Home Liver Diseases Molecular imaging analysis of microvesicular and macrovesicular lipid droplets in non-alcoholic fatty liver disease by Raman microscopy

Molecular imaging analysis of microvesicular and macrovesicular lipid droplets in non-alcoholic fatty liver disease by Raman microscopy

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Molecular imaging analysis of microvesicular and macrovesicular lipid droplets in non-alcoholic fatty liver disease by Raman microscopy

Raman spectra of liver tissues of NASH model mice

Typical Raman spectra of liver tissues of NASH model mice were obtained to reveal the spectral features for Raman spectral analysis. We focused on macrovesicular LDs accumulated near a central vein, as shown in region A in Fig. 1a. The previous studies had shown that the crystallinity of LDs is an important indicator of NAFLD prognosis19. By using the conventional polarization imaging with cross-Nicol configuration, the crystallinity of lipids in LDs can be identified as shown in Fig. 1b,c. We can observe a Maltese cross appearance owing to the polarization modulation by birefringence of a crystalline form of lipids, as indicated by the yellow arrowheads in Fig. 1c. If the Maltese cross is not apparent as indicated by the blue arrowhead in Fig. 1c, the LDs might predominantly contain an amorphous form of lipids. Thus, we investigate the spectral features of the Raman spectra of accumulated LDs with crystalline and amorphous forms of lipids in addition to hepatocytes with non-apparent LDs.

Figure 1

Typical histology of the liver tissue of a NASH model mouse. (a) HE-stained image of the NASH liver around a central vein (CV). (b) White light image of macrovesicular LDs of the NASH liver without any fixation nor staining. (c) Polarization image of the macrovesicular LDs of the NASH liver with cross-Nicol configuration. Blue arrowheads indicate LDs with an amorphous form of lipids. Yellow arrowheads indicate LDs with a crystalline form of lipids. Scale bars of (a), (b), and (c) indicate 200 µm, 10 µm, and 10 µm, respectively.

In the crystalline lipid-rich LDs, main Raman bands were found at 699, 1265, 1302, 1442, 1662, 1677, 1735, 2855, 2875, 2900, 2935, and 2964 cm−1 as shown in Fig. 2 and Table 1. The Raman bands at 1265, 1302, 1442, 2855, and 2900 cm−1 were assigned to CH2-related molecular bonds, indicating the presence of lipid contents in this region. The Raman band at 1662 cm−1 was assigned to C=C double bond stretching mode at the acyl chain, which is an indicator of the unsaturation of the acyl chain of lipids. The Raman bands at 699 and 1677 cm−1 respectively assigned to steroid ring vibration mode and C=C double bond stretching mode at carbon ring, indicating the presence of cholesterol content. As a result, the Raman spectra at the crystalline lipid-rich LDs reflected the molecular features of the LDs, including the cholesterols. The presence of cholesterol in the crystalline lipid-rich LDs was well agreed with the results of the fluorescence imaging with a cholesterol-specific dye in the previous studies19.

Figure 2
figure2

Typical Raman spectra of LDs with crystalline and amorphous forms of lipids and hepatocyte of the liver tissue of a NASH model mouse. The intensity was normalized by the highest intensities of the Raman spectra. LD, lipid droplet.

Table 1 Assignments of the Raman bands of LDs with crystalline and amorphous forms of lipids and hepatocyte of the liver tissue of a NASH model mouse.

In the amorphous lipid-rich LDs, main Raman bands were found at 1265, 1302, 1442, 1662, 1750, 2855, 2875, 2900, 2935, and 2964 cm−1 as shown in Fig. 2 and Table 1. The main Raman bands were similar to that at the crystalline lipid-rich LDs, but the difference was found at 699 and 1677 cm−1, indicating relatively low cholesterol content at the LDs in terms of Raman spectral analysis. This result is also agreed with the result of the fluorescence imaging in the previous studies19. Importantly, Raman microscopy additionally has the potential to quantitatively evaluate the relative content of molecular species and structures without staining by analysing the relative intensity of each Raman band.

In contrast, in the hepatocytes with non-apparent LDs, main Raman bands were found at 746, 1001, 1309, 1448, 1592, 1660, 2855, 2875, 2900, 2935 and 2964 cm−1 as shown in Fig. 2 and Table 1. The Raman spectrum of the hepatocytes was different from that of these two types of LDs. Especially, heme-related Raman bands at 746, 1309, and 1592 cm−1 were observed in the hepatocytes, which might be derived by cytochromes present in mitochondria in hepatocytes or deposited hemoglobin. As a result, the Raman bands of LDs can be confirmed against that of hepatocytes, indicating the Raman spectral analysis of LDs can be performed in the liver tissues of the NASH model mice.

Raman spectral imaging of macrovesicular LDs accumulated in hepatocytes

We also investigated the molecular contents of lipids in terms of the size of LDs. The previous studies had shown that the size of LDs is closely related to the prognosis of NAFLD20. Firstly, we investigated the spatial distribution of molecular contents of lipids in macrovesicular LDs accumulated in hepatocyte of liver tissues of NASH model mice. We obtained two-dimensional Raman images of macrovesicular LDs in region A in Fig. 1a. In this region, macrovesicular LDs with about 10–40 µm in diameter were diffusely distributed, as shown in Fig. 3a. The typical Raman spectra of LDs and hepatocytes in this region were shown in Fig. 3b. As similar results of Fig. 2, the Raman spectrum of LDs was predominantly composed of lipid-related Raman bands, such as 1442, 1662, 1677, and 2855 cm−1, as shown in Fig. 3b. Furthermore, cholesterol-related Raman bands such as 1677 cm−1 were also observed. Raman images of each typical Raman bands were shown in Fig. 3c. The lipid-related Raman bands, such as 1442, 1662, 1677, and 2855 cm−1, clearly reflect the distribution of LDs, which are well agreed with the histology of the liver tissues of the NASH model mice confirmed by the HE-stain image. As the imaging analysis, each LD seems to be separated from each other. In contrast, the Raman bands at 1592 and 2935 cm−1 visualised the distribution of hepatocytes or heme proteins.

Figure 3
figure3

Raman spectral imaging of macrovesicular LDs accumulated in hepatocyte of liver tissues of NASH model mice. (a) HE-stained image of macrovesicular LDs indicated by region A in Fig. 1a. Raman imaging was performed on an unstained tissue section corresponding to the same position as the HE-stained image. (b) Typical Raman spectra of LDs and hepatocytes in this region. (c) Raman images of typical Raman bands. (d) Intensity ratio imaging of Raman bands of 1662 and 2855 cm−1, indicating the unsaturation degree of LDs. (e) Intensity ratio imaging of Raman bands of 1677 and 2855 cm−1, indicating the relative content of cholesterol. These intensity ratio images were obtained at the region with enough high intensity at the Raman band of 2855 cm−1, while the other region was masked. Scale bars of (a), (c), (d), and (e) indicate 50 µm.

To analyse the more specific molecular species and structures of LDs, we performed intensity ratio imaging by using lipid-related Raman bands. The intensity ratio calculation was performed at each pixel to obtain relative contributions of lipid species and structures with the normalization of the amount of lipids. Firstly, we obtained an intensity ratio of 1662 cm−1 against 2855 cm−1, as shown in Fig. 3d, which indicates the unsaturation degrees of lipids. The macrovesicular LDs were exhibited inhomogeneous distribution of the intensity ratio of 1662 cm−1 against 2855 cm−1. Notably, some particulate structures of the intensity ratio were found in the internal structure of LDs, as typically indicated by the arrowheads in Fig. 3d. These results might indicate the inhomogeneous distribution of lipids according to unsaturation degrees of lipids, even in the macrovesicular LDs with morphologically uniform distribution.

We also obtained the intensity ratio of 1677 cm−1 against 2855 cm−1, as shown in Fig. 3e, which indicates the relative content of cholesterol. We found that the inhomogeneous distribution of the intensity ratio of 1677 cm−1 against 2855 cm−1 among LDs. In addition, some of the LDs exhibited a high-intensity ratio partially on the outer wall of the LDs. These results might indicate that the relative content of cholesterol was varied depending on the macrovesicular LDs, and the cholesterol distribution was unevenly distributed even in a single macrovesicular LD.

Raman spectral imaging of microvesicular LDs accumulated in hepatocytes

We also investigated the spatial distribution of molecular contents of lipids in microvesicular LDs accumulated in hepatocyte of liver tissues of NASH model mice. We obtained two-dimensional Raman images of macrovesicular LDs at the dashed squared region of Fig. 4a that are in region B in Fig. 1a. In this region, microvesicular LDs with about a few micrometers or less in diameter were diffusely distributed, as shown in Fig. 4a. The typical Raman spectra of LDs and hepatocytes in this region were shown in Fig. 4b. The Raman spectrum of LDs was exhibited similar to the macrovesicular LDs, such as 1442, 1662, 1677, and 2855 cm−1, as shown in Fig. 3b. However, the relative contribution of 1677 cm−1 was much smaller than that of macrovesicular LDs. Raman images of each typical Raman bands were shown in Fig. 4c. The lipid-related Raman bands, such as 1442, 1662, 1677, and 2855 cm−1, reflect the distribution of LDs, but interestingly, we found the connecting structure between LDs, which was not apparent in the HE-stained image. In contrast, the Raman bands at 1592 and 2935 cm−1 visualised the distribution of hepatocytes or heme proteins as similar to the result around the macrovesicular LDs.

Figure 4
figure4

Raman spectral imaging of microvesicular LDs accumulated in hepatocyte of liver tissues of NASH model mice. (a) HE-stained image of microvesicular LDs indicated by region B in Fig. 1a. Raman imaging was performed on an unstained tissue section corresponding to the dashed squared region of the HE-stained image. (b) Typical Raman spectra of LDs and hepatocytes in this region. (c) Raman images of typical Raman bands. (d) Intensity ratio imaging of Raman bands of 1662 and 2855 cm−1, indicating the unsaturation degree of LDs. (e) Intensity ratio imaging of Raman bands of 1677 and 2855 cm−1, indicating the relative content of cholesterol. These intensity ratio images were obtained at the region with enough high intensity at the Raman band of 2855 cm−1, while the other region was masked. Scale bars of (a), (c), (d), and (e) indicate 50 µm, 10 µm, 10 µm, and 10 µm, respectively.

Intensity ratio imaging was also performed by using lipid-related Raman bands. We obtained the intensity ratio of 1662 cm−1 against 2855 cm−1, as shown in Fig. 4d, which indicates the unsaturation degrees of lipids. The microvesicular LDs have exhibited an almost homogeneous distribution of the intensity ratio of 1662 cm−1 against 2855 cm−1. We also obtained the intensity ratio of 1677 cm−1 against 2855 cm−1, as shown in Fig. 4e, which indicates the relative content of cholesterol. The distribution of the intensity ratio of 1677 cm−1 against 2855 cm−1 was also homogeneous. These results might indicate the unsaturation degree of microvesicular LDs, and the relative content of cholesterol could distribute homogeneously in microvesicular LDs.

Statistical comparison of molecular species and structural features of LDs between microvesicular and macrovesicular LDs

Finally, we investigated the difference of molecular species and structural features of LDs between microvesicular and macrovesicular LDs. We evaluated the intensity ratio of 1662 cm−1 against 2855 cm−1 and the that of 1677 cm−1 against 2855 cm−1 of the central region of the 37 LDs in macrovesicular LDs and 21 LDs in microvesicular LDs. The intensity ratio analyses of these Raman bands were shown in Fig. 5. The mean intensity ratio of 1662 cm−1 against 2855 cm−1 of the microvesicular LDs (0.438 ± 0.038) was significantly higher than that of the macrovesicular LDs (0.425 ± 0.034). This result shows the microvesicular LDs have a higher unsaturation degree of lipids than the macrovesicular LDs. This tendency was also observed by the other indicator of unsaturation degree17 (intensity ratio of 1265 against 1442 cm−1 and 3012 against 2855 cm−1), as shown in Supplementary Fig. 3. The mean intensity ratio of 1677 cm−1 against 2855 cm−1 of the microvesicular LDs (0.308 ± 0.061) was also significantly higher than that of the macrovesicular LDs (0.218 ± 0.104). This result shows the microvesicular LDs have a higher cholesterol content on average. However, the highest intensity ratio of 1677 cm−1 against 2855 cm−1 was found in the macrovesicular LDs, showing the partial aggregation of a higher content of cholesterol.

Figure 5
figure5

Box plot analysis of intensity ratio of Raman bands indicating the unsaturation degree of lipids (1662 cm−1/2855 cm−1) and the relative content of cholesterol (1677 cm−1/2855 cm−1) of microvesicular LDs (Micro LDs) and macrovesicular LDs (Macro LDs). A total of 1270 points for each of 21 microvesicular LDs and 37 macrovesicular LDs were evaluated. The individual data are shown by dots. *, significant difference (p < 0.01).

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