**3. Raman spectroscopic variations**

wide range of sample types. Although being recognized as not as specific and sensitive as other metabolomics techniques, several works already demonstrated the potential of Raman applied to health sciences for metabolic fingerprinting because it is possible with only one spectra to simultaneously analyze carbohydrates, amino acids, fatty acids, lipids, proteins, nucleic acids and polysaccharides with a minimum sample preparation. As Raman is a scattering technique

For diagnostic purposes, it is expected that Raman spectra of biological samples result in quantitative data, so it is essential to define some categorical differentiable classes for data by dividing samples in healthy or disease sample classes. For these purposes, chemometric data

Due to Raman spectroscopy features, it is currently widely used in health sciences for spectral imaging of cells and tissues, for the *in-vivo* and *ex-vivo* diagnosis of tissues, where fiber probes can be used, and for biofluid analysis, contributing to a better knowledge of the disease and disease diagnosis at the molecular level. This chapter describes the most relevant application

Raman spectroscopy is an optical technique based on inelastic scattering of light due to the vibration of the molecules that can provide chemical fingerprints of several samples. In health sciences, Raman can be used in DNA analysis, lipids, proteins and amino acids identification, bacteria classification and recognition, cell responses, cancer diagnosis and prognosis, and dental prosthesis, among others. This wide range of application is due to Raman's capabilities that go far beyond of being a noninvasive and nondestructive method that does not require samples preparation. Raman is compatible with aqueous solutions and is also a technique that produces results in a short time, requiring less than a minute to obtain a good quality spectrum, and no sample preparation is needed. This spectroscopic technique is sensitive to identify many different functional groups that produce weak signals in infrared spectroscopy (C═C; S─S; C─S). Besides that, it has a highly selective fingerprint that allows the discrimination of similar molecules and has high spatial resolution that allows single cell analysis and intracellular imaging.

The ability to use advanced optical technologies in the visible or near-infrared spectral range (lasers, microscopes, and optic fibers) is responsible for the growth of Raman spectroscopy in medical diagnostic. As molecular changes in cells, tissues or biofluids can be detected and quantified by Raman spectroscopy, it is possible to use this technique in the diagnosis of

In fact, Raman spectroscopy can offer chemical fingerprints of cells, tissues (*in vivo* or *in-vitro*), or biofluids. A large range of samples can be used for Raman analysis: formalin-fixed and

Whether a sample is a solid, liquid, powder, slurry, or gas, no sample preparation is normally required, and there is no need to dissolution, grinding, glass formation, or pressing in order

diseases and to study the effects of drugs (treatments) in biosystems.

fresh frozen mammalian tissue, fixed cells and biofluids.

and it is not perturbed by aqueous media, it is suitable to analyze biological samples.

processing is a valuable tool.

276 Raman Spectroscopy

of Raman in biomedical field.

**2. Raman features and sampling**

In recent past, Raman spectroscopy was known as a technique with low signals requiring longer acquisition times. However, recent developments were made to overcome this limitation in the last years. Nonlinear optical effects and metallic nanoparticles are currently used to improve Raman signals, fiber-optic Raman probes were introduced and are used for real-time *in-vivo* experiments, and multimodal integration with other optical techniques increased the acquisition speed and spatial accuracy. These advances in the accuracy allow the application of Raman spectroscopy into clinical diagnosis, and time of analysis allows its clinical use.

Surface enhanced Raman spectroscopy (SERS), Tip Enhanced Raman Scattering (TERS) or nano-Raman and resonance effects increase significantly Raman sensitivity to study biological samples. SERS increases Raman intensity compared to the usual and weak Raman scattering. These improvement features are sufficient to allow even single molecule detection using Raman. SERS is useful in trace material analysis, flow cytometry and other applications where the traditional sensitivity/speed of a Raman measurement is insufficient [2]. Resonance Raman spectroscopy is a variant of Raman spectroscopy that instead of using laser excitation at any wavelength to measure Raman scattering of the laser light, the excitation wavelength is used to overlap with an electronic transition. The overlap results in an extraordinary increase in scattering intensities, thus detection limits and measurement times can be significantly decreased. It is also possible to couple Raman to an optical microscope. Raman microspectroscopy uses visible and near-infrared excitation lasers and allows to extract molecular properties of the samples with diffraction-limited spatial resolution. The typical method to obtain Raman spectral images is by scanning the sample with the laser spot and then applying a uni- or multivariate spectral model to each Raman spectrum [3]. In order to decrease the time of analysis, Raman spectral imaging can be based on line-mapping (laser beam is expanded to form a line spot on the sample surface) [4].

TERS is a chemical imaging technique that is label-free and have enhanced-resolution. TERS imaging is performed with a Raman spectrometer, a scanning probe microscope (SPM) integrated with an optical microspectrometer. The scanning probe microscope provides the means for nanoscale imaging and the optical microscope provides the resources to bring the light to a functionalized probe, and the spectrometer is the sensor analyzing the light output providing chemical specificity. It is possible to increase the signal to obtain high-spatial resolution spectral images for large samples using selective-sampling Raman microspectroscopy. In this approach, it is possible to (1) obtain information about sample spatial features by other optical technique [5] or (2) estimate information in real-time from the Raman spectra [6]. When traditional variations of Raman spectroscopy are used to study tissues, the results are not good due to insufficient penetration depth. The advance of spatially offset Raman spectroscopy (SORS) overcame this limitation enabling spectral measurements until 10–20 mm of the sample and, with this modification, the application of Raman in clinical fields increases by collecting the scattered light away from the point of laser illumination [7]. Transmission Raman spectroscopy (TRS) is the term used when the collection and illumination points are on opposite sides of the sample, and it is quite useful to analyze opaque materials. Besides, it was proved to be very useful to read many millimeters of tissues [8]. To increase the resolution of Raman microspectroscopy, it is possible to use Coherent anti-Stokes Raman spectroscopy (CARS) or stimulated Raman spectroscopy (SRS), both based on nonlinear optical effects that upgrade the spatial resolution. Coherent Raman spectroscopy techniques are based on nonlinear effects to increase the speed and spatial resolution of Raman spectroscopy by inducing coherent molecular vibrations in the sample increasing resolution. CARS is used to obtain image from cells and tissues by exciting the CH stretching vibrations of lipids and proteins and SRS allows to obtain high-speed images [9, 10]. Raman spectroscopy is also adaptable for fiber-optic probes, making it valuable for medical diagnosis *in vivo*, for instance in hollow organs. The probe should have very reduced dimensions to permit the access to body cavities, and the spectra acquisition time should be short to allow accurate measurement [11].
