Stokes polarimetry-based second harmonic generation microscopy for collagen and skeletal muscle fiber characterization
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ORIGINAL ARTICLE
Stokes polarimetry-based second harmonic generation microscopy for collagen and skeletal muscle fiber characterization Nirmal Mazumder 1
&
Fu-Jen Kao 2
Received: 28 May 2020 / Accepted: 10 September 2020 # The Author(s) 2020
Abstract The complete polarization state of second harmonic (SH) light was measured and characterized by collagen type I and skeletal muscle fiber using a Stokes vector-based SHG microscope. The polarization states of the SH signal are analyzed in a pixel-bypixel manner and displayed through two dimensional (2D) Stokes vector images. Various polarization parameters are reconstructed using Stokes values to quantify the polarization properties of SH light. Also, the measurements are extended for different input polarization states to investigate the molecular structure of second harmonic generation (SHG) active molecules such as collagen type I and myosin. Keywords Second harmonic generation (SHG) microscopy . Polarimetry . Ultrafast optics . Collagen type I . Skeletal muscle fiber
Introduction The second harmonic generation (SHG) is a second-order nonlinear coherent optical process via virtual state transitions, widely used for imaging non-centrosymmetric molecules [1, 2]. The technique has been widely applied for structural imaging from the subcellular to tissue level with high penetration depth employing ultrafast pulsed lasers [3–5]. In general, due to the usage of a near-infrared ultrafast laser, it reduces scattering and absorption in bio-tissues that feature deeper imaging of penetration depth up to mm scale [6–8] and maintains high resolution and contrast as well. Most importantly, the localized excitation through a multiphoton process can greatly suppress the out-of-focus scattering, which facilitates optical sectioning capability to image a thick tissue layer by layer and then reconstructs a three-dimensional (3D) image [9]. Alternatively, SHG is a mature imaging tool for characterizing fiber orientation and morphological structure of collagen with 3D submicron spatial resolution [10, 11], in the human dermis * Nirmal Mazumder [email protected] 1
Department of Biophysics, Manipal School of Life Sciences, Manipal Academy of Higher Education (MAHE), Manipal, Karnataka 576104, India
2
Institute of Biophotonics, National Yang-Ming University, 11221, Taipei, Taiwan
[12, 13], keloid [1], cornea [14, 15], and the tumor microenvironment [16–19]. Furthermore, it can determine the degradation degree of type II collagen in cartilage using polarization-resolved measurements (i.e., P-SHG) [20]. SHG microscopy integrated with two-photon excitation fluorescence (TPEF) has also opened new routes towards label-free optical diagnostics [21], particularly for complex cellular assemblies of skin tissue [19] with submicron spatial resolution. It is found that SHG microscopy combined with TPEF is capable of identifying sarcomeric anomalies of skeletal muscle in physiological/biochemical state with significant sensitivity [22–25]. Additional studies found that a combination of coh
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