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Raman Spectroscopy of Living Cells

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Biomedical Applications of Biophysics

Part of the book series: Handbook of Modern Biophysics ((HBBT,volume 3))

Abstract

Raman spectroscopy is an optical technique for the vibrational spectroscopy of molecules. It is a rather unique and powerful technique, because, similar to magnetic resonance imaging (MRI) or X-ray imaging, it is capable of live cell chemical analysis and imaging without destroying or altering the biological materials under investigation. In contrast to these techniques, however, Raman spectroscopy works really well at the cellular and subcellular levels, because it can easily be integrated with optical microscopy. Also, it does not require exogenous probes, such as in fluorescence microscopy, but instead relies fully on an intrinsically generated signal. Raman spectroscopy is based on the fact that a very small number of photons from an intense light source (typically a laser) can inelastically scatter off molecular bonds in a sample, leading todiscrete shifts in the wavelength (or color) of the scattered photons. This chapter will provide a brief introduction to Raman spectroscopy, explain the principles behind it, provide a brief “how-to” guide for interested experimentalists, and discuss applications and recent advancements in the chemical analysis of living cells.

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Author information

Authors and Affiliations

  1. Department of Internal Medicine, and NSF Center for Biophotonics, University of California, Davis, 2700 Stockton Blvd., Suite 1400, Sacramento, CA, 95817, USA

    Tyler Weeks & Thomas Huser

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  1. Tyler Weeks
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  2. Thomas Huser
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Corresponding author

Correspondence to Thomas Huser .

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Editors and Affiliations

  1. Department of Biochemistry and Molecular Medicine, University of California Davis, One Shields Avenue, Davis, CA, 95616, USA

    Thomas Jue Ph.D.

8.1 Electronic Supplementary material

Figure 8.1.

(a) Schematics of an inelastic photon scattering event: incoming photons with energy E i scatter off a molecular bond, thereby depositing energy into the bond vibration. The outgoing, inelastically scattered photons have a slightly lower energy E s . (b) Model of a molecular bond: two atoms with masses M 1 and M 2 are connected by a spring with spring constant k. The energy of the resulting vibration written in spectroscopic terms is shown below, where c is the speed of light and µ = M 1 M 2l(M 1 + M 2) is the reduced mass of both atoms. Please visit http://extras.springer.com/ to view a high-resolution full-color version of this illustration. (PDF 2,774 KB)

Figure 8.2.

(a) Schematics of an inelastic photon scattering event where the photon deposits energy into the bond vibration, and its associated energy diagram. The scattered photon has lost energy relative to the incident photon. This process is called Stokes-shifted Raman scattering. (b) Schematics of an inelastic photon scattering event where the photon scatters off a vibrationally excited bond, and its associated energy diagram. The scattered photon has gained energy relative to the incident photon. This process is called anti-Stokes-shifted Raman scattering. Please visit http://extras.springer.com/ to view a high-resolution full-color antiversion of this illustration. (PDF 2,779 KB)

Figure 8.3.

The energy diagram describing Raman scattering is directly related to the Raman spectrum of a molecule. Here, the energy of the incoming photons (e.g., from a laser) defines the vibrational ground state, while the scattered photons are shifted to the red (blue) depending on the bond vibration and the scattering process. This is shown schematically for the spectrum (red) obtained from an unsaturated fatty acid (oleic acid), where the lipid CH deformation mode results in a peak at 1442 cm–1. Other peaks are not assigned to keep the energy diagram simple. Please visit http://extras.springer.com/ to view a high-resolution full-color version of this illustration. (PDF 2,767 KB)

Figure 8.4.

The fingerprint Raman spectrum of oleic acid results in both Stokes and anti-Stokes scattered photons. The distribution with which each fraction of photons is scattered to the left or right of the laser line (blue) depends on the temperature. Raman spectra are labeled in relative wavenumbers (cm–1) to describe the spectrum in terms independent of the source wavelength. Please visit http://extras.springer.com/ to view a high-resolution full-color version of this illustration. (PDF 2,760 KB)

Figure 8.5.

The strength of the Raman scattering process depends on the wavelength of the source. Since it is a scattering process, the intensity of the inelastically scattered light scales with λ–4. This leads to Raman spectra obtained from the same compound to be more intense when probed with a green laser (box a) rather than a red laser (box b). Also, since green light has a higher photon energy than red light, Raman scattering off the same molecular bond can lead to more “condensed” or “stretched” spectra depending on the laser wavelength. Since Raman scattering by a specific bond always requires the same amount of energy, independent of the wavelength of the source, spectra obtained with a green laser are “denser” relative to those obtained with a red laser (when plotted against a wavelength axis). This is the reason why Raman spectra are typically plotted in terms of the energy difference between the source wavelength and the signal wavelength. These spectra are independent of the wavelength at which they were obtained. Please visit http://extras.springer.com/ to view a high-resolution full-color version of this illustration. (PDF 2,783 KB)

Figure 8.6.

An example of Raman spectra observed from biological samples, in this case bovine sperm cells. (a) Intact, whole sperm cells are deposited onto a quartz cover slip and imaged in a confocal Raman microscope based on their autofluorescence. Specific areas, i.e., the sperm head (I) or sperm tail (II), can then be addressed by point spectroscopy. The resulting spectra and their assigned bond vibrations (see b) show that the sperm head is mostly composed of DNA and proteins (spectrum I), while the major constituent of the tail is just protein (spectrum II). Please visit http://extras.springer.com/ to view a high-resolution full- color version of this illustration. (PDF 2,803 KB)

Figure 8.7.

Modern Raman spectrometers require at least two sets of filters. A laser clean-up (narrow bandpass) filter on the excitation side removes plasma lines and other background contributions that can easily overwhelm the weak Raman signal. On the detection side a steep edge notch filter with high rejection for the laser line (optical density OD > 6) enables detection of both the Stokes and anti-Stokesscattered light. Instead, lower-cost steep-edge longpass filters can also be used, but enable only detection of Stokes-shifted photons. Please visit http://extras.springer.com/ to view a high-resolution full-color version of this illustration. (PDF 2,762 KB)

Figure 8.8.

Diagram of a confocal micro-Raman spectroscopy setup. Light from a continuous wave laser passes a dichroic beamsplitter and is focused onto the sample through a microscope objective lens. The same lens collects the backscattered Raman photons, which are deflected to the left by the dichroic beamsplitter. A spatial filter assembly ensures that only Raman-scattered photons from the very focus of the laser beam at the sample are sent to the detectors, which could utilize a point detector for Raman imaging of a single vibrational mode, or a spectrometer for collecting the entire Raman spectrum. Please visit http://extras.springer.com/ to view a high-resolution full-color version of this illustration. (PDF 2,773 KB)

Figure 8.9.

Calibration of Raman spectra typically utilize a well-characterized substance. Toluene and some alcohols, or mixtures thereof, of strong Raman-active modes spanning most of the fingerprint and high-wavenumber region. Their spectra are also relatively independent of temperature fluctuations, making them good calibration standards. The spectrum shown here was obtained from toluene by gently focusing ~300 μW of power from a red helium-neon laser (632.8 nm wavelength) into a solution of pure toluene. The spectrum was dispersed onto a CCD camera with 1340 pixels by a 600-line/mm grating and the signal was integrated for 30 s. Please visit http://extras.springer.com/ to view a high-resolution full-color version of this illustration. (PDF 2,780 KB)

Figure 8.10.

Rapid Raman imaging by coherent Raman scattering. (a) Energy diagram describing the nonlinear optical process of coherent anti-Stokes Raman scattering. To image bacterial spores by their intense 1013 cm–1 vibration from calcium dipicolinate, two short-pulsed lasers are synchronized and tuned to probe this molecular mode. (c) A pump laser at 750-nm wavelength (for instance) provides two photons that, when combined with a single photon from another laser at 812 nm, coherently probe the 1013 cm–1 vibration. (d) The resulting signal is an intense line at 697 nm composed of a Raman-resonant anti-Stokes Raman signal originating from the molecular vibration, as well as a non-resonant background signal due to four-wave mixing of the photons in the material. (b) The intense signal can then be utilized to obtain an image of bacterial spores prepared on a glass substrate. Please visit http://extras.springer.com/ to view a high imresolution full-color version of this illustration. (PDF 2,798 KB)

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Weeks, T., Huser, T. (2010). Raman Spectroscopy of Living Cells. In: Jue, T. (eds) Biomedical Applications of Biophysics. Handbook of Modern Biophysics, vol 3. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-233-9_8

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