Abstract
We describe experimental results on label free imaging of striated skeletal muscle using second harmonic generation (SHG) and coherent anti-Stokes Raman scattering (CARS) microscopy. The complementarity of the SHG and CARS data makes it possible to clearly identify the main sarcomere sub-structures such as actin, myosin, acto-myosin, and the intact T-tubular system as it emanates from the sarcolemma. Owing to sub-micron spatial resolution and the high sensitivity of the CARS microscopy technique we were able to resolve individual myofibrils. In addition, key organelles such as mitochondria, cell nuclei and their structural constituents were observed revealing the entire structure of the muscle functional units. There is a noticeable difference in the CARS response of the muscle structure within actin, myosin and t-tubule areas with respect to laser polarization. We attribute this to a preferential alignment of the probed molecular bonds along certain directions. The combined CARS and SHG microscopy approach yields more extensive and complementary information and has a potential to become an indispensable method for live skeletal muscle characterization.
Highlights
Muscle characterization using light microscopy has attracted attention as it provides important insights into problems related to contractile physiology [1,2]
The second harmonic generation (SHG) effect is strong in noncentrosymmetric media and has been successfully applied in microscopy applications studying the nonlinear optical response of labeled cell membranes [7], collagen-type-I structures [8], microtubules [18] as well as myosin of the skeletal muscle [19]
SHG images were obtained using picosecond pulses provided by the OPO output at around 816 nm in order to achieve perfect pixelby-pixel overlap with the coherent anti-Stokes Raman scattering (CARS) images
Summary
Muscle characterization using light microscopy has attracted attention as it provides important insights into problems related to contractile physiology [1,2]. Second harmonic generation (SHG) [7,8], third harmonic generation (THG) [9,10] and coherent anti-Stokes Raman scattering (CARS) [11,12,13] are among the key techniques that provide label free imaging of biological media. The SHG effect is strong in noncentrosymmetric media and has been successfully applied in microscopy applications studying the nonlinear optical response of labeled cell membranes [7], collagen-type-I structures [8], microtubules [18] as well as myosin of the skeletal muscle [19]. Ralston et al [23] used a combination of two-photon fluorescence and SHG microscopy to study non-contractile inclusions and sarcomeric anomalies in skeletal muscle. Using CARS microscopy we were able to visualize subsarcolemmal organelles such as mitochondria and cell nuclei, including their substructures
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