研究目的
To develop a method for digital staining of high-definition FT-IR images using deep learning to achieve cellular-level labeling that mimics traditional histology without the need for chemical stains.
研究成果
The proposed CNN-based method successfully achieves high-resolution digital staining of FT-IR images at the cellular level, providing results similar to traditional histology without chemical alterations. It leverages spatial and spectral features to overcome limitations of previous methods, but further validation and integration with clinical practices are needed.
研究不足
The diffraction limit of IR spectroscopy restricts spatial resolution compared to brightfield or fluorescence imaging. The method relies on subjective evaluation and requires extensive validation for clinical use. It may not be compatible with imaging systems using quantum cascade laser sources due to reliance on higher wavenumber bands.
1:Experimental Design and Method Selection:
The study uses a deep convolutional neural network (CNN) to integrate spatial and spectral features from FT-IR images for digital staining. The CNN architecture includes convolutional layers, pooling layers, and fully connected layers to map IR spectra to chemical stain outputs.
2:Sample Selection and Data Sources:
Tissue samples from pig kidney, pig skin, mouse kidney, and mouse brain were fixed, embedded in paraffin, sectioned, mounted on calcium fluoride substrates, deparaffinized, and rehydrated. After FT-IR imaging, samples were chemically stained with H&E or DAPI for ground truth.
3:List of Experimental Equipment and Materials:
Equipment includes a Cary 600 FT-IR spectroscopic microscope (Agilent Technologies), a Nikon optical microscope, calcium fluoride substrates, formalin, ethanol, xylene, paraffin, phosphate buffered saline, H&E stain, DAPI stain. Materials include tissue samples and software like GIMP for image alignment.
4:Experimental Procedures and Operational Workflow:
Samples were prepared and imaged using FT-IR microscopy. Images were preprocessed with baseline correction and PCA. Alignment was performed manually using GIMP. The CNN was trained on 16,000 samples with a batch size of 128, using the Adam optimizer and mean squared error loss.
5:Data Analysis Methods:
Performance was evaluated using mean squared error, coefficient of determination (R2), and structural similarity index (SSIM). Spectra analysis was done using SIproc software.
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Cary 600 FT-IR spectroscopic microscope
Cary 600
Agilent Technologies
Used for FT-IR spectroscopic imaging of tissue samples in the range of 800–4000 cm?1 at 8 cm?1 spectral resolution.
Cary 60 UV-Vis Spectrophotometer
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Tesla k40M GPU
k40M
NVIDIA
Used for training and testing the convolutional neural network to reduce computation time.
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Nikon optical microscope
Nikon
Used for imaging chemically stained samples in brightfield mode for H&E and with specific filters for DAPI staining.
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GIMP software
GNU
Used for manual image alignment between FT-IR and stained images using affine transformations.
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SIproc software
Used for spectral analysis, such as extracting mean spectra from specific regions of the tissue.
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