The combination of Raman microscopy and electron microscopy - Practical considerations of the influence of vacuum on Raman microscopy.

Due to the specific vacuum requirements for scanning electron microscopy (SEM), the Raman microscope has to operate in vacuum in a correlative Raman-SEM, which is a type of microscope combination that has recently increased in popularity. This works considers the implications of conducting Raman microscopy under vacuum, as opposed to operating in ambient air, the standard working regime of this technique. We show that the performance of the optics of the Raman microscope are identical in both conditions, but laser beam-sample interactions, such as fluorescent bleaching and beam damage, might be different due to the lack of oxygen in vacuum. The bleaching of the fluorescent background appears to be mostly unaffected by the lack of oxygen, except when very low laser powers are used. Regarding laser-beam damage, organic samples are more sensitive in vacuum than in air, whereas no definite verdict is possible for inorganic samples. These findings have practical implications for the application of correlative Raman-SEM, as low laser powers, or in extreme cases cryo-methods, need to be used for organic samples that appear only moderately beam sensitive under usual ambient air.