8/13/2019 Sample Analysis - Raman Spectroscopy of Leaf
Section
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Renishaw plcSpectroscopy Products DivisionOld Town,
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Raman spectroscopy of leaf section
Overview
Raman spectra were acquired from a polished section of a leaf.
The sample was strongly fluorescent when
imaged with visible excitation but good Raman spectra were
collected with near-IR excitation.
Raman images were created based on data collected using
Renishaws high speed StreamLine technique
with spectra acquired with 0.7 micrometer step sizes.
Experimental conditions
Model Renishaws inVia Refle Raman microscope
Excitation 532 nm and 785 nm diode lasers
Objective Leica 100x (0.90 NA) and Leica 100x (1.3 NA)
objectives
Scan type Renishaws continuous extended SynchroScan
Renishaws StreamLine high speed Raman imaging
Scan range 100 cm-1
to 3200 cm-1
for extended scanning
500 cm-1
to 1600 cm-1
for StreamLine
Analysis and results
The original sample sent for analysis by Raman spectroscopy was
found to be extraordinarily fluorescent
when excited by the laser. This is probably attributable to
pigments within the leaf. A second sample was
more amenable to analysis; although very fluorescent with
visible excitation (532 nm) there was good
Raman signal (on a fluorescent background) when 785 nm
excitation was used. The background was found
to quench slightly with 785 nm excitation but after 5 minutes
quenching with 532 nm there was still strong
fluorescence background with weak Raman bands observable. Raman
images were created using 785 nm
excitation with the sample immersed in de-ionised water. Figures
2 to 5 show 40 x 40 micrometer Raman
images created from the data collected during StreamLine
measurements. 3,363 spectra were acquired with
step sizes of 0.7 micrometer. Figures 2 and 3 show the
distribution of the cellulose using bands described by
Gierlinger and Schwanniger (2007) at 380 cm-1
and 1097 cm-1
. The latter of these bands forms part of the
composite of overlapping bands in the region 1070 cm-1
to 1190 cm-1
that includes components of both
cellulose and lignin. Figure 4 shows an image where the centres
of the cells are highlighted. The band
around 644 cm-1
was used to image the cell centres, although its origin is
unknown. Figure 5 shows an
image of the cell corners and cell walls, imaged using the band
at 1175 cm-1
which is attributable to lignin.
Figure 6 shows a spectrum from the StreamLine measurement. The
data were pre-processed to removecosmic ray events and then noise
filtered using Renishaws Chemometric package for WiRE 3. Bands
used
to create the images in Figures 3, 4 and 5 are highlighted. The
strongly fluorescent character of the sample
is indicated by the sloping baseline.
8/13/2019 Sample Analysis - Raman Spectroscopy of Leaf
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Raman spectroscopy of leaf section
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Figure 4. Cell centres, 644 cm-1. Figure 5. Cell corners and
cell walls, 1175 cm
-1.
Figure 6. Typical spectrum from StreamLine mapping measurement.
785 nm excitation.
Conclusion
With fully automated control, it takes less than one minute to
switch between excitation wavelengths and fully
optimised spectrometer configurations. With no need to manually
handle optics when working in the visible
to NIR range, both confidence in the performance and
productivity in sample running are increased. Where a
wide range of samples are routinely analysed it becomes very
fast and easy to test the sample using an
alternative excitation.
The leaf samples presented were found to be highly fluorescent
under visible excitation and only one ofthese gave adequate Raman
signal to be imaged. High resolution Raman imaging has been used to
image
different parts of the structure with high quality
(signal:noise) with fast collection times.
1175cm-1
644cm-1
1097cm-1
