Assessment of shifted excitation Raman difference spectroscopy in highly fluorescent biological samples

Shifted excitation Raman difference spectroscopy (SERDS) can be used as an instrumental baseline correction technique to retrieve Raman bands in highly fluorescent samples. Genipin (GE) cross-linked equine pericardium (EP) was used as a model system since a blue pigment is formed upon cross-linking, which results in a strong fluorescent background in the Raman spectra. EP was cross-linked with 0.25% GE solution for 0.5 h, 2 h, 4 h, 6 h, 12 h, and 24 h, and compared with corresponding untreated EP. Raman spectra were collected with three different excitation wavelengths. For the assessment of the SERDS technique, the preprocessed SERDS spectra of two excitation wavelengths (784 nm-786 nm) were compared with the mathematical baseline-corrected Raman spectra at 785 nm excitation using extended multiplicative signal correction, rubberband, the sensitive nonlinear iterative peak and polynomial fitting algorithms. Whereas each baseline correction gave poor quality spectra beyond 6 h GE crosslinking with wave-like artefacts, the SERDS technique resulted in difference spectra, that gave superior reconstructed spectra with clear collagen and resonance enhanced GE pigment bands with lower standard deviation. Key for this progress was an advanced difference optimization approach that is described here. Furthermore, the results of the SERDS technique were independent of the intensity calibration because the system transfer response was compensated by calculating the difference spectrum. We conclude that this SERDS strategy can be transferred to Raman studies on biological and non-biological samples with a strong fluorescence background at 785 nm and also shorter excitation wavelengths which benefit from more intense scattering intensities and higher quantum efficiencies of CCD detectors. This journal i

FLIm and raman spectroscopy for investigating biochemical changes of bovine pericardium upon genipin cross-linking

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm-1. These were validated through density functional theory calculations as cross-linker-specific bands. A multivariate multiple regression model was developed to enhance the biochemical specificity of FLIm parameters fluorescence intensity ratio (R2 = 0.92) and lifetime (R2 = 0.94)) with Raman spectral results. FLIm and Raman spectroscopy detected biochemical changes occurring in the collagenous tissue during the cross-linking process that were characterized by the formation of a blue pigment which affected the tissue fluorescence and scattering properties. In conclusion, FLIm parameters and Raman spectroscopy were used to monitor the degree of cross-linking non-destructively. © 2020 by the authors. Licensee MDPI, Basel, Switzerland

Переработка отходов лесной промышленности Томской области методом гранулирования

Дипломная работа посвящена обзору отходов лесной промышленности и способам их переработки. Проведен обзор методов переработки отходов лесной промышленности. Приведена разработка технологии производства топливных гранул на примере предприятия ООО "СибирьТомЛес".The graduate work is devoted to the review of forest industry wastes and ways of their processing. The review of methods of processing of wastes of forest industry is carried out. The development of technology for the production of fuel pellets is exemplified by the example of the enterprise LLC "SibirTomLes"

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

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.Comment: Accepted and Published by "Sensors" Journal, 19 pages, 8 figure

Raman-Differenzspektroskopie mit zwei verschiedenen Anregungswellenlängen zur untergrundfreien und bildgebenden Untersuchung von biologischen Proben

Mit Hilfe der Raman-Spektroskopie ist es möglich, Informationen über die chemische Zusammensetzung einer Probe zu erhalten, ohne diese zu beschädigen oder im Vorfeld zu markieren. Daher findet die Raman-Spektroskopie immer häufiger Anwendung für die Untersuchung komplexer biologischer Proben wie Zellen, Bakterien und Gewebe. Dabei kann es zur Anregung von Autofluoreszenz in niedrig konzentrierten Substanzen und Chromophoren wie Hämabbauprodukten kommen, welche einen hohen spektralen Untergrund im Raman-Spektrum erzeugt und so zu einer Maskierung der Raman-Banden führen kann. Der Schwerpunkt dieser Dissertation ist die Verwendung der instrumentellen Basislinienkorrekturmethode SERDS (engl. shifted excitation Raman difference spectroscopy) für die Untersuchung von biologischen Proben. Bei dieser Methode werden am gleichen Messpunkt zwei Raman-Spektren mit zwei verschiedenen Anregungswellenlängen gemessen und diese voneinander subtrahiert. Das daraus resultierende SERDS-Spektrum ist idealerweise ein untergrundfreies Differenzspektrum, das der ersten Ableitung eines Raman-Spektrums sehr ähnlich ist. Ziel dieser Dissertation war die Evaluierung und Optimierung der SERDS-Methode und der SERDS-Bildgebung an biologischen Proben. Um die Aufnahme eines SERDS-Bildes zu beschleunigen und den Untergrund zu minimieren, wurde erstmalig die Weitfeld-SERDS-Bildgebung mittels der interlaced nod and shuffle Technik, eine Technik aus der Astrophysik, unter Verwendung sehr schnell wechselnder Laserwellenlängen verwendet