Evaluation of tile artifact correction methods for multiphoton microscopy mosaics of whole-slide tissue sections

Knapp, Thomas G.; Lima, Natzem; Duan, Suzann; Merchant, Juanita L.; Sawyer, Travis W.

doi:10.1117/12.2609634

Cited by 4 publications

(8 citation statements)

References 4 publications

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“…A custom MATLAB frequency filtering step was then used on the maximum projection images. For a more complete writeup on this image processing, please refer to Knapp et al 34 A MATLAB implementation of the structural similarity index (SSIM) 35 was used to determine if image information was retained post-processing. Visual examination of the images was done to confirm the absence of artifacts and the SSIM score was used to quantitatively support the inclusion of the images in our analysis.…”

Section: Methodsmentioning

confidence: 99%

Quantitative Characterization of Duodenal Gastrinoma Autofluorescence using Multi-photon Microscopy

Sawyer

Knapp

2022

Preprint

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Duodenal gastrinomas (DGASTs) are neuroendocrine tumors that develop in the submucosa of the duodenum and produce the hormone, gastrin. Surgical resection of DGASTs is complicated by the small size of these tumors and the tendency for them to develop diffusely in the duodenum. Endoscopic mucosal resection of DGASTS is an increasingly popular method for treating this disease due to its low complication rate but suffers from poor rates of pathologically negative margins. Multiphoton microscopy (MPM) is capable of capturing high-resolution images of biological tissue with contrast generated from endogenous fluorescence (autofluorescence) through two-photon excited fluorescence (2PEF). Second harmonic generation (SHG) is another popular method of generating image contrast with MPM and is a light-scattering phenomenon that occurs predominantly from structures such as collagen in biological samples. Some molecules that contribute to autofluorescence change in abundance from processes related to the cancer disease process (e.g., metabolic changes, oxidative stress, angiogenesis). MPM was used to image 12 separate patient samples of formalin-fixed and paraffinized DGAST slides with a SHG channel 4 2PEF channels, each tuned to capture fluorescence from NADH, FAD, lipofuscin, and porphyrin. We found that there was a significant difference in the relative abundance of signal generated in the 2PEF in comparison to the neighboring tissues of the duodenum. Texture extraction was used to create linear discriminant classifiers for tumor vs all other tissue classes before and after principal component analysis (PCA) of the texture feature dataset. PCA improved the classifier accuracy and reduced the number of features required to achieve maximum accuracy of the classifier. The LDA classifier after PCA distinguished between tumor and other tissue types with an accuracy of 90.6 - 93.8%. These results suggest that MPM 2PEF and SHG imaging is a promising label-free method for discriminating between DGAST tumors and normal duodenal tissue which has implications for future applications of in vivo assessment of resection margins with endoscopic MPM.

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Section: Methodsmentioning

confidence: 99%

Quantitative Characterization of Duodenal Gastrinoma Autofluorescence using Multi-photon Microscopy

Sawyer

Knapp

2022

Preprint

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“…A custom MATLAB frequency filtering step was then used on the maximum projection images. For a more complete writeup on this image processing, please refer to Knapp et al 39 A MATLAB implementation of the structural similarity index (SSIM) 40 was used to determine if image information was retained postprocessing. Visual examination of the images was done to confirm the absence of artifacts and the SSIM score was used to quantitatively support the inclusion of the images in our analysis.…”

Section: Methodsmentioning

confidence: 99%

Quantitative characterization of duodenal gastrinoma autofluorescence using multiphoton microscopy

et al. 2022

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Background: Duodenal gastrinomas (DGASTs) are neuroendocrine tumors that develop in the submucosa of the duodenum and produce the hormone gastrin. Surgical resection of DGASTs is complicated by the small size of these tumors and the tendency for them to develop diffusely in the duodenum. Endoscopic mucosal resection of DGASTs is an increasingly popular method for treating this disease due to its low complication rate but suffers from poor rates of pathologically negative margins. Multiphoton microscopy can capture high-resolution images of biological tissue with contrast generated from endogenous fluorescence (autofluorescence [AF]) through two-photon excited fluorescence (2PEF). Second harmonic generation is another popular method of generating image contrast with multiphoton microscopy (MPM) and is a lightscattering phenomenon that occurs predominantly from structures such as collagen in biological samples. Some molecules that contribute to AF change in abundance from processes related to the cancer disease process (e.g., metabolic changes, oxidative stress, and angiogenesis). Study Design/Materials and Methods: MPM was used to image 12 separate patient samples of formalin-fixed and paraffin-embedded duodenal gastrinoma slides with a second-harmonic generation (SHG) channel and four 2PEF channels. The excitation and emission profiles of each 2PEF channel were tuned to capture signal dominated by distinct fluorophores with well-characterized fluorescent spectra and known connections to the physiologic changes that arise in cancerous tissue. Results: We found that there was a significant difference in the relative abundance of signal generated in the 2PEF channels for regions of DGASTs in comparison to the neighboring tissues of the duodenum. Data generated from texture feature extraction of the MPM images were used in linear discriminant analysis models to create classifiers for tumor versus all other tissue types before and after principal component analysis (PCA). PCA improved the classifier accuracy and reduced the number of features required to achieve maximum accuracy. The linear discriminant classifier after PCA distinguished between tumor and other tissue types with an accuracy of 90.6%−93.8%. Conclusions: These results suggest that multiphoton microscopy 2PEF and SHG imaging is a promising label-free method for discriminating between DGASTs and normal duodenal tissue which has implications for future applications of in vivo assessment of resection margins with endoscopic MPM.

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“…This work is a continuation of the SPIE proceedings paper. 17 Thomas Knapp is a PhD student in the Biomedical Engineering Department at the University of Arizona. He received his BS degree in physiology from the University of Arizona in 2017.…”

Section: Acknowledgmentsmentioning

confidence: 99%

Combined flat-field and frequency filter approach to correcting artifacts of multichannel two-photon microscopy

Knapp,

Lima,

Daigle

et al. 2024

J. Biomed. Opt.

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Significance: Multiphoton microscopy (MPM) is a useful biomedical imaging tool for its ability to probe labeled and unlabeled depth-resolved tissue biomarkers at high resolution. Automated MPM tile scanning allows for whole-slide image acquisition but can suffer from tile-stitching artifacts that prevent accurate quantitative data analysis.Aim: We have investigated postprocessing artifact correction methods using ImageJ macros and custom Python code. Quantitative and qualitative comparisons of these methods were made using whole-slide MPM autofluorescence and secondharmonic generation images of human duodenal tissue.Approach: Image quality after artifact removal is assessed by evaluating the processed image and its unprocessed counterpart using the root mean square error, structural similarity index, and image histogram measurements.Results: Consideration of both quantitative and qualitative results suggest that a combination of a custom flat-field-based correction and frequency filtering processing step provide improved artifact correction when compared with each method used independently to correct for tiling artifacts of tile-scan MPM images.Conclusions: While some image artifacts remain with these methods, further optimization of these processing steps may result in computational-efficient methods for removing these artifacts that are ubiquitous in large-scale MPM imaging. Removal of these artifacts with retention of the original image information would facilitate the use of this imaging modality in both research and clinical settings, where it is highly useful in collecting detailed morphologic and optical properties of tissue.

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Evaluation of tile artifact correction methods for multiphoton microscopy mosaics of whole-slide tissue sections

Cited by 4 publications

References 4 publications

Quantitative Characterization of Duodenal Gastrinoma Autofluorescence using Multi-photon Microscopy

Quantitative Characterization of Duodenal Gastrinoma Autofluorescence using Multi-photon Microscopy

Quantitative characterization of duodenal gastrinoma autofluorescence using multiphoton microscopy

Combined flat-field and frequency filter approach to correcting artifacts of multichannel two-photon microscopy

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