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Alejandra Zegarra-Valverde

Speeding-up Raman microspectroscopy
Leibniz IPHT // Jena, Germany

Q&A Session II // Thursday, October 29 // 1.35 pm – 2.20 pm (CET)

Raman scattering provides high molecular specificity of living organisms without labeling or staining. Due to its low cross section and absorption based sample heating, however, traditional imaging approaches such as confocal microscopy fail to acquire fast high quality images. [1] 

Current developments based on light sheet illumination avoid unnecessary out of focus excitation, reducing sample heating. A Fourier-transform imaging spectrometer based approach has been demonstrated to be five times faster while providing full hyperspectral information. [2] By choosing a low-noise imaging spectrometer method, further speed improvements can be expected. [3,4] 

Here we present a new approach combining light sheet illumination with hyperspectral imaging meeting both optimization criteria: low light load and high signal to noise ratio. [5] Exploiting the astronomical technique integral field spectroscopy [6] we can record 50×50 spectra in parallel at a field of view of 20 μm. The spectral range of our system amounts to the biological fingerprint region from of 800 cm-1 to 2000 cm-1 with a spectral resolution better than 4 cm-1 using an excitation wavelength of 578 nm. This system is expected to be 2500 times faster than a comparable confocal one enabling qualitatively new applications in biomedical as well as intracellular clinical research. 


[1] Krafft C. and Popp J., The many facets of Raman spectroscopy for biomedical analysis. Analytical and Bioanalytical Chemistry (2015); 3, 699-71 

[2] Müller W., Kielhorn M., Schmitt M., Popp J., and Heintzmann R., Light sheet Raman micro-spectroscopy, Optica (2016); 3, 452–457 

[3] Sellar R. G. and Boreman G. D., Comparison of relative signal-to-noise ratios of different classes of imaging spectrometer, Applied Optics (2005); 44, 1614– 1624 

[4] Hauswald W., Thermal illumination limits in 3D Raman microscopy: A comparison of different sample illumination strategies to obtain maximum imaging speed, PLoS ONE (2019); 14, e0220824 

[5] Zegarra-Valverde A., Müller W., and Heintzmann R., Integral Field Light Sheet Raman Microspectroscopy. Frontiers in Optics + Laser Science APS/DLS, OSA Technical Digest (2019); paper FM3F.5. 

[6] Bacon R. et al., The SAURON project-I. The panoramic integral-field spectrograph. Monthly Notices of the Royal Astronomical Society. (2001); 326, 23-35 

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