Mass spectrometry grade chemicals and reagents were used in the present study. Matrix moieties inclusive of 9-aminoacridine (9AA, no. 92817), 2, 5-dihydroxybenzoic acid (DHB, no. 149357), α-cyano-4-hydroxycinnamic acid (CHCA, no. C2020) were obtained from Sigma-Aldrich (St Louis, MO, United States). The internal standard (IS), N-1-naphthylphthalamic acid (NPA), was purchased from Chem Service (West Chester, PA, United States, no. N875498). Ultra-pure grade (18 MU) water was prepared by a Milli-Q system (Millipore Corporation, Billerica, MA, United States). Other reagents include formic acid (FA; Aladdin, no. F112034); Trifluoroacetic Acid (TFA; TEDIA, no. TS4295-013C); Methanol (MeOH; Honeywell, no. AH230-4HC); Ethanol (EtOH; Sigma-Aldrich, no. E7023); Ethanol (Tianjin Fuyu Fine Chemical Co., Ltd.); Acetonitrile (ACN; Honeywell, No. AH015-4HC); Xylene (Tianjin Fuyu Fine Chemical Co., Ltd.); Eosin (Beijing Solarbio Technology Co., Ltd., cat. no. G1100); Neutral gum (Beijing Solarbio Technology Co., Ltd., cat. no. G8590-100 ml), and indium tin oxide (ITO)-coated glass slides (Sigma-Aldrich, no. 578274).

Eleven BAs were selected for analysis: deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), taurodeoxycholic acid (TDCA), taurochenodeoxycholic acid (TCDCA), tauroursodeoxycholic acid (TUDCA), lithocholic acid (LCA), taurolithocholic acid (TLCA), taurocholic acid (TCA), tauromuricholic acid (TMCA), and glycocholic acid (GCA).

All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at the China Medical University. Six to eight weeks old male C57BL/6 mice were purchased from Beijing Huafukang Biotechnology Co., Ltd. The mice were fed with normal circadian circulation for 12 h and had enough food and water. Mice were fed an HFD (TP23520, Tropic diet, China) or a control diet (CD, TP23524, Tropic diet, China) for 4 weeks. There were 6–10 mice in both the CD group and the HFD group. After 4 weeks of feeding, animals were sacrificed for analysis.

After euthanasia, the tissues were quickly removed and all samples should be frozen immediately and stored at −80°C until further analysis. Set the cryomicrotome (Leica CM1950, Nussloch, Germany) to −25°C and cut the samples at a thickness of 10 μm. The slices were pasted on ITO-coated glass slide for analysis by MALDI-MSI. Three to five serial sections (each 10 μm thick) of sampled liver, ileum, and colon tissue were used to evaluate the reproducibility of the iMScope technique. A small droplet of optimal cutting temperature (OCT) compound is applied for mounting the tissue sample on the specimen holder. Because polyvinyl alcohol/polyethylene glycol-based media, such as OCT compound, cause ion suppression during MSI analysis, it is essential to avoid contamination of tissue sections, cryostat tables, or blades by OCT compounds (19). Subsequently, a “two-step matrix application,” which combined with sublimation and airbrushing, was used to coat matrix for tissue sections (18, 20). (1) Sublimation: the electrically conductive glass slide bearing specimen (slice spiked with 10 ng ml^–1 of NPA) was installed in a sample holder, which was then embedded in a vacuum deposition system (SVC-700TMSG iMLayer, Sanyu Electron, Tokyo, Japan). A matrix holder was filled with approximately 300 mg of matrix powder (9AA) and the sample holder and matrix bracket were positioned with 8 cm distance. The matrix power was then heated to the boiling point of the matrix crystals (220°C for 9AA) and the vapor covered the specimen surface for 8 min. The vacuum pressure of the chamber was maintained at 10^–4 Pa in the process of sublimation. (2) Airbrushing: Matrix solution (10 mg ml^–1 of 9AA) was prepared by dissolving matrix power in acetonitrile and distilled water (all containing 0.1% FA) at a ratio of 1:1. The matrix solution (1 ml) was added to the capacity of an artist’s airbrush (MR. Linear Compressor L7/PS270 Airbrush, GSI Creos, Tokyo, Japan). The distance between the tip of the airbrush and the tissue surface was about 8 cm. For the first 3 cycles, the matrix was airbrushed for 2 s at 60 s intervals and, in the following 20 cycles, the matrix was continuously sprayed for 1.0 s at 30 s intervals. The glass slide was then placed in a vacuum dryer to vaporize the solvent for 5 min. After that, the glass slide was vacuum-dried, and then the BA distribution was observed with iMScope.

The parameters of IT-TOF (time-of-flight) MS were set as follows: ion polarity, negative; mass range, 350–550; sample voltage, 3.0 kV; and detector voltage, 1.90 kV. The imaging MS Solution Version 1.12.26 software (Shimadzu, Tokyo, Japan) was used to control the instrument, and the data acquisition, visualization, and quantification were also performed by the same software. The m/z values were externally calibrated using the DHB matrix. The identification of BAs was confirmed by MALDI-TOF-MS/MS with reference to product ion spectra of authentic BA standards.

The iMScope TRIO (Shimadzu, Japan) instrument, a hybrid IT-TOF MS combining an optical microscope and atmospheric pressure MALDI/ionization system, was used to acquire the imaging MS data. One of the most critical processes in MSI is the creation of the region of interest (ROI). The optical microscope embedded in the iMScope permitted us to precisely choose the relevant tissue region prior to performing data acquisition. An ultraviolet laser, tightly focused with a triplet lens, was used to ensure high spatial resolution. Based on the advanced configuration above, a tissue ROI was freely selected via a charge-coupled device (CCD) camera (magnification, ×1.25/×2.5, Olympus Corporation, Tokyo, Japan) and the imaging area was then defined according to the maximum imaging point under a scan pitch of 40 μm. The illumination in the iMScope was operated under the following parameters: light type, trans-illumination; light intensity, 12%. Foci and observation points were controlled with a joystick and the XYZ stage (Kohzu Precision, Kanagawa, Japan) on which the electrically conductive glass slide was fixed. The XYZ coordinates, with position-feedback scales, immediately displayed the ROIs to make position reproducibility possible on a sub-micrometer order. The tissue slices were then irradiated by a focused laser beam in synchrony with stage scanning. The laser in the iMScope system was a diode-pumped 355 nm Nd, YAG laser (Shimadzu Corporation, Kyoto, Japan), and operated under the following parameters: frequency, 1,000 Hz; laser intensity, 55.0; and laser diameter, 3 μm. All the experiments in this work were conducted with the minimum irradiation diameter and irradiated the tissue surface with 100 shots (repetition rate; 1,000 Hz) for each pixel.

Quantitative analysis with MALDI-TOF MS has been demonstrated for compounds of biological interest. For quantification, ISs are necessary to compensate for the poor shot-to-shot reproducibility inherent in the use of MALDI analysis. An ideal IS should be the stable isotope-labeled intact BAs during the qualitative and quantitative analyses of BAs (21). However, it was impractical in the present case due to synthesis challenges and time vs. cost concerns. Therefore, we want to search for some compounds that are chemically similar to BAs, have a quality close to BAs, and are chemically stable during the analysis process as ISs. Unfortunately, we did not find it either. While the NPA chosen for this analysis is chemically different from BAs, it still proved to be effective. NPA has been used to quantify BAs directly from plasma and urine by MALDI-TOF-MS (22, 23). The structure and MS spectrum of NPA are shown in Supplementary Figure 1. We used the average intensity ratio of each BA to NPA to quantitatively describe the relative exposure level of BAs in tissue sections. The average peak intensity ratio was calculated as follows: average peak intensity ratio = BAs average peak intensity/NPA average peak intensity.

After MSI, the tissue sections were stained with hematoxylin and eosin (H&E) for examination based on a protocol (11). In brief, the tissue sections were stained with H&E as follows: the glass slides were gently washed with 70% ethanol to remove the matrix, followed by dipped in 100% ethanol at −20°C for 5 min, and then glass slides were put in distilled water for 2 min. The slides were then immersed in hematoxylin for 5 min, washed with tap water for 5 min, followed by differentiated with 1% hydrochloric acid alcohol for 5 s, and blued in running tap water for 10 min. Staining by eosin for 5 s was followed by a wash with tap water for 5 min. Slides were then dipped in 90% ethanol for 6 min, and 100% ethanol for 2 × 5 min, followed by placing them in three different xylene baths for 5 min each. Finally, The H&E slides were sealed with neutral gum and scanned using an iMScope TRIO (Shimadzu, Japan) instrument.

Data in bar graphs are expressed as mean ± SEM. The Shapiro-Wilcoxon test was used to test the Gaussian distribution of biological parameters. The Student t test was used for comparison between two groups. The Mann-Whitney test was used for variables that were not normally distributed. Correlations were assessed with the Pearson correlation coefficient or as indicated in the graphs. All statistical analysis was performed using SPSS (version 25), considering value of p < 0.05 as statistically significant. ROI analysis by using Imaging MS Solution software (version 1.30). The p of comparison for ROI analyses was assessed via average peak intensities or signals acquired from MS spectra of areas indicated by ROI. Low values of p (p < 0.05) denote significant differences between average peak intensities or signals of targets within the stipulated ROIs.

Supplementary Figure 2 shows approximate values of the optimal laser intensity for each set “Laser Diameter” value and the diameter of the laser in that case. Use this as a guide for the laser intensity and measurement pitch to be set when changing the laser diameter. The example in Supplementary Figure 2 is for the optimal laser intensity when depositing DHB in mouse liver homogenate and measuring lipids in positive ion mode.

As shown in Supplementary Figure 1, the IS (NPA) is seen at m/z 290.082, although NPA is chemically different from BAs, there are an adequate signal and no interference peaks in the mass spectrum of NPA. Therefore, it is a feasible method to quantitatively describe the relative exposure level of BAs in tissue sections by the average intensity ratio of each BA to NPA. Two of the major impediments to the application of MALDI to quantitative experiments are signal suppression and signal interference, especially, of less concentrated components (24). However, this effect can be compensated for the use of an IS and optimization of the experimental parameters (24). NPA, chemically different from BAs, has been proved to be effective in quantifying BAs directly from plasma and urine by MALDI-TOF-MS (22, 23). In our study, we compared the BA content of different groups, we sprayed 10 mg ml^–1 NPA evenly on the tissue sections, and it could be clearly seen through iMScope that NPA exhibits good uniformity and stability. We divided the average peak intensity of BAs by the average peak intensity of NPA to obtain the average peak intensity ratio of each BA. We compared the average peak intensity ratio of each BA in different groups to obtain the final results, which eliminated the influence of signal suppression and signal interference of different groups on the comparison results to a certain extent.

In our study, eleven BAs conjugates were selected for analysis: DCA, CDCA, UDCA, TDCA, TCDCA, TUDCA, LCA, TLCA, TCA, TMCA, and GCA. The structures for these compounds are shown in Supplementary Figure 3. It should be noted that the putative identification using MSI technology is only based on the measured accurate m/z value, so it is impossible to distinguish the isomers of the target compound with the same molecular formula (e.g., m/z 498.2895, putative identification as TCDCA/TUDCA/TDCA). BAs show a high degree of isomerism and similar binding (11, 25), as a result, many BAs share the same molecular formula, so it is impossible to determine the contribution of several isomers (e.g., TCDCA, TUDCA, and TDCA) in the net m/z signal peak intensity (m/z 498.2895 here) and the corresponding m/z image. Based on previous quantitative studies of BAs in rat liver, we assumed that all isomers contributed to the ion abundance (25, 26). In order to distinguish the contribution of each isomer to ion abundance, MSⁿ of commercially available standards of the different isomers should be considered.

The MALDI matrixes, aromatic compounds of low molecular mass, which are crucial for optimal signal-to-noise levels and the quality of data, are used to enhance the ionization efficiency and prevent the analytes from degrading. Owing to the specific ionization property of the individual matrix, the selection of a suitable matrix is understandably critical to MALDI-MS. MALDI matrices are chosen based on their ability to provide sufficient ionization efficiency for a given analytes class (e.g. low molecular weight metabolites, lipids, proteins, polymers, or inorganic compounds) or sub-class (27). Differences in observed analyte sensitivities can be attributed to the physical properties of a matrix such as molecular structure, pH, proton affinity, and peak wavelength absorbance (28). Thus, we investigated three conventional matrices, namely DHB, CHCA and 9AA, to screen the optimum matrix via studying their abilities to form co-crystals onto tissue sections. DHB is the most widely employed and studied MALDI matrix offering sufficient sensitives for many analyte classes in MS analysis (29). 9AA is often used for the analysis of low molecular weight compounds in negative ion mode MS analysis and CHCA ionizes many drugs with high sensitivity in both polarities (30). As shown in Supplementary Figure 4, the ion signal of BAs generated by iMScope is evenly scattered over the tissue sections. The average ion signal intensity of the BA images in Supplementary Figures 4B,C (using CHCA and DHB as the matrix) is much lower than that in Supplementary Figure 4A (using 9AA as the matrix). According to the findings above, 9AA was confirmed to be the most suitable matrix for the analysis of BAs based on iMScope. In conclusion, by using 9AA as the matrix and NPA as IS, we have developed a method for semi-quantitative BAs using MALDI-IMS.

Bile acid synthesis occurs in the liver. Before the synthetic BA enters the duodenum, the liver BAs bind to glycine or taurine. The conjugated BA is stored in the gallbladder until it is released to the duodenum after eating (31). BAs in the human body are mainly conjugated to glycine and less to taurine, while BAs in mice and rats are almost completely conjugated to taurine (6). Here, we detected non-conjugated BAs CDCA/UDCA/DCA and conjugated BAs TCDCA/TUDCA/TDCA and TCA/TMCA in the liver. We found that when compared with the control group MS ion image, the ion intensity of CDCA/UDCA/DCA, TCDCA/TUDCA/TDCA and TCA/TMCA in liver tissue sections of mice fed with HFD for 4 weeks was decreased significantly, especially, TCDCA/TUDCA/TDCA, TCA/TMCA (Figure 1). Supplementary Figures 5A,C,E, respectively, shows the mass spectra of CDCA/UDCA/DCA, TCDCA/TUDCA/TDCA, TCA/TMCA and NPA. NPA-based mean peak intensity ratios also showed that CDCA/UDCA/DCA, TCDCA/TUDCA/TDCA and TCA/TMCA in liver tissue sections of mice fed with HFD for 4 weeks were lower than those in the control group (Supplementary Figures 5B,D,F). BAs have become established as important signaling molecules that enable fine-tuned inter-tissue communication within the enterohepatic circulation. Mounting pieces of evidence indicate that changes in overall BA levels and specific BA metabolites are involved in metabolic diseases, gastrointestinal, and liver cancer (32).

Here, we clearly showed that 1 month of HFD-feeding resulted in visible multiple BAs zonation patterns and might have disturbed the inherent BAs gradients or functional zonal differences in enterohepatic circulation. Taking the spatial information into account is crucial to investigate the underlying mechanisms of injury induced by site-specific BA alterations. Combined with the transport network of BAs in the liver, we analyzed the distribution of TCDCA/TUDCA/TDCA and TCA/TMCA in mouse liver tissue sections at high spatial resolution. As expected, BAs were not evenly distributed in the liver and showed a certain regional aggregation pattern, which had been clearly shown from the MS ion image (Figure 2). It should be pointed out that Figure 2 is derived from the HFD group in Figure 1A. The green arrows indicated bile ducts, whereas the red arrows showed blood vessels. Figure 2 shows MS ion images of the spatial distribution of taurine-conjugated BAs identified in the same tissue section at high spatial resolution. The results showed that there were higher ion signal intensities at m/z 498.2895 ± 0.05 (TCDCA/TUDCA/TDCA) and m/z 514.2844 ± 0.05 (TCA/TMCA) at the bile ducts indicated by the green arrows. The bile duct, portal venule, and portal arteriole form the portal trial, in which the bile duct receives bile from bile canaliculi. Our results have shown that MS ion images in the same liver tissue section at high spatial resolution show that there are zonation patterns and regional differences characteristics of BAs in tissues. Thus, the liver, BAs site of production, displayed physiological and functional zonal differences in the periportal zone versus the centrilobular zone. Therefore, our method can not only explore the spatial distribution characteristics and content variety of BAs in the liver but also intuitively see the distribution characteristics of BAs around blood vessels and bile ducts in liver tissue.

The intestinal tract, modified by the gut microbiota, showed regional differences in BA metabolism (33). After exerting physiological functions, such as dissolving and digesting fat-soluble nutrients, most of BAs are then absorbed by passive diffusion and active transport from the terminal ileum and transported back to the liver via the portal vein, and the rest is excreted with feces. Therefore, the terminal ileum is also the main site of BAs accumulation, especially, conjugated BAs. We took a 3-cm long mouse terminal ileum and rolled it up in a “Swiss roll” (34). Here, we focused on the level of BAs in the ileum. The spatial distribution of TCDCA/TUDCA/TDCA, TCA/TMCA, GCA, and NPA in ileum sections is shown in Figure 3. We found that when compared with the control group MS ion image, the ion intensity of TCDCA/TUDCA/TDCA, TCA/TMCA, and GCA in ileum tissue sections of mice fed with HFD for 4 weeks was decreased significantly, especially TCDCA/TUDCA/TDCA and TCA/TMCA decreased more significantly, which were also proved by the average peak intensity ratio based on NPA (Supplementary Figures 6B,D,F). TCA/TMCA signal strength was the highest. Supplementary Figures 6A,C,E shows the mass spectra of TCDCA/TUDCA/TDCA, TCA/TMCA, GCA, and NPA.

In the colon, bacterial enzymes catalyze the deconjugation and dehydroxylation of primary BAs to produce secondary BAs. Here, we focused on the level of BAs in the colon, eleven BA signals are found in the tissue, as shown in Figure 4. All ion images were created based on the signals from the deprotonated molecules: NPA at m/z 290.0823 ± 0.05, CDCA/UDCA/DCA at m/z 391.2854 ± 0.05, TCDCA/TUDCA/TDCA at m/z 498.2895 ± 0.05, LCA at m/z 375.2905 ± 0.05, TLCA at m/z 482.2946 ± 0.05, TCA/TMCA at m/z 514.2844 ± 0.05, and GCA at m/z 464.3018 ± 0.05. The grating width of the whole tissue section was 700 μm (Figure 4). The spatial distribution of CDCA/UDCA/DCA and NPA in colon sections is shown in Figure 4A. Supplementary Figure 7A shows the mass spectra of CDCA/UDCA/DCA and NPA. We found that when compared with the control group MS ion image, the ion intensity of CDCA/UDCA/DCA in colonic tissue sections of mice fed with HFD for 4 weeks was decreased significantly, which was also proved by the average peak intensity ratio of CDCA/UDCA/DCA based on NPA (Supplementary Figure 7B). The MS ion images of the other eight observed BAs showed the same tendency as CDCA/UDCA/DCA. After feeding with HFD for 4 weeks, the ion intensities of TCDCA/TUDCA/TDCA, LCA, TLCA, TCA/TMCA, and GCA were decreased significantly (Figures 4B–F). The average peak intensity ratio of each BA based on NPA was also significantly different (Supplementary Figures 7D,F,H,J,L), and the trend was consistent with their MS ion images. Supplementary Figures 7C,E,G,I,K shows the mass spectra of TCDCA/TUDCA/TDCA, LCA, TLCA, TCA/TMCA, GCA, and NPA. By overlaying the acquired MS ion images with their respective optical images (Figure 4), it was discovered that the intensities of BAs were diffusely distributed in colonic tissue. Our results indicate that HFD feeding reduced BAs signal in colon sections by impairing the active and passive reabsorption of BAs in the intestine, which resulted in a significant increase in fecal excretion of BAs. In addition, it is worth noting that BAs play a crucial role in limiting bacterial overgrowth through intestinal FXR, so as to protect the intestine from bacterial damage, and the chronic low-grade inflammation of the intestine caused by HFD may be related to the decrease of BAs content in the intestinal tissue sections observed in this study (1–5).

To evaluate the relative abundance and distribution of detectable BAs in the sampled colon tissues, we further analyzed the mass spectra data through multiple ROI analyses of detectable BAs (Figure 5). Several serial sections (each measuring 10 μm) of sampled colon tissues were used to evaluate the reproducibility of iMScope technique. The relative abundance of BAs by their values of p were compared between randomly selected ROIs in submucosa, muscular layer, and adventitia (ROI-1) and mucosa layer (ROI-2) of the sectioned colon tissues. The p in this context is defined as the statistical analyses of average intensities/signals acquired from areas specified by ROI. A small p (p < 0.05) denotes a low likelihood that average intensity values are equal between ROIs. For colonic tissue sections, the relative abundances of TCA/TMCA, TCDCA/TUDCA/TDCA, GCA, and CDCA/UDCA/DCA in the ROI-1 were significantly different from ROI-2 (Figure 5A). The box plot displays the intensity distribution of each measurement point for the applicable m/z value. ROI-1 is displayed on the left and ROI-2 on the right. The upper and lower blue lines are displayed at the position of 25% (25th if there are 100 measurement points) and 75%, respectively. The red line in the center of the “box” is the median. The notches of the “box” represent the difference of the median. The notches indicate the 95% confidence interval (CI) of the median (the median range of the population estimated from the median of samples with a probability of 95%). If the 95% CI of median (notch length) does not overlap between two groups (ROI-1 and ROI-2), the two groups are considered different in the median with a probability of 95% (p < 0.05). However, overlapping does not always indicate that there is no significant difference between the two groups. In other words, if the notches of ROI-1 and ROI-2 are not at the same height, this means that the intensity of ROI-1 and that of ROI-2 have medians that differ by a significance level of 5%. The “whiskers” extending upward and downward from the “box” indicate the range of 1.5× the length of the box. Intensity values beyond the length of the “whiskers” are displayed with a red+ as outliers with significant deviance from the median. If the median is not in the center of the “box,” this means that the intensity distribution is skewed. When checking the test results, the statistical values of the samples (spectrum peak intensity at measurement points) visualized with box plots can be referred to values of p. Figure 5B is the spectrum of each ROI which is set in Figure 5A. The spectra are vertically inverted, with that of ROI-1 above in red and that of ROI-2 below in blue. The abundances of TCA/TMCA and TCDCA/TUDCA/TDCA were higher in the ROI-1, while the relative abundances of GCA and CDCA/UDCA/DCA in the ROI-2 were higher than ROI-1. In addition, there were no significant differences in the distribution of TLCA and LCA in ROI-1 and ROI-2. A significant advantage of the iMScope we used was that we further analyzed the mass spectra data through multiple ROI analyses of detectable BAs. We have identified that the relative abundances of TCA/TMCA, TCDCA/TUDCA/TDCA, GCA, and CDCA/UDCA/DCA in the ROI of the colonic mucosal layer were significantly different from submucosa, muscular layer, and adventitia. Overall, one important future direction of ROI analyses and high spatial resolution is to connect the spatial distribution and signal intensity of BAs with tissue damage in a disease state, so as to provide guidance for us to study the pathogenesis and treatment of gastrointestinal diseases.

Our previous work, and the work of others, showed that only relevant BA changes but not spatial information were obtained since the whole tissue is extracted in one homogenate when traditional MS spectrometry techniques [such as high-performance liquid chromatography (HPLC)-MS/MS] were adopted (2–4, 35–38). However, huge increasing demand on the spatial distribution and content of BAs has become an essential part of metabolic-associated diseases and cancer research (6–10, 32, 33, 39). MALDI-MS can not only analyze and visualize selected molecules but also maintain their spatial distribution and the integrity of the sample, which is an advantage that other MS technologies do not have (40, 41). Genangeli et al. investigated the effect of soyasaponin on the distribution of BAs in duodenum and colon by MALDI-MSI (41). In other study, Kampa et al. (25) investigated the spatial abundances of Amitriptyline, lipids, and BAs in Amitriptyline-treated male rat liver tissue by using MALDI-MSI, and they found two BA signals m/z 498.289 (TCDCA/TDCA) and m/z 514.284 (TCA/TMCA) showed slight downregulation due to Amitriptyline treatment without comment upon the role of BAs homeostasis in the observed toxicological response. Specifically, the fold change of Bas is based on the average peak intensity of the particular monoisotopic peak within the analyzed region (25). Furthermore, MSI, which combines detailed molecular characterization with spatial distribution measurements, has been applied for determining spatial distribution of BAs in mouse liver biliary networks and also has a certain application in the spatial distribution of BAs in the intestine (11, 25, 41). Although there are important discoveries revealed by these studies, there are also limitations. First, less high-resolution spatial location information; second, without quantitative research combined with spatial location information. Thus, further exploration of BAs on quantitative and interaction with histopathological damage should be examined. iMScope is a powerful tool for MSI, which combines detailed molecular characterization with spatial distribution measurement. Especially the combination of optical microscope and mass spectrometer. The microscope area can be enlarged from 1.25 to 40 times with a CCD camera. In the mass spectrum, the combination of the ion trap and TOF enables it to have both the MS capability of the ion trap and the high-precision mass measurement capability based on TOF, which can achieve excellent sensitivity, good repeatability, and stable mass accuracy (42). Therefore, iMScope is ideal to accurately determine the ROI of the liver and intestines. In addition, the MALDI-MSI technique does not require a priori information about the chemical substances present in the tissue section, thus allowing the distribution of many different substances to be mapped in a single experiment (43). Our method, in which 9AA is used as matrix and NPA as IS, can simultaneously measure eleven different BAs by the MALDI-MSI technique. Meanwhile, iMScope can also provide information about the morphology and tissue heterogeneity of specific regions (44, 45). It can increase the applicability of the experimental method.

In summary, in addition to MS ion images that can identify the spatial distribution of BAs with low spatial resolution, the iMScope can also display high spatial resolution MS ion images of the same tissue section. Using a short-term HFD feeding mice model, we fully verified the feasibility and sensitivity of our current method for analyzing the spatial distribution and content of BAs in tissues. Our study further demonstrates even greater potency for a more clear understanding of the mechanism and treatment of gastrointestinal diseases by given spatial distribution and content information of BAs within the enterohepatic circulation.