Multimodal spectral focusing CARS and SFG microscopy with a tailored coherent continuum from a microstructured fiber

Abstract

We report a technologically novel microscopy system for bioimaging based on a 100 fs titanium:sapphire (Ti:Sa) laser pumped coherent continuum from a tailored, 9-cm long, all normal dispersion (ANDi) fiber, enabling concurrent image contrast with (a) spectral focusing coherent anti-Stokes Raman scattering (SF-CARS) (spanning 900-3200 cm −1) and (b) sum frequency generation (SFG). Both modalities were efficiently excited with power levels at the microscope focus compatible with biological samples. Moreover, using the continuum, images were recorded in the back-scattering (epi-detection) geometry, without the necessity for an expensive, computer-controlled, spatial light modulator (SLM), clearly demonstrating the strong signal levels achieved. Image contrast from the multiple modalities provided greater chemical and structural insights than imaging with any single technique in isolation. Numerical simulations supported these developments in regard to both the optimum fiber length for SC generation and the achievement of high spectral resolution in SF-CARS via careful group delay dispersion matching across the pump and Stokes pulses using just an inexpensive sequence of short glass blocks inserted into the Stokes beam. We show bio-images of mouse tissue recorded concurrently via label/stain-free contrast from multiple modalities: CARS, two-photon auto-fluorescence (TPaF) and second harmonic/sum frequency generation (SHG/ SFG). Overall, our approach delivers optimum performance in back-scattered (epi-) detection configuration, suited for thick samples, at reduced complexity and cost. The addition of this simple fiber add-on to lasers already widely used for TPF microscopy can thus extend the capabilities of a significant number of existing microscopy laboratories.

Perspective on the next generation of ultra-low noise fiber supercontinuum sources and their emerging applications in spectroscopy, imaging, and ultrafast photonics

“…The combination of multiphoton microscopy and hyperspectral CARS imaging based on digitally programmable ANDi SC pulses has resulted in an extremely versatile platform for biomedical imaging, offering the potential to translate label-free molecular histopathology for disease diagnosis into routine clinical use 60 . Even without the relatively expensive digital pulse shaping device, broadband multimodal CARS and multi-photon imaging systems based on ANDi SC sources have recently been demonstrated 61 . From our perspective this is really just the beginning of a movement that will see these versatile sources of broadband, low-noise, and ultrashort pulses being used in an increasing numbers of applications which so far were not able to use conventional fiber-based SC sources due to their noise or complex temporal pulse shapes.…”

Recent Advances in Supercontinuum Generation in Specialty Fiber

“…These advances have led to the development of an extremely versatile platform based on digitally programmable ANDi SC pulses, which combines multiple label-free nonlinear imaging and spectroscopy modalities into a single setup, offering the potential to translate this technology into routine clinical use for disease diagnosis [143]. Even without the relatively expensive digital phase shaping device, broadband multimodal CARS and multi-photon imaging systems based on ANDi SC sources have recently been demonstrated [144].…”

Widely-tunable synchronisation-free picosecond laser source for multimodal CARS, SHG, and two-photon microscopy

“…Despite the many attractive features of the spectroscopically specific CARS imaging modality, its uptake is restricted by the cost, size and complexity of the ‘gold-standard’ synchronously pumped OPO systems currently used [ 2 ]. To address the need for sources that output the required few-ps pulse widths considered optimal for CARS bioimaging [ 3 ], progress has been achieved using spectral-focusing CARS [ 4 ] based on either supercontinuum [ 5 ] or a single < 20 fs laser with sufficient bandwidth [ 6 ], but those solutions still have present technical obstacles, so they have not achieved wide acceptance.…”