Abstract
We have developed an epi-detected multimodal nonlinear optical microscopy platform based on a compact and cost-effective laser source featuring simultaneous acquisition of signals arising from hyperspectral coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence, and second harmonic generation. The laser source is based on an approach using a frequency-doubled distributed Bragg reflector-tapered diode laser to pump a femtosecond Ti:sapphire laser. The operational parameters of the laser source are set to the optimum trade-off between the spectral and temporal requirements for these three modalities, achieving sufficient spectral resolution for CARS in the lipid region. The experimental results on a biological tissue reveal that the combination of the epi-detection scheme and the use of a compact diode-pumped femtosecond solid-state laser in the nonlinear optical microscope is promising for biomedical applications in a clinical environment.
Highlights
In recent years, a variety of label-free microscopy techniques have been developed for imaging different kinds of specimens without the use of exogenous stains or fluorophores that may cause problems in studying dynamics and living systems.[1,2,3,4,5,6] Following this trend, label-free imaging is gradually being merged with classical diagnostic tools in many different fields
The performance of the presented compact multimodal nonlinear epi-detected microscope is demonstrated by recording hyperspectral images of a test sample consisting of a mixture of polystyrene and polymethyl methacrylate (PMMA) microspheres of 44- and 11-μm diameter, respectively, immersed in water
The full width at half maximum (FWHM) of the spontaneous Raman signal from PMMA beads at the ∼2950 cm−1 band is ∼40 cm−1, allowing a crude estimate of our spectral resolution
Summary
A variety of label-free microscopy techniques have been developed for imaging different kinds of specimens without the use of exogenous stains or fluorophores that may cause problems in studying dynamics and living systems.[1,2,3,4,5,6] Following this trend, label-free imaging is gradually being merged with classical diagnostic tools in many different fields This mainly becomes possible due to the fact that laser technology used in nonlinear optical imaging has translated into powerful well-established, robust, and turn-key systems, which are commercially available. The only possible way to detect those signals is in the backward (epi) direction which still represents a challenge due to the low intensity of the epipropagation coherent Raman-based and second harmonic signals.[10,11,12,13,14] In terms of image acquisition, speed laser technology
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