On the interpretation of second harmonic generation intensity profiles of striated muscle.
Recently, a supramolecular model was developed for predicting striated skeletal muscle intensity profiles obtained by label-free second harmonic generation (SHG) microscopy. This model allows for a quantitative determination of the length of the thick filament antiparallel range or M-band (M ), and results in M=0.12  μm for single-band intensity profiles when fixing the A-band length (A ) to A=1.6  μm , a value originating from electron microscopy (EM) observations. Using simulations and experimental data sets, we showed that the objective numerical aperture (NA) and the refractive index (RI) mismatch (Δn=n 2ω −n ω ) between the illumination wave (ω ) and the second harmonic wave (2ω ) severely affect the simulated sarcomere intensity profiles. Therefore, our recovered filament lengths did not match with those observed by EM. For an RI mismatch of Δn=0.02 and a moderate illumination NA of 0.8, analysis of single-band SHG intensity profiles with freely adjustable A- and M-band sizes yielded A=1.40±0.04  μm and M=0.07±0.05  μm for skeletal muscle. These lower than expected values were rationalized in terms of the myosin density distribution along the myosin thick filament axis. Our data provided new and practical insights for the application of the supramolecular model to study SHG intensity profiles in striated muscl
Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, Belgium.
Rik
R
Paesen
Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, Belgium.
Sophie
S
Smolders
Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, BelgiumbHasselt University, REVAL-Rehabilitation Research Center, Agoralaan Building A, Diepenbeek 3590, Belgium.
Inez
I
Wens
Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, Belgium.
Kristof
K
Notelaers
Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, Belgium.
José Manolo
JM
de Hoyos Vega
Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, Belgium.
Virginie
V
Bito
Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, BelgiumbHasselt University, REVAL-Rehabilitation Research Center, Agoralaan Building A, Diepenbeek 3590, Belgium.
Bert O
BO
Eijnde
Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, BelgiumbHasselt University, REVAL-Rehabilitation Research Center, Agoralaan Building A, Diepenbeek 3590, Belgium.
Dominique
D
Hansen
Hasselt University, Biomedical Research Institute, Agoralaan Building C, Diepenbeek 3590, Belgium.
Marcel
M
Ameloot
D000465:Algorithms; D000818:Animals; D005260:Female; D007089:Image Enhancement / Q000379:methods*; D007090:Image Interpretation, Computer-Assisted / Q000379:methods*; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008859:Microscopy, Polarization / Q000379:methods*; D018485:Muscle Fibers, Skeletal / Q000648:ultrastructure*; D051381:Rats; D011917:Rats, Inbred Lew; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity