8 research outputs found
Narrowband spectroscopy by all-optical correlation of broadband pulses
High peak power ultrafast lasers are widely used in nonlinear spectroscopy
but often limit its spectral resolution because of the broad frequency
bandwidth of ultrashort laser pulses. Improving the resolution by achieving
spectrally narrow excitation of, or emission from, the resonant medium by means
of multi-photon interferences has been the focus of many recent developments in
ultrafast spectroscopy. We demonstrate an alternative approach, in which high
resolution is exercised by detecting narrow spectral correlations between
broadband excitation and emission optical fields. All-optical correlation
analysis, easily incorporated into the traditional spectroscopic setup, enables
direct, robust and simultaneous detection of multiple narrow resonances with a
single femtosecond pulse.Comment: 5 pages, 4 figures, submitted to PR
Raman spectroscopic analysis of cell differentiation and death modes
Raman spectroscopy provides opportunities for non-invasive, non-destructive, label-free analysis of cell states based on changes in the biochemical composition of cells. We are investigating the suitability of Raman spectroscopy to assess the stages of human embryonic stem cell (hESC) differentiation towards pancreatic insulin-positive cells. Raman microspectrometry analysis has revealed macromolecular composition differences over time that distinguished cell populations differentiating to pancreatic cell types, such as by an increase in the protein-to-nucleic acid signal ratio and to distinguish the presence of insulin. Added insight into these macromolecular changes were provided by principal component analysis (PCA) of the data. However, the application of PCA can be difficult to interpret. The usefulness of non-negative matrix factorization was explored to improve the interpretability of overlapping Raman bands. We demonstrated the utility of this procedure by analyzing spectra to determine the cellular insulin or glucagon content. Thus, Raman spectroscopy can detect such differences in cells to detect the desired product as well as the potential to detect residual hESCs or the emergence of unwanted cells.
We also investigated the suitability of Raman spectroscopy to detect the onset and types of cell death. Apoptotic, necrotic or autophagic Chinese Hamster Ovary cells were compared to uninduced cultures using Raman spectroscopy and PCA. Furthermore, uninduced cells were compared to cells sorted at different stages of apoptosis to determine how early the onset of apoptosis could be detected. Changes were observed in several peaks during the course of cell death, with repeated changes observed in nucleic acid- and lipid-associated peaks, enabling the distinction of cell death modes. Application of such death monitoring capabilities to cellular therapy cultures should be even more useful, given the need for more process analytical technologies to address the often more variable performance of these cultures, especially when adaptive control is needed for primary cell derived manufacturing
Noise auto-correlation spectroscopy with coherent Raman scattering
Ultrafast lasers have become one of the most powerful tools in coherent
nonlinear optical spectroscopy. Short pulses enable direct observation of fast
molecular dynamics, whereas broad spectral bandwidth offers ways of controlling
nonlinear optical processes by means of quantum interferences. Special care is
usually taken to preserve the coherence of laser pulses as it determines the
accuracy of a spectroscopic measurement. Here we present a new approach to
coherent Raman spectroscopy based on deliberately introduced noise, which
increases the spectral resolution, robustness and efficiency. We probe laser
induced molecular vibrations using a broadband laser pulse with intentionally
randomized amplitude and phase. The vibrational resonances result in and are
identified through the appearance of intensity correlations in the noisy
spectrum of coherently scattered photons. Spectral resolution is neither
limited by the pulse bandwidth, nor sensitive to the quality of the temporal
and spectral profile of the pulses. This is particularly attractive for the
applications in microscopy, biological imaging and remote sensing, where
dispersion and scattering properties of the medium often undermine the
applicability of ultrafast lasers. The proposed method combines the efficiency
and resolution of a coherent process with the robustness of incoherent light.
As we demonstrate here, it can be implemented by simply destroying the
coherence of a laser pulse, and without any elaborate temporal scanning or
spectral shaping commonly required by the frequency-resolved spectroscopic
methods with ultrashort pulses.Comment: To appear in Nature Physic
Acoustic cell washing and raman spectroscopy technologies To address cell therapy bioprocess challenges
Many organizations are confronting the challenges of economically ensuring the manufacture of safe and efficacious cell therapy products. These processes often depend on devices and methods that were developed for only related applications, such as blood cell processing or scientific research. Thus, we are in a window of opportunity to tailor innovative technologies to address the emerging specialized needs of cell therapy manufacturing.
The most frequent unit operation is to wash cells between process stages, such as from DMSO containing cryopreservation medium to culture expansion medium. In particular for relatively small-scale autologous cell therapy processing, cell washing is imperfectly performed by closed system blood cell centrifuges or filters. We previously developed an acoustic cell separation device, widely used for over 15 years in CHO cell perfusion cultures. This technology acts as a non-fouling filter for months of operation, by using the forces generated in ultrasonic standing wave fields. These forces separate cells from medium based on differences in density and compressibility. Greater than 99.9% cell washing with 95% washed cell recovery efficiencies have been provided by our device. We also have recently enhanced the acoustic technology to perfuse 100 million cell/mL cultures, maintaining \u3e99% cell separation efficiencies. This provides an alternative high performance closed manufacturing system, to perfuse, concentrate and wash cells, with no physical filter barrier or mechanical moving parts.
While many clinical trials have had few adverse events, the great promise of cellular therapies comes with grave risks, such as from potentially oncogenic pluripotent cells present in embryonic stem cell derived populations. There is an urgent need for process analytical technologies to non-invasively monitor mammalian cell populations and improve the reliability of manufactured cell products. This includes to monitor both the expected differentiation as well as to detect unexpected cells in the process. Recently, technological advances have led to an explosive growth in the capabilities of Raman spectroscopy, increasing the potential for novel applications. We are developing the use of this spectroscopic technique to track cell development, by measuring macromolecular changes in cell samples from cultures where stem cells are differentiated towards insulin-producing cells for the treatment of diabetes. Raman spectroscopy has great potential to provide continuous on-line assessment of cell quality during the manufacture of cell-derived therapeutic cells
Process Analytical Utility of Raman Microspectroscopy in the Directed Differentiation of Human Pancreatic Insulin-Positive Cells
Continued
advances toward cell-based therapies for human disease
generate a growing need for unbiased and label-free monitoring of
cellular characteristics. We used Raman microspectroscopy to characterize
four important stages in the 26-day directed differentiation of human
embryonic stem cells (hESCs) to insulin-positive cells. The extent
to which the cells retained spectroscopic features of pluripotent
cells or developed spectroscopic features suggestive of pancreatic
endocrine cells, as well as assessing the homogeneity of the cell
populations at these developmental stages, were of particular interest.
Such information could have implications for the utility of Raman
microspectroscopy process analysis for the generation of insulin-positive
cells from hESCs. Because hESC seeding density influences the subsequent
pancreatic development, three different seeding density cultures were
analyzed. Transcription factor and other marker analyses assessed
the progress of the cells through the relevant developmental stages.
Increases in the Raman protein-to-nucleic acid band ratios were observed
at the final endocrine stage analyzed, but this increase was less
than expected. Also, high glycogen band intensities, somewhat unexpected
in pancreatic endocrine cells, suggested the presence of a substantial
number of glycogen containing cells. We discuss the potential process
analytical technology application of these findings and their importance
for cell manufacturing
Spin alignment and violation of the OZI rule in exclusive omega and phi production in pp collisions
Exclusive production of the isoscalar vector mesons omega and phi is measured with a 190 GeV/c proton beam impinging on a liquid hydrogen target. Cross section ratios are determined in three intervals of the Feynman variable x(F) of the fast proton. A significant violation of the OZI rule is found, confirming earlier findings. Its kinematic dependence on xF and on the invariant mass M-pV of the system formed by fast proton p(fast) and vector meson V is discussed in terms of diffractive production of p(fast) V resonances in competition with central production. The measurement of the spin density matrix element rho(00) of the vector mesons in different selected reference frames provides another handle to distinguish the contributions of these two major reaction types. Again, dependences of the alignment on x(F) and on M-pV are found. Most of the observations can be traced back to the existence of several excited baryon states contributing to omega production which are absent in the case of the phi meson. Removing the low-mass M-pV resonant region, the OZI rule is found to be violated by a factor of eight, independently of x(F). (C) 2014 CERN for the benefit of the COMPASS Collaboration. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).DFG [1102]; ICTP programme for Training and Research in Italian Laboratories (TRIL); German Bundesministerium fur Bildung und Forschung; Czech Republic MEYS Grants [ME492, LA242]; SAIL (CSR), Govt. of India; CERN-RFBR Grants [08-02-91009, 12-02-91500]; Portuguese FCT - Fundacao para a Ciencia e Tecnologia [CERN/FP/109323/2009, CERN/FP/116376/2010, CERN/FP/123600/2011]; MEXT; JSPS [18002006, 20540299, 18540281]; Daiko Foundation; Yamada Foundation; DFG cluster of excellence 'Origin and Structure of the Universe'; EU FP7 (HadronPhysics3) [283286]; Israel Science Foundation; Polish NCN Grant [DEC-2011/01/M/ST2/02350
Piezoelectric, dielectric and pyroelectric properties of 0-3 ceramic-polymer composites
The circular samples of composites were made from PZT ceramic grains dispersed in polymers (epoxy or rubber). Pressure dependencies of the hydrostatic piezoelectric coefficient dh and permittivity ε were measured in the range up to 60 MPa. The hydrostatic strain coefficient dh, the hydrostatic voltage coefficient gh and figure of merit dh·gh were determined using the static method. The pressure dependence of the hydrostatic coefficients was measured for several successive cycles. Experimental dependence of the charge on pressure and temperature was fitted by multi parameter function. The pressure and temperature dependencies of the hydrostatic dh and pyroelectric p3 coefficients were determined. It was found that the hydrostatic strain coefficient dh and hydrostatic voltage coefficient gh decreased and pyroelectric coefficient p3 increased with pressure at constant temperature.DFG [1102]; ICTP programme for Training and Research in Italian Laboratories (TRIL); German Bundesministerium fur Bildung und Forschung; Czech Republic MEYS Grants [ME492, LA242]; SAIL (CSR), Govt. of India; CERN-RFBR Grants [08-02-91009, 12-02-91500]; Portuguese FCT - Fundacao para a Ciencia e Tecnologia [CERN/FP/109323/2009, CERN/FP/116376/2010, CERN/FP/123600/2011]; MEXT; JSPS [18002006, 20540299, 18540281]; Daiko Foundation; Yamada Foundation; DFG cluster of excellence 'Origin and Structure of the Universe'; EU FP7 (HadronPhysics3) [283286]; Israel Science Foundation; Polish NCN Grant [DEC-2011/01/M/ST2/02350