Polarization Second Harmonic Generation Microscopy for Individual Muscle and Collagen Fibril Ultrastructure Determination

Intrinsic ultrastructural information about living tissues can be obtained using second harmonic generation microscopy (SHG). SHG can visualize and structurally interrogate collagen and myosin in tissues. Both collagenous and muscle tissues consist of multilevel hierarchical structures and researchers have focused on using SHG to understand their structure at the tissue level. However, it is beneficial to study both collagen and muscle at their fundamental structural form, individual fibrils, to understand how changes in the ultrastructure of fibrils can affect the arrangement of tissues. Here we investigate collagen and myosin fibrils using the polarization-resolved SHG technique, polarization-in, polarization-out (PIPO) SHG, while using a high numerical aperture (NA) objective in hopes of better understanding the SHG signals observed from collagenous and muscle tissues. Collagen fibrils were extracted from a section of adult bovine lateral digital extensor tendon obtained from a local abattoir. The tendon section was hydrated and the fibrils were extracted by scraping the tissue with tweezers [1]. The fibril solution was deposited onto a glass coverslip and left for 45 minutes. The fibrils were then washed for 1 minute, dried using N2 gas for 5 minutes and left to air dry overnight. Myofibrils were extracted from the indirect flight muscle of Oregon Red Drosophila melanogaster. A phosphate buffer saline solution containing the muscle was agitated to separate the muscle into myofibrils. The myofibrils were then fixed with formaldehyde. The solution was stirred, centrifuged and rinsed twice. The PIPO SHG microscope consisted of an ultrafast pulsed laser (1030 nm wavelength, 5 MHz repetition rate, and 290 fs pulse duration). Galvanometric scan mirrors raster scanned the laser across the sample with a pixel dwell time of 12 µs creating an 18×18 µm image with 100×100 pixels [2]. A 0.8 NA air immersion objective lens focused the laser beam onto the sample while a 0.85 NA objective lens collected SHG signal in the transmission direction. Different laser linear polarization orientations were obtained using a linear polarizer followed by a half-wave plate in a mechanical rotator placed before the objective lens. Polarization measurements of the SHG signal were performed with a linear polarizer, known as the analyzer, in a second mechanical rotator located after the collection objective lens. SHG signal was measured using a single-photon-counting photomultiplier detector with an interference filter (⁠515±5nm) on it. SHG signal was obtained using a data acquisition card (NI). A PIPO SHG measurement consisted of recording 8 emission polarization angles for each of 8 half-wave plate angles resulting in 64 SHG images in total. Analysis of the PIPO SHG data was performed assuming a laboratory Cartesian coordinate system (XYZ) where Y is the propagation direction of the laser and XZ is the imaging plane. Additionally, the collagen fibrils and myofibrils were assumed to have C6 symmetry and χzxx(2)=χxxz(2)⁠. With these assumptions the following equation was used [4]: where I2ω is the SHG intensity, and ρ and κ are the laboratory frame second-order nonlinear optical susceptibility ratios, χZZZ(2)/χZXX(2) and χXYZ(2)/χZXX(2)⁠, respectively. Previously, ρ has been related to the helical pitch angle of SHG emitters [3] while κ has been related to the nonlinear molecular chirality [4]. The angles θ′ and φ′ are defined as: θ′=θ−δ and φ′=φ−δ where θ is the laser electric field polarization orientation, φ is the orientation of the analyzer and δ is the in-plane angle between the crystal axis and the laboratory Z-axis. A is a constant of proportionality and F accounts for noise. SHG intensity images of individual collagen and myo-fibrils were obtained. The SHG intensity from a collagen fibril was constant along the fibril while from a myofibril the SHG intensity was striated due to periodic centrosymmetric regions present in the myofibril (i.e. the Z line). Fitting of PIPO SHG data of collagen and myosin fibrils was performed. A gradient in the ρ values transverse to the axis of the fibrils was observed varying from ∼1.5-4 for collagen fibrils and ∼0.3-0.7 for myofibrils. However, when the gradient values were neglected by only averaging the middle 3-4 pixels, ρ values of 2.0±0.1and 0.5±0.1 were obtained, typical for dry collagen [5] and myosin [3]. A gradient in the ρ values was previously observed for collagen fibrils and occurs due to a high degree of circular polarization present near the lateral edge of the focal volume of a high NA objective [5]. Focal volume modeling has shown that even moderate NA focusing results in significant opposite circular polarizations on each side of the focal volume. Furthermore, modeling was used to show that the gradient in the ρ values only occurs when the sample has a significant imaginary χXYZ(2) component, which interacts with the circular polarization [5]. Therefore, a gradient in the ρ values is expected to occur for structures with diameters smaller than the focal volume such as collagen fibrils (∼200 nm). In the present study we find that a gradient in the ρ values also occurs in myofibrils, which have diameters larger than the focal volume (∼1000 nm), indicating a gradient in ρ values should always be expected wherever the SHG emitting structure only fills the edge of a focal volume, and has a complex χxyz(2) component. Also, it indicates that myosin has a complex χxyz(2) component. Previously, experimental results of the gradient in the ρ values of collagen fibrils have been compared to numerical simulations to estimate parameters related to the structure and chirality of the collagen fibril [5]. Future work includes performing similar numerical simulations to determine these parameters for myofibrils. These results are expected to lead to a better understanding of PSHG results from tissues.

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