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Confocal Raman Microscopy with Adaptive Optics

Confocal Raman microscopy is a precise and label-free technique for analyzing thick samples at the microscale, but its use is often limited by weak Raman signals. Sample inhomogeneities introduce wavefront aberrations, further diminishing signal strength and requiring longer acquisition times. In this study, we present the first application of Adaptive Optics in confocal Raman microscopy to correct these aberrations, achieving substantial improvements in signal intensity and image quality. This approach integrates seamlessly with commercial microscopes without the need for hardware modifications. It utilizes a wavefront sensorless method, relying on an optofluidic, transmissive spatial light modulator attached to the microscope nosepiece to measure and correct aberrations. Experimental validation shows effective correction of aberrations in artificial scatterers and mouse brain tissue, enhancing spatial resolution and increasing signal intensity by up to 3.5 times. These results establ...
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Raman Intensity Calibration from 500 to 1100 nm laser excitation

For repetitive and reproducible results is required a suitable calibration considering the y-axis or intensity axis. The standard calibration method is using a standard calibration lamp and experimental measurement of lamp spectrum, which holds the same wavenumber range as the samples. 

The first plot corresponds to the reference spectrum along the wavelength axis from 500 to 1100 nm.

The second step is measuring the spectrum of Halogen Lamp as the next plot shows.

The calibration factor is determined considering a normalization between the standard data and the experimental lamp spectrum. The wavelength axis is converted to wavenumber for the normalization between the same units. The result is a numerical factor for compensating and calibrating further spectra.

An example of a sample before intensity calibration is shown in the next figure.

The calibrated spectrum of the same sample is shown in the next figure. The calibration shows that the high wavenumber holds less positive compensation than the lower wavenumbers. The final result holds a flatter baseline and with stronger intensity amplitudes. 


Acknowledgments.

Dr. Christoph Krafft from Leibniz Institute of Photonic Technology provided me all the necessary files and explanations regarding the Raman intensity calibration. 



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