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Label‐Free Analysis of Cellular Biochemistry by Raman Spectroscopy and Microscopy

Iwan W. Schie, Thomas Huser

10.1002/cphy.c120025

Source: Volume 3, Issue 2, April 2013

Published online: April 2013

Full Article on Wiley Online Library



Abstract

We review the biomedical applications of Raman spectroscopy at the single cell and tissue level. Raman scattering is the inelastic scattering of light by molecular bonds resulting in a wealth of spectral bands, which enable the identification of biological materials and the nondestructive analysis of dynamic changes in their biochemistry. We briefly review the basics behind highly sensitive Raman spectroscopy and highlight recent applications to biomedical research. We discuss advanced chemometrics methods that are utilized to analyze Raman spectral data and which permit one, for example, to distinguish between normal and diseased cells or which enable one to follow the differentiation of stem cells without perturbing the cellular biochemistry. We also discuss advanced coherent Raman scattering techniques, such as coherent anti‐Stokes Raman scattering and stimulated Raman scattering, which allow for the molecularly specific imaging of cells, tissues, and entire organisms in vitro and in vivo. © 2013 American Physiological Society. Compr Physiol 3:941‐956, 2013.

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Figure 1. Figure 1.

Schematic description of several Raman scattering processes important for biomedical applications. (upper left‐hand box) Stokes‐shifted Raman scattering. Here, a photon with frequency is scattered by a molecular bond in the vibrational ground state (). As a result, the molecule‐photon pair spends a very short amount of time in a virtual state, where they are indistinguishable, only to then release a red‐shifted photon (Stokes‐shifted, frequency ) after which the molecule then remains in an exited vibrational state (). (upper right‐hand box) If the molecule is already in an exited vibrational state (), the scattering event leads to an even higher virtual state, from where a blue‐shifted photon (Stokes‐shifted, frequency ) is released returning the molecule to the vibrational ground state. (Lower box) Coherent anti‐Stokes Raman scattering: here, the interaction of a pump photon with a Stokes‐photon results in the molecule being elevated to the excited vibrational state, from where it can scatter another pump photon resulting in anti‐Stokes emission. Due to the high pulse energies required for this process, many molecules with the same resonant molecular group undergo this transition simultaneously, resulting in the coherent emission of anti‐Stokes photons.
Figure 2. Figure 2.

Raman spectra of bovine sperm cells. The image to the left was obtained by scanning the cells with a confocal laser‐scanning microscope at 488 nm wavelength while collecting the cellular autofluorescence. The box on the right hand side shows spectra obtained from either the head region of the cells (resulting in protein and DNA Raman spectra), as well as spectra obtained from the tail of the sperm cells (resulting in pure protein spectra). Raman spectra were also acquired with 488‐nm‐excitation wavelength and the broad fluorescence background has been subtracted.
Figure 3. Figure 3.

Raman spectra of human monocytes (THP‐1 cell line, American Type Tissue Culture) stained with the blue‐fluorescent nuclear live cell stain Hoechst 33342. The inset shows a fluorescence micrograph of an optically trapped, Hoechst‐stained monocyte. The red spectrum is the spontaneous Raman spectrum obtained from a Hoechst‐stained cell, resulting in the presence of additional stain‐related peaks when compared to an unstained monocyte (black spectrum). This observation leads us to conclude that these cells are trapped by their nucleus. The difference spectrum between the two cases is shown at the bottom of the figure.
Figure 4. Figure 4.

Cultured hepatocytes treated with fatty acids, resulting in the accumulation of large lipid droplets in the cell's cytoplasm. The spectra on the right hand side of the figure were obtained directly from a lipid droplet and from the cytoplasm in an area where no large lipid droplets were visible as indicated in the image acquired by coherent anti‐Stokes Raman scattering microscopy.
Figure 5. Figure 5.

Raman spectra of saturated palmitic acid, monounsaturated oleic acid and polyunsaturated linoleic acid, and linolenic acid. The dark orange bands highlight peaks of interest. Peaks 2 and 5 are associated with the degree of fatty acid bond instauration and show an increase in the spectra for mono and polyunsaturated fatty acids, whereas peaks 2, 3, 4, and 6 are decreasing with an increase in the number of unsaturated bonds. The change in the fatty acid chain packing structure can be seen in peak 1, where the solid palmitic acid exhibits very distinct peaks, and liquid unsaturated fatty acids have more convoluted peaks.
Figure 6. Figure 6.

Epi‐detected CARS image of adipocytes derived from mesenchymal stem cells. The signals from the bulk of the large droplets are mostly forward scattered, which is why we detect little to no CARS signals in the epi‐direction. Only in the circumference of the droplets is some of the signal emitted in the epi‐direction. Here, the polarization‐dependent nature of the CARS signal is also evident by the fact that only parts of the circumference where the molecules are aligned parallel to the polarization of the CARS excitation beam are visible.
Figure 7. Figure 7.

CARS image of a living C. elegans nematode scanned at the 2845 cm−1 lipid‐sensitive CH2 vibration. The image shows a single optical section through the worm revealing a large number of lipid droplets distributed throughout the organism. The image on the upper right hand side shows the same CARS image, but in binary form, threshold‐adjusted to reveal only the lipid droplets resulting in a 7.2% lipid area fraction. The lower right hand side shows a cross section of the worm as indicated in the CARS image revealing the average diameter of the droplets to be around 1 μm.
Figure 8. Figure 8.

Combined multiphoton image of rat cartilage with spontaneous Raman spectroscopy from points of interest. The green colored image shows the distribution of the collagen in the extracellular matrix acquired with second harmonic generation. The blue colored image shows the distribution of the CH2 vibration, primarily found in cellular lipid droplets and the cellular lipid bilayer. Successive Raman spectroscopy shows the chemical difference from various points of interest in the sample. Spectra from the cellular lipid droplets indicate the presence of highly unsaturated fatty acids in the lipid droplets, where as the spectra of the cartilage show peaks typically present in collagen.


Figure 1.

Schematic description of several Raman scattering processes important for biomedical applications. (upper left‐hand box) Stokes‐shifted Raman scattering. Here, a photon with frequency is scattered by a molecular bond in the vibrational ground state (). As a result, the molecule‐photon pair spends a very short amount of time in a virtual state, where they are indistinguishable, only to then release a red‐shifted photon (Stokes‐shifted, frequency ) after which the molecule then remains in an exited vibrational state (). (upper right‐hand box) If the molecule is already in an exited vibrational state (), the scattering event leads to an even higher virtual state, from where a blue‐shifted photon (Stokes‐shifted, frequency ) is released returning the molecule to the vibrational ground state. (Lower box) Coherent anti‐Stokes Raman scattering: here, the interaction of a pump photon with a Stokes‐photon results in the molecule being elevated to the excited vibrational state, from where it can scatter another pump photon resulting in anti‐Stokes emission. Due to the high pulse energies required for this process, many molecules with the same resonant molecular group undergo this transition simultaneously, resulting in the coherent emission of anti‐Stokes photons.



Figure 2.

Raman spectra of bovine sperm cells. The image to the left was obtained by scanning the cells with a confocal laser‐scanning microscope at 488 nm wavelength while collecting the cellular autofluorescence. The box on the right hand side shows spectra obtained from either the head region of the cells (resulting in protein and DNA Raman spectra), as well as spectra obtained from the tail of the sperm cells (resulting in pure protein spectra). Raman spectra were also acquired with 488‐nm‐excitation wavelength and the broad fluorescence background has been subtracted.



Figure 3.

Raman spectra of human monocytes (THP‐1 cell line, American Type Tissue Culture) stained with the blue‐fluorescent nuclear live cell stain Hoechst 33342. The inset shows a fluorescence micrograph of an optically trapped, Hoechst‐stained monocyte. The red spectrum is the spontaneous Raman spectrum obtained from a Hoechst‐stained cell, resulting in the presence of additional stain‐related peaks when compared to an unstained monocyte (black spectrum). This observation leads us to conclude that these cells are trapped by their nucleus. The difference spectrum between the two cases is shown at the bottom of the figure.



Figure 4.

Cultured hepatocytes treated with fatty acids, resulting in the accumulation of large lipid droplets in the cell's cytoplasm. The spectra on the right hand side of the figure were obtained directly from a lipid droplet and from the cytoplasm in an area where no large lipid droplets were visible as indicated in the image acquired by coherent anti‐Stokes Raman scattering microscopy.



Figure 5.

Raman spectra of saturated palmitic acid, monounsaturated oleic acid and polyunsaturated linoleic acid, and linolenic acid. The dark orange bands highlight peaks of interest. Peaks 2 and 5 are associated with the degree of fatty acid bond instauration and show an increase in the spectra for mono and polyunsaturated fatty acids, whereas peaks 2, 3, 4, and 6 are decreasing with an increase in the number of unsaturated bonds. The change in the fatty acid chain packing structure can be seen in peak 1, where the solid palmitic acid exhibits very distinct peaks, and liquid unsaturated fatty acids have more convoluted peaks.



Figure 6.

Epi‐detected CARS image of adipocytes derived from mesenchymal stem cells. The signals from the bulk of the large droplets are mostly forward scattered, which is why we detect little to no CARS signals in the epi‐direction. Only in the circumference of the droplets is some of the signal emitted in the epi‐direction. Here, the polarization‐dependent nature of the CARS signal is also evident by the fact that only parts of the circumference where the molecules are aligned parallel to the polarization of the CARS excitation beam are visible.



Figure 7.

CARS image of a living C. elegans nematode scanned at the 2845 cm−1 lipid‐sensitive CH2 vibration. The image shows a single optical section through the worm revealing a large number of lipid droplets distributed throughout the organism. The image on the upper right hand side shows the same CARS image, but in binary form, threshold‐adjusted to reveal only the lipid droplets resulting in a 7.2% lipid area fraction. The lower right hand side shows a cross section of the worm as indicated in the CARS image revealing the average diameter of the droplets to be around 1 μm.



Figure 8.

Combined multiphoton image of rat cartilage with spontaneous Raman spectroscopy from points of interest. The green colored image shows the distribution of the collagen in the extracellular matrix acquired with second harmonic generation. The blue colored image shows the distribution of the CH2 vibration, primarily found in cellular lipid droplets and the cellular lipid bilayer. Successive Raman spectroscopy shows the chemical difference from various points of interest in the sample. Spectra from the cellular lipid droplets indicate the presence of highly unsaturated fatty acids in the lipid droplets, where as the spectra of the cartilage show peaks typically present in collagen.

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Article Title: Label‐Free Analysis of Cellular Biochemistry by Raman Spectroscopy and Microscopy
 

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Iwan W. Schie, Thomas Huser. Label‐Free Analysis of Cellular Biochemistry by Raman Spectroscopy and Microscopy. Compr Physiol 2013, 3: 941-956. doi: 10.1002/cphy.c120025