molecules Review Overview of Popular Techniques of Raman Spectroscopy and Their Potential in the Study of Plant Tissues Aneta Saletnik, Bogdan Saletnik * and Czesław Puchalski Department of Bioenergetics, Food Analysis and Microbiology, Institute of Food Technology and Nutrition, College of Natural Sciences, University of Rzeszów, Cwikli´nskiej2D,´ 35-601 Rzeszów, Poland; [email protected] (A.S.); [email protected] (C.P.) * Correspondence: [email protected]; Tel.: +48-(17)-785-49-65 Abstract: Raman spectroscopy is one of the main analytical techniques used in optical metrology. It is a vibration, marker-free technique that provides insight into the structure and composition of tissues and cells at the molecular level. Raman spectroscopy is an outstanding material identification technique. It provides spatial information of vibrations from complex biological samples which renders it a very accurate tool for the analysis of highly complex plant tissues. Raman spectra can be used as a fingerprint tool for a very wide range of compounds. Raman spectroscopy enables all the polymers that build the cell walls of plants to be tracked simultaneously; it facilitates the analysis of both the molecular composition and the molecular structure of cell walls. Due to its high sensitivity to even minute structural changes, this method is used for comparative tests. The introduction of new and improved Raman techniques by scientists as well as the constant technological development of the apparatus has resulted in an increased importance of Raman spectroscopy in the discovery and defining of tissues and the processes taking place in them. Citation: Saletnik, A.; Saletnik, B.; Keywords: Raman spectroscopy; spontaneous Raman scattering; stimulated Raman scattering; Puchalski, C. Overview of Popular surface-enhanced Raman scattering (SERS); tip-enhanced Raman scattering (TERS); confocal Raman Techniques of Raman Spectroscopy microscopy; chemical imaging and Their Potential in the Study of Plant Tissues. Molecules 2021, 26, 1537. https://doi.org/10.3390/ molecules26061537 1. Introduction Academic Editor: Grazyna˙ Neunert Biological material is characterized by a complex structure and varied chemical com- position. Often the molecules tested are found in the sample at very low concentrations [1]. Received: 17 February 2021 Instrument-based methods that can be used in the analysis of cell wall structure in plants Accepted: 9 March 2021 are UV-VIS, fluorescence, infrared, Raman spectroscopy, and nuclear magnetic resonance Published: 11 March 2021 spectroscopy [2]. Spectroscopic methods are analytical techniques in which a spectrum is generated as a result of the interaction of electromagnetic radiation with matter. They in- Publisher’s Note: MDPI stays neutral clude, among other techniques, molecular spectroscopy, which deals with the interaction of with regard to jurisdictional claims in electromagnetic radiation with molecules resulting in complex changes in the energy states published maps and institutional affil- of the molecules. As a result of absorption or emission of a quantum of electromagnetic iations. radiation, transitions between the existing energy levels of atoms and molecules take place. As a result of the absorption of electromagnetic radiation by the molecule, the neces- sary energy levels are excited. This will result in changes in the spectrum of the excitation radiation (absorption spectrometry) or an emission spectrum is generated due to the return Copyright: © 2021 by the authors. of the molecule to the ground state. The distinction is made between the spectrum associ- Licensee MDPI, Basel, Switzerland. ated with the change of rotation, the vibration and the excitation levels of electrons. Raman This article is an open access article spectroscopy examines the rotational and oscillatory-rotational spectra of molecules [3–7]. distributed under the terms and Raman light scattering was first observed in 1928 and was used to explore the vibrational conditions of the Creative Commons state of molecules in the 1930s [5–7]. Raman applications on plants began in the 1984 with Attribution (CC BY) license (https:// the investigation of cellulose. Raman spectroscopy has undergone a renaissance in the past creativecommons.org/licenses/by/ decade [8]. 4.0/). Molecules 2021, 26, 1537. https://doi.org/10.3390/molecules26061537 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x 2 of 16 Molecules 2021, 26, 1537the 1984 with the investigation of cellulose. Raman spectroscopy has undergone a re- 2 of 16 naissance in the past decade [8]. 2. Principle, Instrumentation 2. Principle, Instrumentation Raman spectroscopy is used to study the structure, dynamics of changes, and func- tions of biomolecules.Raman In spectroscopy combination is with used mi tocroscopy, study the data structure, with dynamicshigh spatial of changes,resolution and functions is acquired of[8]. biomolecules. Raman spectroscopy In combination is a vibrational with microscopy, technique data that with does high not spatial utilize resolution is markers. It thusacquired enables [8]. an Raman insight spectroscopy into the structure is a vibrational of tissues technique and cells that and does permits not utilize an markers. investigationIt of thus their enables composition an insight at into the themolecular structure level of tissues thus playing and cells a and very permits important an investigation role in biologicalof their research composition [9,10]. at Raman the molecular spectroscopy level thus is playingdesigned a veryto measure important the role fre- in biological quency shiftresearch of inelastic [9,10 scattered]. Raman light spectroscopy when a photon is designed of incident to measure light hits the frequencya particle and shift of inelastic produces a scatteredscattered lightphoton when [11–16]. a photon A Ra ofman incident spectrum light hitsconsist a particle of bands and which produces are a scattered caused by anphoton inelastic [11– scattering16]. A Raman from spectrumthe chemically consist bonded of bands structures which are [17]. caused The byob- an inelastic tained Ramanscattering spectrum from is composed the chemically of band bondeds whose structures position [ 17depends]. The obtained on the vibration Raman spectrum is frequency ofcomposed the sample of bandscomponents. whose Each position organic depends compound on the vibrationand functional frequency groups of the sample have a characteristiccomponents. vibration Each organicfrequency compound visualized and in functionalthe Raman groups spectrum have in a the characteristic form of vibration frequency visualized in the Raman spectrum in the form of a peak [6]. The characteristic a peak [6]. The characteristic spectral pattern is the so-called fingerprint, owing to which spectral pattern is the so-called fingerprint, owing to which we can identify the compound, we can identify the compound, while the intensity of the bands can be used to calculate while the intensity of the bands can be used to calculate its concentration in the analyzed its concentration in the analyzed sample [8]. sample [8]. In the light scattered by the test medium there is, for the most part, a component of In the light scattered by the test medium there is, for the most part, a component of the same frequency as in the incident light (Rayleigh scattering, elastic scattering) [18,19], the same frequency as in the incident light (Rayleigh scattering, elastic scattering) [18,19], while in a minority of cases there are variable frequency components associated with the while in a minority of cases there are variable frequency components associated with the change in photon energy (inelastic scattering, Raman scattering). The outgoing scattered change in photon energy (inelastic scattering, Raman scattering). The outgoing scattered light can be a photon with a frequency lower than the incident photon and in such cases light can be a photon with a frequency lower than the incident photon and in such cases we we call it Stokes Raman scattering, or it is of a frequency that is higher, and then it is call it Stokes Raman scattering, or it is of a frequency that is higher, and then it is known known as anti-Stokesas anti-Stokes Raman Raman scattering scattering [20]. [The20]. Stokes The Stokes band bandforms formswhen whenthe molecule, the molecule, after after interactinginteracting with the with excitation the excitation radiation, radiation, shifts shifts to a tohigher a higher vibratory vibratory level level and and the the scattered scattered photonphoton has has energy energy lower lower by by the the energy energy difference difference between between the levels ofof vibrationalvibra- energy. tional energy.On On the the other other hand, hand, the the anti-Stokes anti-Stokes band band may may appear appear if if thethe moleculemolecule waswas at the excited the excited oscillatoryoscillatory level level before before the the impact impact of of the the excitation excitation radiation—that radiation—that way way there there is a high is a high probabilityprobability that that it will it will return return to tothe the basic basic oscillatory oscillatory level. level. The The scattered scattered photon photon will have will have anan energy energy greater greater by by the the differenc differencee in in energy of thethe oscillatingoscillating energyenergy levelslevels [ 21]. In anti- [21]. In anti-StokesStokes Raman scattering, the the photon photon will will collect collect energy energy from from the the bond