Visualizing hydrocarbon chain size and fatty acid saturation in mouse liver by combining near-infrared hyperspectral imaging and machine studying.


Chloroform, methanol, acetyl chloride, and hexane have been bought from Fujifilm Wako Pure Chemical Co. (Osaka, Japan). Heptadecanoic acid (C17:0) was bought from Sigma-Aldrich (St. Louis, MO, USA). Hydrochloric acid (2%) in methanol (2% HCl/MeOH) was ready by mixing acetyl chloride (5 mL) and methanol (35 mL) on ice.

Animals and gather liver samples

All protocols have been carried out utilizing mice whereas minimizing the quantity and struggling of mice used. In accordance with nationwide and institutional tips beneath approval by the Animal Care and Use Committees of Tokyo College of Science (Approval No.: K22004) and Osaka Metropolitan College (Approval No.: 2021-088). All routes are reported in response to accessibility tips. C57BL/6NJcl and BALB/c mice have been bought from CLEA Japan, Inc. (Tokyo, Japan) and Japan SLC, Inc. (Shizuoka, Japan), respectively. Mice have been housed beneath pathogen-free situations with a 12 h/12 ​​h gentle/darkish cycle. They have been fed both ND (AIN-76A; Analysis Diets, Inc., NJ, USA), or a high-fat eating regimen (HFD) (D12492; 60% vitality fats with enriched lard; Analysis Diets, Inc.), eating regimen. Excessive ldl cholesterol (HCD) (D12336; 35% vitality fats with cocoa butter, coconut oil and 1.5% added ldl cholesterol; Analysis Diets), or modified HFD with 2% LA (D17011206; Analysis Diets, Inc.) or 12% LA ( D17011207; Analysis Diets Inc.) for five days to 32 weeks (Complement Desk S1). HFDs, with 2% and 12% LA, have been designed as diets to analyze liver carcinogenesis by NAFLD.29, ready by changing lard and soybean oil with particular proportions of coconut oil (wealthy in saturated fatty acids with low LA) and safflower oil (excessive LA). Such variety in eating regimen causes mice to harbor a variety of liver lipid parts with completely different properties, equivalent to HCL and DS. Liver samples have been remoted from mice beneath anesthesia with 2.0% isoflurane and subjected to NIR-HSI evaluation. Hyperspectral NIR photographs (wavelength: 760–1885 nm; vary interval: 2.2 nm) have been captured for every liver lobe.

Liver most cancers attributable to chemical substances

Male mice have been handled with 7,12-dimethylbenz.a) Anthracene (DMBA) (Sigma) for carcinogenesis experiments as beforehand described30. Briefly, 50 μl of 0.5% DMBA in acetone was utilized to the dorsal floor on postnatal day 4–5. Neonatal mice utilized in carcinogenesis experiments have been blended with littermates when grouping to keep away from any tendency to litter. Subsequent, the mom and pups have been fed an HFD. Pups have been weaned at 4 weeks of age and fed constantly with an HFD containing 12% LA till euthanized at 34 weeks of age.

Fats extraction and complete fats weighing

Liver samples have been weighed utilizing an digital scale and subjected to NIR-HSI earlier than lipid extraction. Complete lipids have been extracted from every pattern utilizing the Folch methodology16, which is a longtime protocol for isolating lipids from animal tissue. Briefly, the pattern was minced utilizing a BioMasher (Nippi, Inc., Tokyo, Japan) in 7 mL of a 2:1 (v/v) combination of chloroform and methanol. After including 2 ml of distilled water to the combination containing the fats, it was centrifuged at 1400×.g For 7 minutes. The natural fraction was then positioned in a glass tube and heated to 60 °C till the natural solvent utterly evaporated. The remaining fats within the glass tubes was weighed utilizing an digital scale.

GC evaluation

The fatty acid content material of lipids extracted from every liver lobe pattern was analyzed. The focus of every fatty acid was analyzed by GC of lipid samples extracted from every liver lobe collected from ND-fed and advert libitum-fed mice to acquire consultant data on a collection of fatty acids.18. After lipid extraction, heptadecanoic acid (1 mg) was added to the response combination as an inside normal. The extracted lipids have been dissolved in 2% HCl/MeOH (2 ml) at 55°C for 20 min for methylation. Then distilled water (1 ml) and nHexane (3 ml) was added to the fatty acid methyl esters and vortexed. Samples have been centrifuged at 3000×g For 10 minutes, the n-hexane fraction is concentrated by evaporation beneath low strain. Fatty acid methyl esters have been extracted with chloroform and analyzed by gasoline–liquid chromatography utilizing a gasoline chromatograph (Nexis GC-2030; Shimadzu Co., Kyoto, Japan) outfitted with a flame ionization detector (FID) and a break up injection system (break up ratio 1:50) outfitted with a column. Capillary (Supelco SPB-1; 30 m size × 0.25 mm i.d.; Sigma-Aldrich). The preliminary column temperature was 180 °C for 30 min after which elevated to 210 °C at a fee of 60 °C/min. The column temperature was maintained at 210 °C for 29.5 min. The injector and detector have been operated at 250°C. Helium was used because the service gasoline at a stream fee of 1.4 ml/min. Fatty acid peaks have been recognized by evaluating retention instances with these of identified requirements (Suppl. Determine S1). Every fatty acid was quantified utilizing 1 mg of inside and exterior requirements.

NIR-HSI and information evaluation by SVR

A line-scanning NIR-HSI system was used to acquire hyperspectral photographs of the samples, as proven schematically in Determine 1. The system features a NIR reflectance imaging spectrometer (ImSpector N17E, Specim, Oulu, Finland) coupled to a NIR digicam (XEVA-1.7-640, Xenics nv, Leuven, Belgium) and NIR goal lens (SWIR 83-160 collection, focal size 25 mm, Edmund Optics, NJ, USA), gold-plated rotation mirror (40 x 100 mm)2) for the y-axis scan, a halogen lamp (LA-150UE, Hayashi-Repic Co., Ltd., Tokyo, Japan) as the sunshine supply, and a pc (JFE Techno-Analysis Co., Tokyo, Japan)13 (Determine 1b). The NIR digicam features a 2D indium gallium arsenide (InGaAs) sensor array that captures 512 x 640 pixel photographs that correspond to the wavelength and place of NIR gentle (900–1700 nm; wavelength decision: 2.2 nm) on this coupled system. In a single shot utilizing a NIR-HSI system, optical density values ​​for every wavelength band are captured at every pixel on the road. The y-axis line scan reconstructs depth maps (reflectance ratio) for every wavelength on the xy pixel array. The facility of the halogen lamp was managed in order that the pattern temperature was secure. HSI information acquisition for liver samples was carried out in a darkish room at a temperature and humidity of roughly 22–24 °C and 50–60%, respectively. Acquisition was carried out after recording the darkish and white references to acquire the reflectance ratio for every wavelength vary, and the info have been then pre-processed as described beforehand.13. Area of curiosity sizes have been 1500–3000 pixels per pattern. NIR reflectance photographs have been normalized to find out the relative reflectance R(to) utilizing the next equation:

$$start{array}{c}Rleft(lambda proper)=frac {{I}_{uncooked}left(lambda proper)-{I}_{darkish}left( lambdaright)} {{I}_{white}left(lambdaright) – {I}_{darkish}left(lambdaright)}finish{array},$$


the place R() is the relative reflectance calculated for every , Iuncooked(×) is the preliminary depth of a given pixel, Idarkish(π) is the darkish reference picture for changing depth photographs into reflectance photographs by eradicating the impact of darkish present, and Iwhite(π) is the depth obtained from the white reference picture. After eradicating spikes within the NIR photographs, all pixels belonging to every liver pattern have been chosen. Moreover, the reflectance spectrum of every liver pattern was averaged from the element pixel reflectance spectra and used to signify the liver samples. As well as, the absorption spectra corresponding to those reflectance spectra, that are outlined as log(1/R(to), have been used for evaluation. Spectra wavelength ranges have been eliminated <1000 نانومتر و> 1400 nm of study because of the low sensitivity of the NIR digicam and the excessive absorption by water in these ranges.

Pixel reflectance spectral information have been analyzed by SVR, a non-parametric supervised machine studying algorithm.31 It may well rework a nonlinear regression drawback right into a linear regression by implementing a kernel perform32. Since SVR has been proven as an appropriate regression mannequin for analyzing NIR-HSI information by evaluating the outcomes with these obtained utilizing partial least squares regression (PLSR).13,SVR was additionally used on this research. To compensate for the impact of scattering on the pattern floor and the darkish present noise of the digicam, the NIR reflectance spectra have been normalized by SNV utilizing the imply and normal deviation of the spectrum of curiosity.33 As follows:

$$start{array}{c}z =frac{x-mathrm{imply}left(xright)}{mathrm{std}left(xright)},finish{array} $$


the place s And Z are the unique and reworked SNV spectra, respectively, and the means (s) and sexually transmitted ailments (s) are the imply and normal deviation of s Over all the wavelength vary. Inside validation was carried out utilizing a leave-one-out cross-validation methodology to create SVR calibration fashions. SNV may normalize information for every set of experiments to match all the information set. Coaching information have been generated by matching NIR spectral information with common values ​​of complete lipid content material, HCL, and DS for fatty acids recognized utilizing the Folch and GC extraction methodology. Fashions have been evaluated utilizing R2 Validation, outlined as follows:

$$start{array}{*{20}c} {R^{2} = 1 – frac{{mathop sum nolimits_{i}^{n} left( {hat{y}_ {i} – y_{i} } proper)^{2} }}{{mathop sum nolimits_{i}^{n} left( {hat{y}_{i} – overline{ {y_{i} }} } proper)^{2} }}} finish{array} , $$


the place (hat{r}_{i}) And yI are the measured and predicted values ​​of I-liver pattern, respectively, n is the variety of samples, and (high line {r}) It’s the common of the measured values ​​for all samples.

Schematic diagram of HCL/DS

To visualise modifications in liver fatty acid content material attributable to completely different diets, the imply values ​​(for every eating regimen group) of HCL and DS for liver fatty acids estimated by NIR-HSI and validated by GC have been plotted on a biplot. Dimensional airplane with HCL and DS represented on the x and y axes respectively.

Evaluation of tumor-bearing mice by SVR

Livers have been harvested beneath anesthesia from tumor-bearing mice and NIR-HSI evaluation carried out. Hyperspectral NIR photographs have been acquired for every liver lobe. Reflectance spectral information of tumor-bearing mice have been analyzed by SVR utilizing the 102-sample information set proven within the Appendix. Desk S1 to visualise the distribution of complete lipid content material, HCL and DS.

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