A novel sensitive coherent Raman imaging method for cellular imaging of photodynamic agents

Abstract

Label free imaging techniques are becoming increasingly popular, with coherent Raman techniques among those leading the way. A shortcoming of coherent Raman techniques is that they have a high limit of detection, being unable to detect low concentrations like those found within cells for certain components. A few enhancement techniques for coherent Raman methodologies exist however these are underdeveloped in the literature. Here a resonance coherent Raman technique will be developed and applied. Resonance Raman is a long established Raman enhancement technique that uses electronic transitions to enhance the signal. Coherent resonance Raman was established over 45 years ago and since then very little biological imaging has been carried out with this technique. In this work a pre-resonance enhanced coherent anti-Stokes Raman scattering (CARS) methodology was developed and optimised. Multiple photodynamic agents with different absorption properties were analysed. Specifically, these were, protoporphyrin IX (PPIX), indocyanine green (ICG) and rhodamine 800 (Rh800). It was discovered that Rh800 and ICG showed pre-resonance enhancement. An improvement in the limit of detection of an order of magnitude was observed for ICG and Rh800. When applied to cellular detection it shows great promise for the robust detection of ICG and Rh800 within cells. Pre-resonance imaging of Rh800 allowed mitochondria to be imaged and mapping of bound water within cells was possible. The results for Rh800 showed a better signal to noise ratio. Using the knowledge gained about pre-resonance CARS enhancement, a further study was conducted to see if pre-resonance enhancement could be applied to a hyperspectral CARS imaging system using the spectral-focussing technique. A home built setup using a NKT-1050-NEG1 fibre was used which provided both broadband pump and Stoke pulses. The popular dyes Methylene blue and Nile blue were selected for use in this study as their absorption falls within the calculated ideal pre-resonance enhancement region. Cellular imaging of these compounds was successful. Overall this work demonstrates a step forward for CARS imaging, where it is getting closer to fluorescence spectroscopy in terms of limit of detection. Although currently it has only been shown with dyes, in principle, pre-resonance and resonance CARS could be applied to autofluorophores in biological cells. The additional advantages over fluorescence, such as better multiplexing with a greater number of different dyes, could see this technique being used in various applications going forward.

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Identifiers

ORCID for James Arthur Read: orcid.org/0000-0001-5923-1688

ORCID for Sumeet Mahajan: orcid.org/0000-0001-8923-6666

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