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Evaluation of shifted excitation Raman difference spectroscopy and comparison to computational background correction methods applied to biochemical Raman spectra
Raman spectroscopy provides label-free biochemical information from tissue samples without complicated sample preparation. The clinical capability of Raman spectroscopy has been demonstrated in a wide range of in vitro and in vivo applications. However, a challenge for in vivo applications is the simultaneous excitation of auto-fluorescence in the majority of tissues of interest, such as liver, bladder, brain, and others. Raman bands are then superimposed on a fluorescence background, which can be several orders of magnitude larger than the Raman signal. To eliminate the disturbing fluorescence background, several approaches are available. Among instrumentational methods shifted excitation Raman difference spectroscopy (SERDS) has been widely applied and studied. Similarly, computational techniques, for instance extended multiplicative scatter correction (EMSC), have also been employed to remove undesired background contributions. Here, we present a theoretical and experimental evaluation and comparison of fluorescence background removal approaches for Raman spectra based on SERDS and EMSC.
Multimode Fabry-Perot Interferometer Probe based on Vernier Effect for Enhanced Temperature Sensing
New miniaturized sensors for biological and medical applications must be adapted to the measuring environments and they should provide a high measurement resolution to sense small changes. The Vernier effect is an effective way of magnifying the sensitivity of a device, allowing for higher resolution sensing. We applied this concept to the development of a small-size optical fiber Fabry–Perot interferometer probe that presents more than 60-fold higher sensitivity to temperature than the normal Fabry–Perot interferometer without the Vernier effect. This enables the sensor to reach higher temperature resolutions. The silica Fabry–Perot interferometer is created by focused ion beam milling of the end of a tapered multimode fiber. Multiple Fabry–Perot interferometers with shifted frequencies are generated in the cavity due to the presence of multiple modes. The reflection spectrum shows two main components in the Fast Fourier transform that give rise to the Vernier effect. The superposition of these components presents an enhancement of sensitivity to temperature. The same effect is also obtained by monitoring the reflection spectrum node without any filtering. A temperature sensitivity of −654 pm/°C was obtained between 30 °C and 120 °C, with an experimental resolution of 0.14 °C. Stability measurements are also reported.
Application of High-Throughput Screening Raman Spectroscopy (HTS-RS) for Label-Free Identification and Molecular Characterization of Pollen
Pollen studies play a critical role in various fields of science. In the last couple of decades, replacement of manual identification of pollen by image-based methods using pollen morphological features was a great leap forward, but challenges for pollen with similar morphology remain, and additional approaches are required. Spectroscopy approaches for identification of pollen, such as Raman spectroscopy has potential benefits over traditional methods, due to the investigation of the intrinsic molecular composition of a sample. However, current Raman-based characterization of pollen is complex and time-consuming, resulting in low throughput and limiting the statistical significance of the acquired data. Previously demonstrated high-throughput screening Raman spectroscopy (HTS-RS) eliminates the complexity as well as human interaction by incorporation full automation of the data acquisition process. Here, we present a customization of HTS-RS for pollen identification, enabling sampling of a large number of pollen in comparison to other state-of-the-art Raman pollen investigations. We show that using Raman spectra we are able to provide a preliminary estimation of pollen types based on growth habits using hierarchical cluster analysis (HCA) as well as good taxonomy of 37 different Pollen using principal component analysis-support vector machine (PCA-SVM) with good accuracy even for the pollen specimens sharing similar morphological features. Our results suggest that HTS-RS platform meets the demands for automated pollen detection making it an alternative method for research concerning pollen.
A new strategy for silver deposition on Au nanoparticles with the use of peroxidase-mimicking DNAzyme monitored by Localized Surface Plasmon Resonance technique
Peroxidase-mimicking DNAzyme was applied as a catalyst of silver deposition on gold nanoparticles. This DNAzyme is formed when hemin binds to the G-quadruplex-forming DNA sequence. Such a system is able to catalyze a redox reaction with a one- or two-electron transfer. The process of silver deposition was monitored via a localized surface plasmon resonance technique (LSPR), which allows one to record scattering spectrum of a single nanoparticle. Our study showed that DNAzyme is able to catalyze silver deposition. The AFM experiments proved that DNAzyme induced the deposition of silver shells of approximately 20 nm thickness on Au nanoparticles (AuNPs). Such an effect is not observed when hemin is absent in the system. However, we noticed non-specific binding of hemin to the capture oligonucleotides on a gold NP probe that also induced some silver deposition, even though the capture probe was unable to form G-quadruplex. Analysis of SEM images indicated that the surface morphology of the silver layer deposited by DNAzyme is different from that obtained for hemin alone. The proposed strategy of silver layer synthesis on gold nanoparticles catalyzed by DNAzyme is an innovative approach and can be applied in bioanalysis (LSPR, electrochemistry) as well as in material sciences.
Microfluidic Cultivation and Laser Tweezers Raman Spectroscopy of E. coli under Antibiotic Stress
Analyzing the cells in various body fluids can greatly deepen the understanding of the mechanisms governing the cellular physiology. Due to the variability of physiological and metabolic states, it is important to be able to perform such studies on individual cells. Therefore, we developed an optofluidic system in which we precisely manipulated and monitored individual cells of Escherichia coli. We tested optical micromanipulation in a microfluidic chamber chip by transferring individual bacteria into the chambers. We then subjected the cells in the chambers to antibiotic cefotaxime and we observed the changes by using time-lapse microscopy. Separately, we used laser tweezers Raman spectroscopy (LTRS) in a different micro-chamber chip to manipulate and analyze individual cefotaxime-treated E. coli cells. Additionally, we performed conventional Raman micro-spectroscopic measurements of E. coli cells in a micro-chamber. We found observable changes in the cellular morphology (cell elongation) and in Raman spectra, which were consistent with other recently published observations. The principal component analysis (PCA) of Raman data distinguished between the cefotaxime treated cells and control. We tested the capabilities of the optofluidic system and found it to be a reliable and versatile solution for this class of microbiological experiments.
UV absorption spectroscopy in water-filled antiresonant hollow core fibers for pharmaceutical detection
Due to a worldwide increased use of pharmaceuticals and, in particular, antibiotics, a growing number of these substance residues now contaminate natural water resources and drinking supplies. This triggers a considerable demand for low-cost, high-sensitivity methods for monitoring water quality. Since many biological substances exhibit strong and characteristic absorption features at wavelengths shorter than 300 nm, UV spectroscopy presents a suitable approach for the quantitative identification of such water-contaminating species. However, current UV spectroscopic devices often show limited light-matter interaction lengths, demand sophisticated and bulky experimental infrastructure which is not compatible with microfluidics, and leave large fractions of the sample analyte unused. Here, we introduce the concept of UV spectroscopy in liquid-filled anti-resonant hollow core fibers, with large core diameters and lengths of approximately 1 m, as a means to overcome such limitations. This extended light-matter interaction length principally improves the concentration detection limit by two orders of magnitude while using almost the entire sample volume—that is three orders of magnitude smaller compared to cuvette based approaches. By integrating the fibers into an optofluidic chip environment and operating within the lowest experimentally feasible transmission band, concentrations of the application-relevant pharmaceutical substances, sulfamethoxazole (SMX) and sodium salicylate (SS), were detectable down to 0.1 µM (26 ppb) and 0.4 µM (64 ppb), respectively, with the potential to reach significantly lower detection limits for further device integration.
Raman Signal Enhancement Tunable by Gold-Covered Porous Silicon Films with Different Morphology
The ease of fabrication, large surface area, tunable pore size and morphology as well surface modification capabilities of a porous silicon (PSi) layer make it widely used for sensoric applications. The pore size of a PSi layer can be an important parameter when used as a matrix for creating surface-enhanced Raman scattering (SERS) surfaces. Here, we evaluated the SERS activity of PSi with pores ranging in size from meso to macro, the surface of which was coated with gold nanoparticles (Au NPs). We found that different pore diameters in the PSi layers provide different morphology of the gold coating, from an almost monolayer to 50 nm distance between nanoparticles. Methylene blue (MB) and 4-mercaptopyridine (4-MPy) were used to describe the SERS activity of obtained Au/PSi surfaces. The best Raman signal enhancement was shown when the internal diameter of torus-shaped Au NPs is around 35 nm. To understand the role of plasmonic resonances in the observed SERS spectrum, we performed electromagnetic simulations of Raman scattering intensity as a function of the internal diameter. The results of these simulations are consistent with the obtained experimental data
Noise Sources and Requirements for Confocal Raman Spectrometers in Biosensor Applications
Raman spectroscopy probes the biochemical composition of samples in a non-destructive, non-invasive and label-free fashion yielding specific information on a molecular level. Nevertheless, the Raman effect is very weak. The detection of all inelastically scattered photons with highest efficiency is therefore crucial as well as the identification of all noise sources present in the system. Here we provide a study for performance comparison and assessment of different spectrometers for confocal Raman spectroscopy in biosensor applications. A low-cost, home-built Raman spectrometer with a complementary metal-oxide-semiconductor (CMOS) camera, a middle price-class mini charge-coupled device (CCD) Raman spectrometer and a laboratory grade confocal Raman system with a deeply cooled CCD detector are compared. It is often overlooked that the sample itself is the most important “optical” component in a Raman spectrometer and its properties contribute most significantly to the signal-to-noise ratio. For this purpose, different representative samples: a crystalline silicon wafer, a polypropylene sample and E. coli bacteria were measured under similar conditions using the three confocal Raman spectrometers. We show that biosensor applications do not in every case profit from the most expensive equipment. Finally, a small Raman database of three different bacteria species is set up with the middle price-class mini CCD Raman spectrometer in order to demonstrate the potential of a compact setup for pathogen discrimination.
Wide Field Spectral Imaging with Shifted Excitation Raman Difference Spectroscopy Using the Nod and Shuffle Technique
Wide field Raman imaging using the integral field spectroscopy approach was used as a fast, one shot imaging method for the simultaneous collection of all spectra composing a Raman image. For the suppression of autofluorescence and background signals such as room light, shifted excitation Raman difference spectroscopy (SERDS) was applied to remove background artifacts in Raman spectra. To reduce acquisition times in wide field SERDS imaging, we adapted the nod and shuffle technique from astrophysics and implemented it into a wide field SERDS imaging setup. In our adapted version, the nod corresponds to the change in excitation wavelength, whereas the shuffle corresponds to the shifting of charges up and down on a Charge-Coupled Device (CCD) chip synchronous to the change in excitation wavelength. We coupled this improved wide field SERDS imaging setup to diode lasers with 784.4/785.5 and 457.7/458.9 nm excitation and applied it to samples such as paracetamol and aspirin tablets, polystyrene and polymethyl methacrylate beads, as well as pork meat using multiple accumulations with acquisition times in the range of 50 to 200 ms. The results tackle two main challenges of SERDS imaging: gradual photobleaching changes the autofluorescence background, and multiple readouts of CCD detector prolong the acquisition time.
Volume fraction determination of binary liquid mixtures by measurement of the equalization wavelength
A method for determination of the volume fraction in binary liquid mixtures by measurement of the equalization wavelength of intermodal interference of modes LP01 and LP11 in a liquid core optical fiber is presented in this paper. This method was studied using a liquid core optical fiber with fused silica cladding and a core made up of a binary silicon oil/chloroform liquid mixture with different volume fractions of chloroform. The interference technique used allows us to determine the chloroform volume fraction in the binary mixture with accuracy better than 0.1%. One of the most attractive advantages of presented method is very small volume of investigated mixture needed, as only a few hundred picoliters are necessary for reliable results. © 2010 by the authors.