Raman scattering is the result of an interaction between monochromatic light radiation and molecular vibrations. It was first
discovered by C.V. Raman in liquids in 1928 [4], and 2 years
later, Raman won the Nobel Prize in Physics for his outstanding
contributions. During Raman scattering, molecules are excited to
the short-lived higher energy levels through direct absorption of a
photon from the incident monochromatic light. The excited molecules can further scatter a photon and return to the stable ground
state energy levels. Inelastic Raman scattering refers to the situation
in which the energy of scattered photons is different from that of
absorbed photons. Raman spectra plots depict the intensity of the
scattered light as a function of its frequency difference to the
incident light, and Raman imaging generates false-color images of
component distribution by acquiring and interrogating Raman
spectra at every pixel of the image. In reality, Raman scattering is
a rare phenomenon with an extremely low probability of occurrence. As a result, many advanced techniques are being developed
to enhance Raman signal intensity, such as surface-enhanced
Raman
spectroscopy
(SERS),
resonance
Raman
(RR) spectroscopy, and stimulated Raman spectroscopy (SRS) [5].
Raman spectroscopy has been widely used for characterizing
biological materials, as Raman spectra and images provide plentiful
information about the material [6]. Furthermore, Raman bioanalysis is noninvasive and nondestructive to the sample and requires
little sample preparation [7]. A review of applications of Raman
spectroscopy in single cell analysis has been published by Huser and
colleagues [8], and examples of common applications include identification of cells [9], drug-cell interaction [10], and discrimination
between healthy and unhealthy cells [11]. Recently, Raman spectroscopy is emerging as a powerful technique for characterizing the
freezing responses of cells cryopreserved with various cryoprotectants [12–14], defining morphology and size of ice crystals formed
in different freezing solutions [15–17], as well as analyzing phase
compositions of complex frozen samples [18, 19]. In this chapter,
we will demonstrate the manner by which low-temperature Raman
spectroscopy can be employed to analyze single cells during freezing and to reveal the spatial distribution of cellular composition, ice
crystals, and cryoprotectants.
2 Materials
2.1 Confocal Raman
Microscopy/
Spectroscopy
1. A WITec alpha300 R Confocal Raman Imaging system (with a
motor-driven scanning stage), equipped with a UHTS300
spectrometer and a DV401 CCD detector with 600/mm
grating (WITec, Ulm, Germany).
352
Guanglin Yu et al.
discovered by C.V. Raman in liquids in 1928 [4], and 2 years
later, Raman won the Nobel Prize in Physics for his outstanding
contributions. During Raman scattering, molecules are excited to
the short-lived higher energy levels through direct absorption of a
photon from the incident monochromatic light. The excited molecules can further scatter a photon and return to the stable ground
state energy levels. Inelastic Raman scattering refers to the situation
in which the energy of scattered photons is different from that of
absorbed photons. Raman spectra plots depict the intensity of the
scattered light as a function of its frequency difference to the
incident light, and Raman imaging generates false-color images of
component distribution by acquiring and interrogating Raman
spectra at every pixel of the image. In reality, Raman scattering is
a rare phenomenon with an extremely low probability of occurrence. As a result, many advanced techniques are being developed
to enhance Raman signal intensity, such as surface-enhanced
Raman
spectroscopy
(SERS),
resonance
Raman
(RR) spectroscopy, and stimulated Raman spectroscopy (SRS) [5].
Raman spectroscopy has been widely used for characterizing
biological materials, as Raman spectra and images provide plentiful
information about the material [6]. Furthermore, Raman bioanalysis is noninvasive and nondestructive to the sample and requires
little sample preparation [7]. A review of applications of Raman
spectroscopy in single cell analysis has been published by Huser and
colleagues [8], and examples of common applications include identification of cells [9], drug-cell interaction [10], and discrimination
between healthy and unhealthy cells [11]. Recently, Raman spectroscopy is emerging as a powerful technique for characterizing the
freezing responses of cells cryopreserved with various cryoprotectants [12–14], defining morphology and size of ice crystals formed
in different freezing solutions [15–17], as well as analyzing phase
compositions of complex frozen samples [18, 19]. In this chapter,
we will demonstrate the manner by which low-temperature Raman
spectroscopy can be employed to analyze single cells during freezing and to reveal the spatial distribution of cellular composition, ice
crystals, and cryoprotectants.
2 Materials
2.1 Confocal Raman
Microscopy/
Spectroscopy
1. A WITec alpha300 R Confocal Raman Imaging system (with a
motor-driven scanning stage), equipped with a UHTS300
spectrometer and a DV401 CCD detector with 600/mm
grating (WITec, Ulm, Germany).
352
Guanglin Yu et al.