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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 Wavenumber Calibration by paracetamol

The calibration of an instrument is important because the measurements must be reproducible anywhere. 
Raman calibration requires a well-known sample such as polystyrene and silica, paracetamol. However, for precise measurements, cyclohexane and neon are better options. The below figure shows the Raman spectrum of paracetamol.

The figure is taken from: https://www.chem.ualberta.ca/~mccreery/ramanmaterials.html

The instrument collects the signature of paracetamol like the next figure, which shows the pixels instead of wavenumber.
The procedure for calibration relies on finding the matching peaks of the pixels and the real peaks. For example, for this calibration the results were:

real_wavenumber = [329.2, 390.9, 465.1, 651.6, 797.2, 857.9, 968.7, 1168.5, 1236.8, 1278.5, 1323.9, 1371.5, 1561.6, 1648.4]

pixels = [18.50, 35.99, 57.38, 112.66, 157.36, 175.98, 211.23, 275.66, 298.39, 312.20, 328.16, 343.84, 410.35, 441.57]

After finding the matching pixels and wavenumber, polynomial regression is required (usually a third-order polynomial works properly). The next figure shows the calibration plot with an error of 0.99998. The error is improved using other samples like the cyclohexene and neon since they do not hold crystallinity properties.

After finding the coefficients is required to convert the pixels to wavenumber. Then plotting again with the calibrated axis the final result is shown in the next figure.



References

Richard L. McCreery. Raman Spectroscopy for Chemical Analysis. John Wiley & Sons. 2005.


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