研究目的
To develop and validate a 2D Discrete Fourier Transform (DFT) based method for quantitative analysis of collagen fiber structure from Second Harmonic Generation (SHG) images, with application to the human optic nerve head and other tissues.
研究成果
The presented DFT-based method with PPSID correction effectively quantifies collagen fiber structure from SHG images, preserving peripheral data and providing accurate parameters for biomechanical modeling. It was validated against WAXS and applied to various tissues, demonstrating its robustness and potential for 3D analysis in future studies.
研究不足
Limitations include mechanical disruption from microtome sectioning, underestimation of out-of-plane fibers, reliance on laser power ramping for signal normalization, elliptical polarization bias in the microscope, and inability to analyze at single-pixel level or track individual fibers compared to spatial domain methods.
1：Experimental Design and Method Selection:
The study used SHG microscopy for imaging collagen fibers and a bespoke MATLAB-based DFT analysis method to quantify fiber orientation, aligned content, and dispersion. The PPSID algorithm was incorporated for edge artefact correction.
2：Sample Selection and Data Sources:
Human optic nerve head tissues from two donors (ages 86 and 69), avian cornea from adult chickens, rat tail tendon from Wistar rats, and actin cytoskeleton in cultured bovine scleral fibroblasts were used. Tissues were prepared by sectioning and mounting for imaging.
3：List of Experimental Equipment and Materials:
SHG imaging was performed using a Zeiss LSM 880 META NLO microscope with a Coherent Cameleon Vision II laser. WAXS experiments used Diamond Light Source beamline I03 with a Pilatus-6MF detector. MATLAB software was used for image analysis. Materials included paraformaldehyde, PBS/glycerol mixture, and various biological tissues.
4：Experimental Procedures and Operational Workflow:
Tissues were sectioned, mounted, and imaged with SHG. Images were processed in MATLAB: partitioned into tiles, DFT applied with PPSID correction, and fiber parameters extracted. Validation was done through correlation with WAXS data and application to other tissues.
5：Data Analysis Methods:
DFT power spectra were analyzed to compute fiber orientation distributions, aligned collagen content, and degree of fiber recruitment. Correlation coefficients were calculated for validation against WAXS.
独家科研数据包，助您复现前沿成果，加速创新突破
获取完整内容-
LSM 880 META NLO microscope
LSM 880 META NLO
Zeiss
Used for non-linear laser scanning multiphoton microscopy and SHG imaging of collagen fibers.
-
Cameleon Vision II laser
Cameleon Vision II
Coherent
Ultrafast titanium-sapphire tuneable infrared laser used to generate SHG signals from collagen.
-
Pilatus-6MF detector
Pilatus-6MF
Dectris
Silicon pixel detector used in WAXS experiments to record scattering patterns.
-
MATLAB software
MathWorks
Used for developing bespoke image analysis routines, including DFT computation and fiber orientation analysis.
-
HM440E microtome
HM440E
Microm
Sledge cryo-microtome used to cut tissue sections for SHG imaging.
-
FX 3000 tensile system
FX 3000
Flexcell International
Used to apply equibiaxial cyclic load to cultured fibroblasts for actin cytoskeleton studies.
-
BioFlex culture plates
BioFlex
Dunn Labortechnik
Culture plates coated with type I collagen for cell seeding in fibroblast experiments.
-
Alexa-488 phalloidin
Alexa-488
Invitrogen
Fluorescent label used to stain F-actin stress fibers in fibroblasts for confocal imaging.
-
登录查看剩余6件设备及参数对照表
查看全部
