Organelle specific simultaneous Raman/green fluorescence protein microspectroscopy for living cell physicochemical studies

Abstract

We demonstrate a novel bio-spectroscopic technique, "simultaneous Raman/GFP microspectroscopy". It enables organelle specific Raman microspectroscopy of living cells. Fission yeast, Schizosaccharomyces pombe, whose mitochondria are green fluorescence protein (GFP) labeled, is used as a test model system. Raman excitation laser and GFP excitation light irradiate the sample yeast cells simultaneously. GFP signal is monitored in the anti-Stokes region where interference from Raman scattering is negligibly small. Of note, 13 568 Raman spectra measured from different points of 19 living yeast cells are categorized according to their GFP fluorescence intensities, with the use of a two-component multivariate curve resolution with alternate least squares (MCR-ALS) analysis in the anti-Stokes region. This categorization allows us to know whether or not Raman spectra are taken from mitochondria. Raman spectra specific to mitochondria are obtained by an MCR-ALS analysis in the Stokes region of 1389 strongly GFP positive spectra. Two mitochondria specific Raman spectra have been obtained. The first one is dominated by protein Raman bands and the second by lipid Raman bands, being consistent with the known molecular composition of mitochondria. In addition, the second spectrum shows a strong band of ergosterol at 1602 cm −1 , previously reported as "Raman spectroscopic signature of life ofanti-Stokes Raman spectroscopy, mitochondria, Raman spectroscopic signature of life, Schizosaccharomyces pombe, simultaneous Raman/GFP microspectroscopy, yeast

Online Liquid Chromatography–Raman Spectroscopy Using the Vertical Flow Method

“…Various methods for the analysis of the 2D data have been proposed for chemometrics. − Among them, the multivariate curve resolution alternate least square (MCR-ALS) analysis was employed to retrieve the spectral ( V l ′) and temporal ( U l ′) components from the 2D data for comparison with the SVD and reconstruction method . The spatial distribution of several compounds exhibiting a unique spectral pattern is taken from the Raman mapping image using the MCR-ALS method . The Frobenius norm ∥ M – U l ′ V l ′∥ 2 and the L1 norms of U l ′ and V l ′ multiplied by 10 –5 were minimized by the least square method under the non-negativity constraint for U l ′ and V l ′.…”

“…36 The spatial distribution of several compounds exhibiting a unique spectral pattern is taken from the Raman mapping image using the MCR-ALS method. 38 The Frobenius norm ∥M−U l ′V l ′∥ 2 and the L1 norms of U l ′ and V l ′ multiplied by 10 −5 were minimized by the least square method under the non-negativity constraint for U l ′ and V l ′. Figure 6a,b shows the deduced spectral and temporal vectors of the three positional isomers.…”

A novel portable Raman scattering platform for antibiotic screening in pig urine

“…Multivariate curve resolution-ALS was performed using the IGOR Pro (WaveMatrics, Inc., Oregon, USA). More than 300 spectra from three datasets of enrofloxacin in water, spiked pig urine, and urine samples were analyzed using MCR-ALS to verify the concentration of antibiotics [ 24 ]. Standard antibiotics Raman spectra were used as references for the analysis.…”

A Comprehensive Review on Raman Spectroscopy Applications

“…As reported by Li et al [161], this approach could be used to evaluate the functionalities of lipid droplets and bacteria by analyzing the finger print region showing the relative abundance of chemical functions, as reported in Figure 6. This approach was also used to monitor the organelle in vivo [162,163], the chromosomes [164], and for the evaluation of the hemoglobin in red blood cells and for the density of water inside the cells [165]. This last study is particularly interesting because it proved the presence of a strong and highly organized hydrogen bond network in the cytosol.…”

“…Nasdala et al [169] reported that this technique can investigate inside the inner structure of mineralogic samples in different temperature and pressure conditions. These in This approach was also used to monitor the organelle in vivo [162,163], the chromosomes [164], and for the evaluation of the hemoglobin in red blood cells and for the density of water inside the cells [165]. This last study is particularly interesting because it proved the presence of a strong and highly organized hydrogen bond network in the cytosol.…”