorption spectra of the overtones and combinations of infrared bands are recorded and evaluated, it is necessary to discuss if and how NIR Raman spectra are disturbed by the NIR absorption spectra. The main component of living tissues is water, therefore it seems to be sufficient to discuss the properties of the absorption spectrum of water in this range (Fig. 1). We see that the exciting radiation of 1064 nm is situated at a minimum of the water absorption spectrum. This has two consequences: • The penetration depth of the exciting radiation is several cm (37% transmission for a thickness of 4 cm), therefore large volumes may be illuminated uniformly, also, the danger of overheating the sample by the exciting radiation is minimal. • The depth, from which Raman signals are

B. Schrader et al. / Journal of Molecular Structure 480–481 (1999) 21–32

Fig. 2. Modification of a ZEISS inverted microscope Axiovert 135 to a Raman microscope. The beam of a Nd : YAG laser (1064 nm) is introduced by a holographic beam splitter (KAISER). Left: KOEHLER arrangement: When the laser beam is focused at the inside focus of the objective, the sample is illuminated by a parallel ‘soft’ beam. Raman radiation of a relatively large area of the sample is transported to the spectrometer. Right: Illumination by focused exciting radiation. By imaging of the Raman radiation on a pinhole, a confocal arrangement is set up.

observed, is for the range 700–1800 cm ⫺1 only some millimeters – this means that mainly signals from near the illuminated surface are observed. However, signals with Raman shifts smaller than 700 cm ⫺1 may give information of deeper layers.

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microscope, AXIOVERT 135 to a Raman microscope (Fig. 2), using a Nd : YAG laser mounted directly and coupled by a holographic beam splitter (KAISER OPTICAL SYSTEMS INC.). This instrument can be used in the KOEHLER and the confocal mode. A ‘soft’ illumination is possible by the KOEHLER principle. By focusing the laser beam at the internal focal point of the objective, a ‘soft’ parallel beam leaves the objective, allowing the excitation of Raman spectra of the sample with a polarized laser beam. Using the KOEHLER mode, the spectrometer is illuminated with a large optical conductance [18]. Alternatively, the sample can be excited with a laser beam focused at the sample point of interest. Employing a pinhole in the beam of the Raman radiation, a confocal arrangement is set up, allowing to discriminate sample points of different depths. Most other samples were investigated in a ‘sample cup’, a cylinder of 10 mm diameter, 10 mm long, of aluminum with a parabolic highly polished cavity, covered with a 1 mm thick CaF2 window. The Raman radiation was collected by an aspheric lens. Just behind the lens a holographic notch filter reflects the exciting radiation back to the sample. Thus a multireflection cell was arranged, resulting in a somewhat higher intensity compared to ordinary arrangements, similar to the spherical cuvette [18,19].

3. Results and discussion 3.1. Identification and qualification of animal tissues

The Raman spectra in this paper were mainly recorded using the NIR FT Raman Spectrometer BRUKER RFS 100 with a diode-pumped Nd : YAG laser, emitting at 1064 nm and a germanium detector, cooled with liquid nitrogen. Most spectra were recorded with a resolution of 4 cm ⫺1, most plant spectra with 8 cm ⫺1 with a laser power of less than 300 mW using an unfocused laser beam. For the investigation of tissues in situ we used a fiber-optical arrangement with complementary filters in order to suppress the Raman scattering of the fibers, similar to the arrangement provided by Bruker (Raman fiber probe R 361). For investigations of bones and other biomineralized material we modified a ZEISS inverse

The main disadvantage of the investigation of biomaterials by infrared spectroscopy is the large concentration of the strongly absorbing water. Since the Raman bands of water are quite weak most tissues can be investigated by Raman spectroscopy without the need of drying processes. Keller [15] did not observe any heating of samples of homogenized liver after 4 h of illumination with Nd : YAG laser radiation of 300 mW. Open cuvettes only showed some drying and oxidation effects. Keller was able to identify the tissues of different rabbit organs [15]. Raman spectra of human livers showed significant differences when pathological findings were present: Fat liver or liver cirrhosis or some kinds of leukemia

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Fig. 3. Raman spectrum of (a) a medullar carcinoma and, (b) a murine adenocarcinoma.

could be identified [5–11,15], this might be useful for the qualification of a liver before a transplantation. 3.2. Investigation of different tumors There are no bands, which definitely prove the existence and the nature of a tumor. However, there are definite changes of the spectra of tissues, for example a decreased intensity of cholesterol and increased intensities of nucleic acids in case of a brain tumor [15]. The same tumor of different patients showed quite similar spectra, the spectra of tissues with different tumors showed definite differences [15].

There are essentially two types of breast tissues, showing either mainly protein or lipid structures. Very often different tissues are heavily mixed. [17]. The differences between different tumors may be small, but significant. In Fig. 3a the spectrum of a medullar carcinoma is compared to the spectrum of an undifferentiated murine carcinoma. In Fig. 3b the band at 1234 cm ⫺1 within the amide III range shows a larger concentration of pleated sheet structures. The bands in the 700–900 cm ⫺1 range show vibrations of DNA [17]. A cluster analysis of the protein tissues as well as factor analysis shows typical differences between benign and malignant states. The spectra of

Fig. 4. Raman spectrum of different parts of an infiltrating ductal carcinoma with (a) high concentration of tumor cells, (b) desmoplastic part, (c),(d), parts with large lipid concentration.

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be similar, however, the amide III band shows the different collagen types, and the relative intensities of other bands can be used to determine the lipid or water content, as well as that of the different DNA bases [13,20]. Cluster analysis definitely helps in evaluating Raman spectra with regard to benign or malignant skin lesions (Fig. 6) [20]. The water content of hair, nails or skin can be determined by the relative intensities of the integrated bands at 3250 and 2940 cm ⫺1. In wet tissues the proteine backbone vibration at 932 cm ⫺1 increases, indicating (a -helical proteins, also the n (C–S–S–C) at 510 cm ⫺1 increases [35]. The aim of the current research is to completely eliminate the need of biopsy of samples by employing Raman spectroscopy [20–22]. 3.4. Identification of cells and microorganisms Fig. 5. Raman spectra of (A) normal skin, (B) psoriatic skin, (C) eczema, (D) chronic dermatitis and, (E) Kaposi’s sarcoma.

tumors are dependent on the site of the investigated sample (Fig. 4) [17]. 3.3. Investigations of skin and skin lesions by NIR FT Raman spectroscopy NIR FT Raman spectra of different skin lesions are shown in Fig. 5 [13]. At first sight the spectra seem to

Fig. 6. Results of a cluster analysis of the spectra of Fig. 5.

The Raman spectra of blood cells may be used to differentiate different types of leukemia [14]. Bacteria and fungi may be characterized and even identified by NIR FT Raman spectroscopy [15,16]. The spectra allow the observation of several properties of living microorganisms: sporogenesis, accumulation of storage compounds as well as the detection of resistance [11,16]. When combined with IR spectra the potential of identification will be considerably enhanced [23,24]. 3.5. Investigation of processes of biomineralization Raman spectra of teeth and bones show simultaneously the inorganic and the organic parts of the tissue. It is now possible to determine the quality and the state of the biomineralized structures [15] as in teeth. Using the inverted microscope (Fig. 2) NIR FT Raman spectra of bone tissues can be recorded without the need of the usual preparation. Prostheses covered with tricalciumphosphate by plasma spray have been implanted in the hip of a dog. The biomineralization of the bone tissue can be followed by the intensity of the band of hydroxyapatite (HA) relative to that of tricalciumphosphate (TCP) in different distance of an endoprosthesis (Figs. 7 and 8) [12,25]. In the immediate vicinity of a HA-coated prosthesis a large HA content could be observed, that descreased to a minimum towards the periphery of the coating and increased at the site of the ongrown bone. NIR

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Fig. 7. Raman spectrum of the hydroxyapatite coating of a prosthesis implanted in the hip of a dog and the ongrown bone at different lateral positions.

Raman spectroscopy is a promising method to investigate the development of the mineralization of bone and the biological interction between HA coatings and bone with minimal sample preparation. 3.6. NIR Raman spectroscopy of plants

Fig. 8. Relative intensity of the Raman lines of tricalciumphosphate (TCP) versus hydroxyapatite, determined by a band contour analysis of a bone grown on a prosthesis covered with tricalciumphosphate.

The most important compound of plants, chlorophyll, could not be investigated by ‘classical’ Raman spectroscopy owing to thermal degradation, luminescence and photooxidation [26]. However, spectra, excited with radiation of 1064 nm show an enhancement of the intensity of the chlorophyll bands by a pre-resonance Raman effect [27]. Thus, analyses of natural products in green plants is not a problem anymore. Urlaub et al. [28] studied the distribution of alkaloids in liana plants. Conifers can be classified by the Raman spectra of single needles [29] (Figs. 9 and 10). A correlation between the spectra and the taxonomy of the conifers is possible. The main differences between the spectra are a result

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Fig. 9. Raman spectra of the needles of conifers: (1): Tsuga canadensis, (2): Abies concolor, (3): Cedrus atlantica ‘Glauca’, (4): Picea abies, (5): Picea glauca ‘Conica’, (6): Picea omorica, (7): Picea pungens ‘Glauca’, (8): Pinus cembra, (9): Pinus mugo mughus, (10): Pinus mugo ‘Mops’, (11): Pinus nigra nigra, (12): Pinus strobus.

of the terpenes showing their bands all over the spectrum with minimal intensity, but quite typically in the range above 1600 cm ⫺1. Essential oils are substances typically used for the qualification of herbs and fruits. Usually they are determined by steam distillation combined with gas chromatography. In cooperation with the Bundesanstalt fu¨r Zu¨chtungsforschung we are devloping ways for the nondestructive analysis of the essential oils in fruit and leaves by NIR Raman spectroscopy. Fig. 11 shows the Raman spectra of, (1) green dill leaves, (2) dried leaves, (3) dill seeds and, (4) anethol, the main terpene molecule of dill. Some bands are present in all spectra, probably because of the smelling principle [30]. Others are only present in the green plants, the fresh as well as the freeze-dried dill. The spectrum of fresh dill shows several strong bands (Fig. 12) which

are also present in Fig. 13, where the Raman spectra of fresh herbs are collected (Fig. 13 (2)–(7)), relatively to the spectrum of ordinary meadow grass, (1) and nerol, (8), the main terpene of dianthus. Besides the same strong bands as shown by grass the spectra of the herbs show several bands of lower intensity, which seem to characterize the essential oil, especially in the range above 1600 cm ⫺1 [19]. Fig. 14 shows the tentative assignment of the bands of the grass spectrum. Some bands of low intensity at 1603, 1326, 1286 and 746 cm ⫺1 can be assigned to chlorophyll [26,29]. The strongest bands however are because of carotenoids: at 1155 and 1525 cm ⫺1. Carotenes are detected quite easily employing these bands. In Fig. 15, (1) is the spectrum of a petal of a tagetes flower, recorded with 100 mW, unfocused, within 30 min; (2) of a red paprika (200 mW, 30 min) and;

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Fig. 10. Cluster analysis of two preparations of 1 needle of the conifers of Fig. 9. Ward’s algorithm, correlation with scaling to 1st range, frequency ranges (weights) 1676–1569 cm ⫺1 (1.0), 1489–1178 cm ⫺1 (1.0), 965–640 cm ⫺1 (1.0).

Fig. 11. Raman spectra of (1) fresh dill leaves, (2) freeze-dried dill leaves, (3) dill seeds and, (4) anethol.

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Fig. 12. Wavenumbers of the Raman bands of fresh dill leaves.

Fig. 13. Raman spectra of fresh herbs: (1) grass, (2) rosemary, (3) sage, (4) lemon balm, (5) wormwood, (6) lavender, (7) basil, (8) Raman spectrum of eugenol.

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Fig. 14. Raman spectrum of fresh grass with tentative assignment of the bands: CHL chlorophyll, CAR carotinoids, CH2 deformation vibration of CH bonds, PHE phenylalanine.

Fig. 15. Raman spectra of plant parts showing carotenoid vibrations: (1) petal of tagetes flower, (2) fruit of red paprika and, (3) safran (stamen of Crocus).

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(3) of safran, the stamen of crocus (50 mW unfocused, 30 min).

4. Conclusions • Employing the NIR-FT technique and excitation with radiation of 1064 nm Raman spectra may be recorded from any kind of biomaterial without denaturation of the sample. • Problems of biomineralization and biocompatibility can be studied using an inverted Raman microscope [12,25]. • Cancer and pre-cancer states can be detected in skin, cells and of tissues [13,17,20,31,32,34,35]. However, in order to employ the small but significant differences between normal and pathological samples as a diagnostic tool the present results have to be confirmed by many more experiments and a data bank has to be set up, containing spectra of many pathologic and normal tissues, combined with evaluation programs providing the diagnoses in the language of medical doctors. • Microorganisms and even fungi can be identified by their Raman spectra [7,11,16]. Properties of the living cells can be studied: sporogenesis, accumulation of storage compounds and detection of resistance [16]. The results can be confirmed in combination with the infrared spectra [23,24]. • NIR Raman spectra of any plant material: flowers, leaves, fruit, may be applied for the study of the distribution of natural products, to taxonomy, plant breeding and control of food [10,15,28,29]. • Products and artifacts of biomaterials can be analyzed nondestructively – this can be of help in characterization, dating, for restoration and conservation, even in archaeology [33]. In biochemistry the fluorescent compounds are essential compounds which are not permitted to be removed. The only way to avoid fluorescence is to employ excitation at 1064 nm. Then the ‘sleeping giant’ [2,36] can be brought to productive activity of highest importance.

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Acknowledgements We like to thank Prof. Dr. de Groot for supporting our work at the Universita¨tsklinikum Essen and Dieter Naumann for a fruitful cooperation in combining the evaluation of IR and Raman spectra of microorganisms. Financial help of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged.

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