Phase-sensitive detection in Raman Tweezers: biological applications

In the biomedicine field, scientists act as detectives working hard to unravel the mysteries surrounding cells. To this end, Raman spectroscopy has revealed itself particularly useful, providing information concerning both the chemical composition and the structural conformation of the investigated samples. If compared to many other techniques devoted to the identification of microorganisms (as for instance fluorescence spectroscopy), Raman spectroscopy presents the relevant advantage of providing sharp peaks correlated to vibrational modes of the investigated sample. Moreover, it is a noninvasive technique, not requiring the addition of chemical agents or labels for a sample identification. Therefore, Raman spectra behave as the fingerprints of the analyzed sample. Hence, the Raman spectroscopy technique represents a powerful tool to detect cellular transformations in real time, contributing to elucidate many biochemical processes even in living cells. However, the analytical capabilities of Raman spectroscopy are limited by its inability to manipulate, and therefore analyze the samples without making physical contact or disturbing their unique environment. This limitation has been resolved by coupling Raman spectroscopy to a technology called Optical Tweezers (OT). The new method, termed Raman Tweezers (RT), employs Optical Tweezers to trap a microsized object in order to confine its motion for Raman spectroscopic analysis. Optical Tweezers are based on the force exerted on micrometer-sized particles by a strongly focused laser beam. They allow trapping and manipulation of single particles without any mechanical contact. The technique, which was first developed by Arthur Ashkin et al. in 1970, has the ability to control objects ranging in size from 5 nm to over 100 micron, among which atoms, viruses, bacteria, proteins, cells, or other biological molecules. In such a way, a single selected particle can be spectroscopically analyzed in its natural environment, without any need to fix it to a substrate. Therefore, Raman Tweezers have the capability to analyze a molecule from each angle and therefore provide more accurate information about identity, structure, and conformation. In addition, sample confinement in the laser beam focus maximizes the excitation and collection of Raman scattering and, at the same time, reduces the intrinsic interference effects due to the cover plate. This optical trap also allows Raman Tweezers to easily separate molecules for an isolated study, such as their response to different conditions and/or treatments. The first publication on using Raman Tweezers for living cells dates back to 2002. In their work, Ajito and Torimitsu, by combining the laser trapping technique with near-infrared Raman (NIR) spectroscopy, analyzed single cellular organelles in the nanometer range. The samples were synaptosomes, nerve-ending particles (about 500-700 nm in diameter) isolated from a neuron in a rat brain, dispersed in the phosphate buffer solution. The NIR laser Raman trapping system captured a single synaptosome without photochemical damage and provided a Raman spectrum of the sample with less fluorescence background. Over the course of the next six years, the techniques of Raman Tweezers have made unique and revolutionary contributions to the experimental studies in the fields of biophysics. Raman Tweezers has been applied for the identification and characterization of single optically trapped microparticles, red blood cells, yeast cells, bacterial cells, and liposomal membranes. For instance, a Raman Tweezers system was used to control and to characterize the coagulation of two liquid aerosol droplets and to investigate the formation and properties of multiphase aerosol. Recently, RT has also been reported to identify the bacterial spores in aqueous solution, in which bacterial spores are discriminated from non-biological particles, such as polystyrene spheres, on the basis of Raman signature. Unilamellar phospholipid vesicle were optically trapped and the effect of optical forces on the lipid membrane shape was investigated by using confocal Raman microscopy. Near infrared laser tweezers Raman spectroscopy system was also applied to study the effect of alcohol on single human red blood cells and to distinguish activation-dependent phases of T cells. As matter of fact, Raman Tweezers has the potential to become an incredibly effective diagnostic tool, and therefore holds great promise in the field of biomedicine for distinguishing between normal and diseased cells. In my PhD-work, mainly performed in the Laser Spectroscopy and Optical Manipulation group of Prof. A. Sasso, I have started a new research activity consisting of the Raman analysis of single particles of bio-technological interest. To achieve this goal, the following steps have been followed: - Build-up of a RT system allowing Raman analysis of an optically trapped cell in a non-invasive way. - Implementation of a novel electronics method, allowing an optimum suppression of scattering signal from the environment surrounding the optically trapped object. - Applicability test of the Raman Tweezers technique to model experiments. In particular, I have focused my research on the characterization of normal and thalassemic red blood cells. Since the aim of my thesis is to study living cells in vivo, with particular reference to red blood cells (RBCs), the main challenges have been to find correct laser wavelength and intensity, alignment and calibration of the Raman Tweezers system and finally its application to demonstrate its usefulness. Erythrocyte are used as model organism in these Raman Tweezers experiments. In fact, the human RBCs represent a very simple cell, they are devoid of the nucleus and are rich in hemoglobin (Hb), a protein composed of four globular protein with an embedded heme group. The heme group presents a strong Hb-absorption band in the visible region. This feature gives rise to a resonant enhancement of the Hb-signal, when it is an excitation wavelength closed to the heme electronic absorption bands. In such a way, it becomes possible to acquire the Raman spectrum of the Hb inside a single erythrocyte without any influence of the surrounding globin or other parts of the RBC crossed by the Raman probe. This is a quite interesting issue for the characterization of Hb-related blood diseases, such as thalassemias. Therefore, it has been chosen a doubled frequency Nd:YVO laser (at 532 nm) to excite the Raman spectra. The most widespread RT systems use two separate beams for trapping and the Raman excitation with a single objective. This configuration has the advantage that the wavelength and the power of the trapping beam and the Raman probe can be adjusted independently. Also in the present work, it has been used a two-wavelength Raman Tweezers system, where an IR laser (Nd:YAG at 1064 nm) has been used to trap the sample. This selection has revealed to be quite useful, mainly because it becomes possible to study living cells (RBCs) for long time periods, keeping the level of photo-damage low. After building the RT set-up, it has been possible to characterize the system by acquiring the Raman spectra of trapped polystyrene beads. By this preliminary study, it has been clear the presence of a general problem affecting both Raman spectrometers and Raman Tweezers systems, i.e. the background caused by the environment surrounding the sample under investigation. It is possible to find many practical experimental situations in which the inelastic as well as elastic scattering from the solvent, in which the sample is embedded, can affect severely the reliability and reproducibility of the measurements. This happens, for instance, when the solvent has strong Raman-active bands that spectrally overlap with the sample Raman features. In this case, reasonable but often distorted spectra are created using either manual or automatic subtraction procedures. This drawback is particularly relevant when Raman spectroscopy is used to monitor the health state of single microorganism. In fact, the diagnosis of cellular disorders is often performed by monitoring the relative intensities of selected Raman peaks. Therefore, it is required a high accuracy in subtracting the solvent contribution. To tackle this problem, during my PhD work, I have implemented a novel method that allows acquiring Raman spectra of a single trapped particle free from any background contribution. The method is based on the use of two collinear and copropagating laser beams. The first is devoted to trapping (trap laser), while the second one is used to excite the Raman transitions (pump laser). The trap laser moves the trapped particle periodically, by means of a galvomirror, back and forth across the pump laser. The back-scattered photons are analyzed by a spectrometer and detected by a photomultiplier. Then, the resulting signal is sent to a lock-in amplifier for phase-sensitive detection. The obtained results demonstrate that our Raman Tweezers system may find valuable applications in rapid sensing of biological samples in aqueous solutions. Finally, it has been demonstrated the potential of the developed Raman Tweezers system as a diagnostic tool to study a specific disease related to oxygenation capability of individual red blood cells, known as thalassemia. Thalassemia is the name of a group of genetic blood disorders. In thalassemia, the genetic defect results in reduced rate of synthesis of one of the globin chains that make up the Hb. If there is a reduced synthesis of one of the globin chains, the RBCs do not form properly and cannot carry sufficient oxygen. By way of a resonant excitation of Hb Raman bands, it has been examined the oxygenation capability of both normal and thalassemic erythrocytes. A reduction of this fundamental erythrocyte function for thalassemia has been shown. Raman spectroscopy has been also used to draw hemoglobin distribution inside single erythrocytes. The results have confirmed the characteristic anomaly (target shape), occurring in thalassemia and some other blood disorders. Finally, the deformability of thalassemic erythrocytes has been quantified by measuring the membrane shear modulus using an Optical Stretcher (double-trap system). The success of the spectroscopical and mechanical characterization of normal and thalassemic RBCs reported in this thesis provide an interesting starting point to explore the application of Raman Tweezers systems in the analysis of several blood disorders