Thermal illumination limits in 3D Raman microscopy: A comparison of different sample illumination strategies to obtain maximum imaging speed

Abstract

Confocal Raman microscopy is a powerful tool for material science and biomedical research. However, the low Raman scattering cross-section limits the working speed, which reduces the applicability for large and sensitive samples. Here, we discuss the fundamental physical limits of Raman spectroscopy with respect to signal-to-noise, sample load and how to achieve maximal imaging speed. For this, we develop a simple model to describe arbitrary far field light microscopes and their thermal influence on the sample. This model is used to compare the practical applicability of point- and line-confocal microscopes as well as wide-field-, light sheet- and light line illumination, for the measurement of 3D biological samples. The parallelization degree of the illumination can positively affect the imaging speed as long as it is not limited by thermal sample heating. In case of heat build-up inside the sample, the advantages of parallelization can be lost due to the required attenuation of excitation and the working speed can drop below that of a sequential method. We show that for point like illumination, the exposure time is thermally not as critical for the sample as the irradiance, while for volume like illumination, the exposure time and irradiance result in the same thermal effect. The results of our theoretical study are experimentally confirmed and suggest new concepts of Raman microscopy, thus extending its applicability. The developed model can be applied to Raman imaging as well as to other modes (e.g. two- or three- photon imaging, STED, PALM/STORM, MINFLUX) where thermal effects impose a practical limit due to the high irradiance required.

Principles and Applications of Vibrational Spectroscopic Imaging in Plant Science: A Review

“…The optimization of the measurement conditions is a continuously developed topic. Recently, Hauswald et al. (2019) dissected the existing strategies for sample illumination and proposed a novel approach that improves Raman imaging quality versus the thermal illumination limit.…”

“…Furthermore, it enables practical confocal resolution, enabling acquisition of information from beneath sample surface in a controlled manner. Care needs to be taken when examining specimens sensitive to thermal decomposition; recently a comprehensive dissection of this issue was published ( Hauswald et al., 2019 ). This technique is fully suitable to study moist samples, although green plant material may pose a challenge because of interfering fluorescence signal.…”

“…The study evaluated the practical applicability of point-and line-confocal microscopes as well as widefield-, light sheet-, and light line illumination, based on the developed models describing the fundamental physical limits of Raman spectroscopy with respect to SNR, sample load and maximum imaging speed. These accomplishments may help to develop new concepts of Raman microscopy, by extending its applicability for the three-dimensional measurement of biological samples including large and sensitive specimens (Hauswald et al, 2019). Fluorescence imaging occupies a particular spot across the field of plant-oriented studies.…”

Noise Sources and Requirements for Confocal Raman Spectrometers in Biosensor Applications

“…In principle, different light spectrometer classes (dispersive, filter based, Fourier transform) are available. For Raman spectroscopy in the wavelength window of silicon matrix detectors, dispersive spectrometers are the instruments of choice due to their efficiency and noise performance [ 12 , 13 ]. However, the use of a prism or grating-based spectrometer implies a reduced field-of-view due to the necessary entrance slit.…”