Design of a Multimodal Imaging System and Its First Application to Distinguish Grey and White Matter of Brain Tissue. A Proof-of-Concept-Study  
Annabell Heintz  
1  
Sebastian Sold  
1  
Felix Wuhler  
1  
Julia Dyckow  
2  
Lucas Schirmer  
2,3,4  
Thomas Beuermann  
5,  
as the standard dye promotes the identification of certain structures since acidophilic components such as cell cytoplasm, fiber structures, and interstitial stromal materials appear pale to deep pink, whereas basophilic components of the cell structure such as RNA and DNA are colored in blue [3; 5]. Further morphological imaging techniques for biomedical applications are, for instance, optical coherence (OC) tomography [6; 7; 8; 9], laser scanning microscopy [10; 11; 12], second-harmonic generation (SHG) microscopy [13; 14], and two-photon excited fluorescence (TPEF) microscopy [15; 16]. Those techniques have in common that they highlight structures within the tissue. In contrast, functional imaging techniques visualize functional parameters such as the local chemical composition [4]. One of these imaging techniques is Raman-based microscopy. This technique is a combination of Raman spectroscopy and microscopy. It was first proposed by Delhaye and Dhamelincourt [17] in 1975 and has been commercially sold by companies since the 1990s [18]. Thereby, a microscope objective with a high numerical aperture (NA) focuses the excitation light to a small spot with a diameter of a few micrometers and enables a high collection efficiency of the Raman signal from a microscopic area [18]. The so-called spontaneous Raman effect was predicted by Adolf Smekal [19] in 1923 and experimentally confirmed by Raman and Krishnan [20] in 1928. Raman spectroscopy is an important vibrational spectroscopic method based on inelastic scattering of light by molecules. Hence, the frequencies of an incident photon, which interacts with a molecule, and the resulting scattered photon differ from each other. Raman scattered light with a lower frequency than the incident frequency is known as Stokes Raman scattering, and vice versa, anti-Stokes Raman scattering is Raman scattered light with a higher frequency. This frequency shift is indicative of discrete vibrational modes associated with chemical bonds within the molecule, independent of the wavelength of the excitation light source (laser). Consequently, qualitative measurements of chemical compositions can be obtained. Thus, Raman spectroscopy provides a label-free highly molecular specific technique. However, a change in polarizability of a chemical bonding orbital is crucial for the excitation of the fundamental vibration in a molecule and the observation of Raman spectra. Furthermore, Raman scattering is a weak effect with an occurrence probability of approximately 1 in \(10^{8}\) incident photons [21; 22] compared to Rayleigh scattering where the light frequency remains constant (elastic scattering). In consequence, Raman spectroscopy requires intensive monochromatic light like lasers as a light source. The choice of laser wavelength and power plays an important role in Raman spectroscopy. The Raman intensity is linear to the laser's power and proportional to the fourth power of the laser's frequency [23]. This results in higher Raman signals for shorter excitation wavelengths. In contrast, short laser wavelengths cause fluorescence signal that interferes with the Raman spectrum [24]. This is why an excitation wavelength in the red or NIR region is recommended for Raman spectroscopy of fluorescent biological material [23; 25]. Another approach is the reduction of the detection volume with a confocal detection principle [18]. In this case, the image of a spot in the focal plane is detected through a pinhole in front of the detector [26]. Further fluorescence background in Raman microscopy is caused by standard glass microscope slides [27], the substrate for biological specimens.