Hyperspectral multimodal CARS microscopy in the fingerprint region

Abstract

A simple scheme for multimodal coherent anti-Stokes Raman scattering (CARS) microscopy is based on the spectral focusing of ultrafast-oscillator-derived pump/probe light and synchronous photonic crystal fiber (PCF) fiber-generated broadband Stokes light. To date, such schemes allowed rapid hyperspectral imaging throughout the CH/OH high frequency region (2700-4000 cm(-1) ). Here we extend this approach to the middle (1640-3300 cm(-1) ) and fingerprint regions (850-1800 cm(-1) ) of the Raman spectrum. Our simple integrated approach to rapid hyperspectral CARS microscopy in the fingerprint region is demonstrated by applications to label-free multimodal imaging of cellulose and bulk bone, including use of the phosphate resonance at 960 cm(-1) .

Spectrally-broad coherent anti-Stokes Raman scattering hyper-microscopy utilizing a Stokes supercontinuum pumped at 800 nm

“…While this approach works to some degree, the need to change the pump wavelength significantly away from 800 nm is problematic in that it requires changing various routing and collection filters; requires detection of anti-Stokes wavelengths above 820 nm where alkali PMTs are precipitously inefficient; and requires a broadly-tunable laser oscillator source. Furthermore, the need to reduce the input power for the SCG module meant a corresponding reduction in generated Stokes power available for imaging [17]. Here, we find that by using longer pulses (or ones that are already chirped) we can generate significant Stokes power in the spectral region between 800 nm and 945 nm.…”

“…One consequence of efficient supercontinuum generation is that access to frequencies near that of the pump is limited. For example, in a prior demonstration of CARS microscopy at fingerprint (<1800 cm 1 ) frequencies using the same PCF [17], the Stokes generation efficiency was reduced by lowering the pump power into the PCF, while concurrently shifting the pump wavelength above 900 nm, closer to the 945 nm ZDW. While this approach works to some degree, the need to change the pump wavelength significantly away from 800 nm is problematic in that it requires changing various routing and collection filters; requires detection of anti-Stokes wavelengths above 820 nm where alkali PMTs are precipitously inefficient; and requires a broadly-tunable laser oscillator source.…”

“…the probed CARS frequency-can be simply scanned by sweeping the relative temporal overlap between the two pulses. Because it utilizes single-wavelength detection, SF-CARS is predominantly an imaging-first technique; and since the spectrum can be scanned in a matter of seconds-tominutes [5,10,16,17], it can yield good-quality hyperspectral image stacks at a rate that challenges multiplex-CARS techniques.…”

“…To date, an approach that requires only a single set of optics that can be used for broadband SF-CARS microscopy spanning both the fingerprint and the CH/OH vibrational frequencies has not been demonstrated. For example, in some recent implementations of SF-CARS, different optics [16], pump/probe wavelengths [17], or totally different experimental setups [18] have been used to separately access the fingerprint and the CH/OH frequencies.…”

“…This makes it difficult to detect vibrational frequencies below 1900 cm 1 . While this limitation can be overcome by tuning the pump/probe wavelength to above 900 nm (<11110 cm 1 ) [17], the resulting Stokes supercontinuum is insufficiently broad to enable access to the important CH/OH vibrational region. In this work, we show that by using conveniently-chirped (i.e.…”