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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 -1 -1 Scan range 100 cm to 3200 cm for extended scanning -1 -1 500 cm to 1600 cm 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 -1 -1 Gierlinger and Schwanniger (2007) at 380 cm and 1097 cm . The latter of these bands forms part of the -1 -1 composite of overlapping bands in the region 1070 cm to 1190 cm that includes components of both cellulose and lignin. Figure 4 shows an image where the centres of the cells are highlighted. The band -1 around 644 cm was used to image the cell centres, although its origin is unknown. Figure 5 shows an -1 image of the cell corners and cell walls, imaged using the band at 1175 cm which is attributable to lignin. Figure 6 shows a spectrum from the StreamLine measurement. The data were pre-processed to remove cosmic 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.

Raman spectroscopy of leaf section

532 nm after 300 s quench

785 nm

Figure 1. 532 nm and 785 nm Raman spectra from leaf sample. Raw data, offset for clarity.

Figure 2. Cellulose distribution, 380 cm-1

Figure 3. Cellulose distribution, 1097 cm-1

Raman spectroscopy of leaf section

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 of these 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. 3

644 cm-1

1097 cm-1

1175 cm-1

Raman spectroscopy of leaf section

References Gierlinger, N. & Schwanniger, M. (2007) The potential of Raman microscopy and Raman imaging in plant research. Spectroscopy 21, 69-89.