2.1.

Tissue Specimens

Human glioblastoma multiforme tumor diagnosed as malignancy stage grade IV, acoustic neuroma benign, pituitary adenoma benign and malignant meningioma diagnosed as malignancy stage grade III, benign minigioma by postoperative histopathology and immunohistochemistry analysis and normal meningeal brain tissues were obtained from the General Hospital of the Air Force, Beijing, China. The malignancy stages are according to the World Health Organization (WHO) standard. The experimental procedures were approved by the committee of the General Hospital of the Air Force, Beijing, China. The tissue specimens came from patients from 27 to 56 years old, 70% of them were over 50 years, and 29% of the human brain tissues were from female patients.

Specimens were not chemically treated prior to the spectroscopic studies. The tissue specimens arrived frozen and kept under snap-frozen condition, uncut, and in irregular shapes. They were thawed to ambient room temperature for the spectroscopic studies. Most specimens were measured within 36 h post-surgery. Specimens of random shapes were mounted on a quartz plate for Raman spectral measurement.

2.2.

Acquisition of RR Spectra of Brain Tissues

The RR spectra were collected using a multichannel modular triple Raman system (JY-HR800 France) with confocal laser micro-Raman spectrometer at the 532-nm excitation wavelength. The excitation light beam was directly shining on the surface of specimen. The exposure time was 60 s for one scan region, and for the full region (four local regions), the scan time was $4\times 60\mathrm{s}$. The average size of the samples was approximately $10\times 5\times 2\mathrm{mm}$. A $100\times$ microscope objective lenswas used for focusing the laser beam and collection of the scattered signals. The spot diameter of the focused laser beam on the sample position is about 1 μm. The solid-state diode laser (532 nm) from Coherent Company-Verdi-2 with 3.5 mW was used as an excitation source. The laser power on the sample was kept with 0.9 mW. All measurements were taken at room temperature. The final spectral resolution was $2{\mathrm{cm}}^{-1}$ in the range of interest (200 to $4000{\mathrm{cm}}^{-1}$).

A total of 43 Raman spectra were acquired from seven subjects using the confocal micro-Raman system. In the inhomogeneous lesion, the spectral collection needs at least seven scan sites for each local area—e.g., fat part (yellow), hemorrhage area (red), and regular parts—were measured carefully. The results of spectroscopic measurements were then analyzed and compared to the pathology and immunohistochemistry report.

2.3.

Analysis Method of Support Vector Machine

Since RR spectra are sampled into a higher-dimensional space, training of a classifier might be suffered from the “curse of dimensionality.” By the principal component analysis (PCA), the spectral data can be project onto the subspace spanned by a small number of principal components (PCs), thus significantly reducing dimensionality while reserving most power of data. Specifically, the matrix of Raman spectral data $\mathbf{X}=[\mathbf{x}(1),\dots ,\mathbf{x}(N)]$ of all $N$ samples are decomposed into

A support vector machine (SVM) with a linear kernel can be expressed as

3.1.

RR Spectra of Glioblastoma Multiforme Grade IV, Acoustic Neuroma, and Pituitary Adenoma

Figure 1 shows the RR spectra of glioblastoma multiforme tumor tissue diagnosed as grade IV (top), benign human acoustic neuroma lesion (center), and benign human pituitary adenoma lesion (bottom) that were collected in vitro using the confocal micro-Raman system with an excitation wavelength of 532 nm within a scan region from 500 to $4000{\mathrm{cm}}^{-1}$. In all of the three RR spectra in Fig. 1 at the top, center, and bottom plots, the amide I, amide II, and amide III peaks were clearly enhanced. The amide II band near $1547{\mathrm{cm}}^{-1}$ exhibited intense resonance enhancement at excitation 532 nm, possibly duo to the amide II mode in active at this excitation wavelength. The results showed that for the amides (amide I near $1639{\mathrm{cm}}^{-1}$, amide III near $1226{\mathrm{cm}}^{-1}$) have demonstrated selectively enhanced by the excited wavelength at 532-nm amide $\pi \to \pi *$ electronic transition, while the amide I and amide III show less enhancement from this transition.

Fig. 1

The RR spectra recorded from three types of brain tissues. The top of Raman spectrum was from glioblastoma multiforme, grade IV tissue. The center of Raman spectrum was from acoustic neuroma benign tissue, and the bottom of Raman spectrum was from pituitary adenoma benign tissues.

Typically, glioblastoma cells display somatic gene alterations, which products have a negative impact on the cellular regulatory processes of growth; the cells’ response to extracellular signals; and cell-cycle control. The RR spectra collected from the gliomas showed in the top of Fig. 1. The Raman spectrum of glioblastoma multiforme grade IV tissue (Fig. 1, top) displayed a total of 22 peaks, 10 of which demonstrated strong enhanced modes at 752, 1004, 1172, 1337, 1358 1547, 1587, 1606, 2896, and $2938{\mathrm{cm}}^{-1}$. Characteristic enhanced Raman peaks at 1547 (amide II), 1587, 1606, and $1004{\mathrm{cm}}^{-1}$ were assignable to proteins and collagen types I and IV showing the enhancement comparison with the previous reports.^23–26

The RR spectra collected from the benign acoustic neuroma (vestibular schwannomas) lesion (Fig. 1, center), which is a noncancerous lesion that develops on the nerve that connects the ear to the brain and usually grows slowly. RR spectra collected from benign acoustic neuroma tissue displayed 18 resonance-enhanced peaks, with 12 strong enhanced peaks at 648, 757, 1004, 1155, 1338, 1357, 1551 cm, 1585, 1607, 1639, 2888, and $2934{\mathrm{cm}}^{-1}$.

Displayed at the bottom of Fig. 1 is a typical RR spectrum from the benign pituitary adenoma lesion. A pituitary tumor is an abnormal growth in the pituitary gland, the part of the brain where hormones are regulated and the body’s balance of hormones are controlled. These lesions are most noncancerous (benign). The RR spectra were measured from pituitary adenoma benign tissue. Thirteen Raman peaks were observed in which nine distinct vibrational modes at 750, 1005, 1130, 1173, 1543, 1585, 1667, 2882, and $2932{\mathrm{cm}}^{-1}$ were clearly enhanced. The RR spectra of amide I band of 1645 to $1668{\mathrm{cm}}^{-1}$ observed in the spectra that may be assigned to active pituitary adenoma tissue.^27–29

3.2.

RR Spectra of Cancer, Benign, and Normal Meningeal Brain Tissues

The RR spectra from three types of the meningeal brain tissues are represented in Fig. 2. The RR spectra from the malignant menigioma grade III (top, cancer), the benign menigioma (center), and the normal (bottom) menigngeal tissues were measured within the region from 500 to $4000{\mathrm{cm}}^{-1}$ using a confocal micro-Raman system. Table 1 lists the position of Raman spectra in wavenumbers of the observed peaks and their assignments.^{18,20,30–33} The RR spectra with the relative enhanced peaks at 750, 1004, 1156, 1358, 1548, 1587, 1605, and $1639{\mathrm{cm}}^{-1}$ and the broad peaks near $2800{\mathrm{cm}}^{-1}$ to $3000{\mathrm{cm}}^{-1}$ were observed in cancerous tissue (Fig. 2, top). When comparing the spectra collected from the malignant meningioma tissue with that collected from the benign lesions (Fig. 2, center) and the normal meningeal (Fig. 2, bottom) tissues, the RR spectra give a higher signal to noise ratio and more intense enhancement, as has been previously reported.^{14,16,18,21,25}

Fig. 2

The RR spectra recorded from three types of meningeal tissues. The Raman spectra showed from top to bottom as the Raman spectrum of malignant meningioma tissue: grade III (a); the Raman spectrum of meningioma benign tissue (b); and Raman spectrum of normal meningeal tissue (c).

Table 1

Raman band frequencies and assignments of Brain Tissue.

We identified an enhancement mode of amide II. In the RR spectra, the excitation wavelength of 532 nm makes the amide II mode active. Amide II is one of the nine amide bands of protein, which was resonance enhanced in the three types of meningeal tissues (usually the amide II band is weak or absent in the nonRR system) was shown in Fig. 2. The amide I and amide III bands show fewer enhancements from this transition or explained in less active. Near $1004{\mathrm{cm}}^{-1}$ is assigned to the phenyl ring-breathing mode of phenylalanine. The spectral peak intensities were normalized to the intensity near the $1004{\mathrm{cm}}^{-1}$ peak, assigned to the breathing mode of phenylalanine, for calculation and comparisons amongst peak intensities (Fig. 2), due to the relative stability of intensity and position of the peak due to different environments. A clear and obvious increased in the intensity ratios of $1587{\mathrm{cm}}^{-1}$ (arising from cytochrome c/mitochondria) to $1605{\mathrm{cm}}^{-1}$ (arising from phenelalanine and tyrosine) were calculated from Fig. 2 as 0.97:0.89:0.88, which displayed the differences between the cancer, benign and normal meningeal tissues. This phenomenon may propose that the differences (increased the intensity in cancer of Raman peak at $1587{\mathrm{cm}}^{-1}$) arise during the cancer cells transformations, the cytochrome c might be released from the mitochondrial membrane since the cancer cells cause of the mutation of the mitochondria in the cancer tissue.

Methyl ($─{\mathrm{CH}}_3$) symmetric stretching was observed as a strong band in Raman at $2935{\mathrm{cm}}^{-1}$. The peak intensity ratio of $2935{\mathrm{cm}}^{-1}$ (methyl bond ${\mathrm{CH}}_3$) to $2880{\mathrm{cm}}^{-1}$ (methylene bond ${\mathrm{CH}}_2$), and the width of board profile range between 2850 and $3000{\mathrm{cm}}^{-1}$ were calculated for the three types of the meningeal tissues from the spectra displayed in Fig. 2. An increased in the width (border) of board profile in the range between 2800 and $3000{\mathrm{cm}}^{-1}$ was observed for malignant meningeoam. The ratios of the width (border) of board profile at cancer: benign: normal were found to be $1:0.98:0.95$, respectively. A decrease in the methyl to methylene intensity ratios was calculated from Fig. 2 to be $1.25:1.47:1.79$, for the spectra collected from the cancerous, benign and normal meningeal tissues, respectively, which suggests the changes of the molecular conformation order. It is supposed that the cancer cells are an abnormal mass in which the cells grow faster with multiple uncontrollably and invading control normal cells. The growth of a tumor also takes up more space within the skull and interferes with normal brain activity. This may cause disorder in tumors as the observed variation in the ${\mathrm{CH}}_3/{\mathrm{CH}}_2$ ratio. Normal tissue cells have a higher molecular conformation order coefficient than tumors. Normal tissue cells should exhibit a more stable biochemistry and metabolism than will tumor cells.^20,34–36

3.3.

RR Spectral Data Analysis of Cancer, Benign and Normal Meningeal Brain Tissues by SVM Method

The statistical analyses of the RR data collected from the tissues using the PCA, SVM, and ROC methods^37,38 yielded a diagnostic sensitivity of 90.9% and specificity of 100% for ROC using two PCs, when compared with the histopathology and immunohistochemical analysis (the “gold standard”) for the classification amongst the spectra of malignant meningioma brain tissue; meningeal benign lesions, and normal meningeal tissues. Direct comparisons between the spectra data collected are displayed in Fig. 3 and in Table 2. In Fig. 3, the separating lines were calculated using the SVM algorithm, that yields a diagnostic sensitivity of 90.9% and a specificity of 100% for classifying the spectra collected from cancerous tissues from the spectra from benign and normal brain tissue. Table 2 illustrates how the performance of the SVM classifier is improved as the power of data samples located in the subspace spanned by the selected PCs increases. The identifications are shown to be effective for the Raman spectral diagnosis and classification of brain tissues.

Fig. 3

The scatterplot of the posterior probability of classification for the malignant, benign, and normal brain tissues were using PC1 and PC4 as diagnostically significant components. The separating lines were calculated using SVM algorithm, which yields a diagnostic sensitivity of 90.9% and specificity of 100% for identifying cancer from benign and normal brain tissue.

Table 2

Performance in classifications.