Single source CARS-based multimodal microscopy system for biological tissue imaging [Invited]

Abstract

A coherent anti-Stokes Raman scattering (CARS)-based multimodality microscopy system was developed using a single Ti:sapphire femtosecond laser source for biological imaging. It provides three complementary and co-registered imaging modalities: CARS, MPM (multiphoton microscopy), and RCM (reflectance confocal microscopy). The imaging speed is about 1 frame-per-second (fps) with a digital resolution of 1024 × 1024 pixels. This microscopy system can provide clear 2-dimensional and 3-dimensional images of ex-vivo biological tissue samples. Its spectral selection initiates vibrational excitation in lipid cells (approximately 2850 cm^-1) using two filters on the pump and Stokes beam paths. The excitation can be tuned over a wide spectral range with adjustable spectral filters. The imaging capability of this CARS-based multimodal microscopy system was demonstrated using porcine fat, murine skin, and murine liver tissue samples.

Fig. 1.

Block diagram of CARS-based multimodal microscopy system using a single laser source and a single objective lens. FI (Faraday isolator), BS (beam splitter), Attenuator (a half wave plate and a Glan-Laser Calcite Polarizer), SCG-800 (supercontinuum generation kit), RELP (razor edge long pass filter), LP (long pass filter), OBJ (objective lens), SP (short pass filters), BP (band pass filters), DM (dichroic mirror), L (Lens), and PMT (photomultiplier). APD: avalanche photodiode.

Fig. 2.

CARS image of ex vivo porcine adipose tissue with field of view of 400 µm (FOV = 400 µm).

Fig. 3.

CARS imaging of ex vivo murine ear skin with field of view of 200 µm (FOV = 200 µm). (a) sebaceous gland (b) subcutaneous fat layer.

Fig. 4.

CARS imaging of ex vivo murine liver with field of view at the same depth but at different locations (FOV = 200 µm).

Fig. 5.

(a) CARS video of a murine liver tissue (FOV = 200 µm), see Visualization 1 for playing the video; (b) Volumetric CARS 3D image of a murine liver tissue at depth 40 µm (FOV = 200 µm), see Visualization 2.

Fig. 6.

Multimodal microscopic images of a murine ear tissue sample (FOV = 200 µm) (a) CARS image; (b) RCM image; (c) MPM image; (d) two-color composite image of CARS and RCM (green: CARS, red: RCM); (e) two-color composite image of CARS and MPM (green: CARS, red: MPM).

Fig. 7.

Multimodal microscopic images of a murine liver sample (FOV = 200 µm) (a) CARS image; (b) RCM image; (c) MPM image; (d) two-color composite image of CARS and RCM (green: CARS, red: RCM); (e) two-color composite image of CARS and MPM (green: CARS, red: MPM).