partially overlap. In order to render accurate Raman images of
these different components in the sample, it is essential to only
use Raman peaks unique to each component and not shared by
other components.
10. Most of the time frozen cells can be identified in the brightfield mode of Raman microscopy. However, a rounded small
ice crystal or air bubble sometimes resembles a frozen cell.
Spectrally verifying the identity of a frozen cell in the ROI
using the “oscilloscope” can prevent the error of imaging an
ice crystal or air bubble mistaken for a cell.
11. It takes approximately 10 min to image a single Jurkat cell
based on the image size and resolution given in this demonstration. As a result, it might not be appropriate to investigate
transient phenomena, such as transmembrane motion of water
and cryoprotectants during freezing or thawing, using Raman
imaging. It is still feasible to use Raman spectra to study
transient phenomena, as it can take less than 1 s to acquire
Raman spectra adjustable by the integration time. Geometry of
ROI can be adjusted to fit the size of each frozen cell. Points
per line and lines per image can be adjusted accordingly to
ensure at least three data points collected per micron of the
image.
Acknowledgments
This work was partially supported by funding from the National
Institute of Biomedical Imaging and Bioengineering of the
National Institutes of Health under award number
R01EB023880. Raman spectroscopy in this demonstration was
carried out in the Characterization Facility, University of Minnesota, which receives partial support from the National Science
Foundation through the MRSEC program.
References
1. Diller KR, Cravalho EG (1971) A cryomicroscope for the study of freezing and thawing
processes in biological cells. Cryobiology
7:191–199
2. Dong J, Malsam J, Bischof JC, Hubel A, Aksan
A (2010) Spatial distribution of the state of
water in frozen mammalian cells. Biophys J
99:2453–2459
3. Yu G, Yap YR, Pollock K, Hubel A (2017)
Characterizing intracellular ice formation of
lymphoblasts using low-temperature Raman
spectroscopy. Biophys J 112:2653–2663
4. Raman CV, Krishnan KS (1928) A new type of
secondary radiation. Nature 121:501–502
5. Das RS, Agrawal YK (2011) Raman spectroscopy: recent advancements, techniques and
applications. Vib Spectrosc 57:163–176
6. Butler HJ, Ashton L, Bird B, Cinque G,
Curtis K, Dorney J, Esmonde-White K, Fullwood NJ, Gardner B, Martin-Hirsch PL,
Walsh MJ, McAinsh MR, Stone N, Martin FL
(2016) Using Raman spectroscopy to characterize biological materials. Nat Protoc
11:664–687
360
Guanglin Yu et al.
these different components in the sample, it is essential to only
use Raman peaks unique to each component and not shared by
other components.
10. Most of the time frozen cells can be identified in the brightfield mode of Raman microscopy. However, a rounded small
ice crystal or air bubble sometimes resembles a frozen cell.
Spectrally verifying the identity of a frozen cell in the ROI
using the “oscilloscope” can prevent the error of imaging an
ice crystal or air bubble mistaken for a cell.
11. It takes approximately 10 min to image a single Jurkat cell
based on the image size and resolution given in this demonstration. As a result, it might not be appropriate to investigate
transient phenomena, such as transmembrane motion of water
and cryoprotectants during freezing or thawing, using Raman
imaging. It is still feasible to use Raman spectra to study
transient phenomena, as it can take less than 1 s to acquire
Raman spectra adjustable by the integration time. Geometry of
ROI can be adjusted to fit the size of each frozen cell. Points
per line and lines per image can be adjusted accordingly to
ensure at least three data points collected per micron of the
image.
Acknowledgments
This work was partially supported by funding from the National
Institute of Biomedical Imaging and Bioengineering of the
National Institutes of Health under award number
R01EB023880. Raman spectroscopy in this demonstration was
carried out in the Characterization Facility, University of Minnesota, which receives partial support from the National Science
Foundation through the MRSEC program.
References
1. Diller KR, Cravalho EG (1971) A cryomicroscope for the study of freezing and thawing
processes in biological cells. Cryobiology
7:191–199
2. Dong J, Malsam J, Bischof JC, Hubel A, Aksan
A (2010) Spatial distribution of the state of
water in frozen mammalian cells. Biophys J
99:2453–2459
3. Yu G, Yap YR, Pollock K, Hubel A (2017)
Characterizing intracellular ice formation of
lymphoblasts using low-temperature Raman
spectroscopy. Biophys J 112:2653–2663
4. Raman CV, Krishnan KS (1928) A new type of
secondary radiation. Nature 121:501–502
5. Das RS, Agrawal YK (2011) Raman spectroscopy: recent advancements, techniques and
applications. Vib Spectrosc 57:163–176
6. Butler HJ, Ashton L, Bird B, Cinque G,
Curtis K, Dorney J, Esmonde-White K, Fullwood NJ, Gardner B, Martin-Hirsch PL,
Walsh MJ, McAinsh MR, Stone N, Martin FL
(2016) Using Raman spectroscopy to characterize biological materials. Nat Protoc
11:664–687
360
Guanglin Yu et al.