Advanced wide-field CARS microscopy

The intention of the project is to improve an earlier developed unique wide-field coherent anti-Stokes Raman scattering (CARS) microscope for obtaining a higher efficiency, to extend the accessible range in which Raman resonances can be imaged, and to increase the signal to background contrast. This will provide an important tool for chemically selective imaging in biomedical applications and material research. The efficiency of a CARS signal is a limiting factor for the identification of microscopic amounts of chemical substances in biological samples and material research. Therefore we plan to advance our wide-field CARS setup which we developed in an earlier FWF project. The main idea is to change the previous CARS excitation geometry by implementing a specially designed phase grating, or a diffractive optical element (DOE) into the pump beam path. This produces a modified directional distribution of the incoming pump light which now optimally satisfies the so-called wave matching condition. Numerical calculations suggest that a ten fold increase of the CARS signal intensity might be achievable when using the same excitation power. Furthermore we plan to upgrade our system with optimally suited equipment for generating more efficient CARS signals, which we can specify now after 5 years experience with wide-field CARS imaging. Particularly the optimization of our laser system for obtaining higher excitation power, and a new camera system which has a both a higher sensitivity in the imaged wavelength range and an increased spatial resolution, promise a significant improvement of the imaging capabilities. Overall, the technical advances should allow to obtain highly contrasted images of intrinsic chemical species in biological cells, like lipid distribution, proteins, or ingested extrinsic substances like nanoparticles, and thus help to promote our special CARS variant as a complementary method of advanced bioimaging technologies.

Coherent anti-Stokes Raman scattering (CARS) microscopy is a recently developed imaging method enabling chemical mapping. Upon irradiation with laser beams, targeted chemical constituents in the sample become visible by emitting light that is shifted towards shorter wavelength, which is favorable for detection. This happens whenever the frequency difference between the incident lasers matches a vibrational transition of the chemical molecules of interest. In contrast to fluorescence microscopy, CARS-microscopy does not require any staining of the sample, which makes it a good alternative or complementary imaging method in biomedical or and material science research, when a label-free method required.The FWF-project Advanced wide-field CARS-microscopy has developed a second generation WF-CARS Microscope of increased efficiency, sensitivity, and also flexibility. This was made possible by built-in optical wave-front shaping with gratings or for maximal flexibility- with a spatial light modulator (SLM). The wave-front control allows for convenient tuning of the chemical resonances and of the phase-matching governing the efficiency of the signal generation. The fine-tuning of the effective phase-matching may be employed, for example, to detect small particles embedded in a substance of similar spectral response.After demonstrating the capabilities of the new WF-CARS microscope on simple samples, it was used in two biomedical applications. Firstly as a label-free control imaging modality while studying the triggered release of fluorescent markers from nano-carriers in inner ear cells, and secondly, in ongoing research on mouse brain slices to detect alpha-synuclein vesicles as a signature of neurodegenerative disease such as Alzheimers or Parkinsons.