Application of an advanced maximum likelihood estimation restoration method for enhanced‐resolution and contrast in second‐harmonic generation microscopy

• Published in: Journal of microscopy (Oxford) Vol. 267; no. 3; pp. 397 - 408
• Main Authors: SIVAGURU, MAYANDI, KABIR, MOHAMMAD M., GARTIA, MANAS RANJAN, BIGGS, DAVID S.C., SIVAGURU, BARGHAV S., SIVAGURU, VIGNESH A., FRIED, GLENN A., LIU, GANG LOGAN, SADAYAPPAN, SAKTHIVEL, TOUSSAINT, KIMANI C.
• Format: Journal Article
• Language: English
• Published: England Wiley Subscription Services, Inc 01-09-2017
• Subjects: Algorithms; Animals; Chickens; Coherent scattering; Collagen; Computed tomography; Deconvolution; Diagnosis; Diffraction; Extracellular matrix; Fluorescence; Fourier analysis; image processing; Image Processing, Computer-Assisted; Image restoration; Imaging, Three-Dimensional - methods; Iterative methods; Light diffraction; Likelihood Functions; Magnetic resonance imaging; Maximum likelihood estimation; Medical diagnosis; Medical imaging; Mice; Microscopy; Microscopy - methods; Microscopy - standards; Myocardium; Myosin; NMR; nonlinear microscopy; Nuclear magnetic resonance; Organs; Photons; Point spread functions; Refractivity; Sarcomeres; second‐harmonic generation; Signal to noise ratio; Spatial discrimination; Spatial resolution; Tendons; image processing; Deconvolution; second-harmonic generation; nonlinear microscopy
• ISSN: 0022-2720; 1365-2818
• DOI: 10.1111/jmi.12579

Summary
Second‐harmonic generation (SHG) microscopy has gained popularity because of its ability to perform submicron, label‐free imaging of noncentrosymmetric biological structures, such as fibrillar collagen in the extracellular matrix environment of various organs with high contrast and specificity. Because SHG is a two‐photon coherent scattering process, it is difficult to define a point spread function (PSF) for this modality. Hence, compared to incoherent two‐photon processes like two‐photon fluorescence, it is challenging to apply the various PSF‐engineering methods to improve the spatial resolution to be close to the diffraction limit. Using a synthetic PSF and application of an advanced maximum likelihood estimation (AdvMLE) deconvolution algorithm, we demonstrate restoration of the spatial resolution in SHG images to that closer to the theoretical diffraction limit. The AdvMLE algorithm adaptively and iteratively develops a PSF for the supplied image and succeeds in improving the signal to noise ratio (SNR) for images where the SHG signals are derived from various sources such as collagen in tendon and myosin in heart sarcomere. Approximately 3.5 times improvement in SNR is observed for tissue images at depths of up to ∼480 nm, which helps in revealing the underlying helical structures in collagen fibres with an ∼26% improvement in the amplitude contrast in a fibre pitch. Our approach could be adapted to noisy and low resolution modalities such as micro‐nano CT and MRI, impacting precision of diagnosis and treatment of human diseases.
Layman's (Sivaguru et al.)
Second‐harmonic generation imaging is an emerging microscopic tool to understand molecular organisation of unlabelled biological structures such as collagen. Conventionally, visualising them using a two‐photon microscopy is a gold standard in understanding their dynamics at a resolution theoretically limited by the diffraction of light. However, due to scattering, absorption and changes in refractive indices, it is hard to reach that theoretical resolution limit. Here we implement a custom‐built iterative image restoration algorithm titled advanced maximum likelihood algorithm (AdvMLE) on SHG images. The algorithm uses deconvolution to restore blurred photons to their original state and recover high spatial frequencies using Fourier analysis. The AdvMLE‐processed images of chicken tendon collagen and mouse heart sarcomeres shows substantial improvement in both lateral and axial resolution, which helps in visualising and analysing the structures with better precision. We believe that this algorithm could be modified in future to restore images from other optical modalities where a conventional resolution improvement is not possible such as magnetic resonance imaging (MRI) or computer assisted tomography (CATScan), and obtain better precision in diagnosis and treatment.