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Imaging Fibrosis and Separating Collagens using Second Harmonic Generation and Phasor Approach to Fluorescence Lifetime Imaging.

Ranjit S, Dvornikov A, Stakic M, Hong SH, Levi M, Evans RM, Gratton E - Sci Rep (2015)

Bottom Line: In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur.FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy.Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California Irvine, California.

ABSTRACT
In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur. This is a label free approach towards fast automatable detection of fibrosis in tissue samples. FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy. Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated.

No MeSH data available.


Related in: MedlinePlus

Fluorescence and SHG signals of the gel prepared from the mixture of collagen I and III.The fluorescence image was selected either for the high intensity (Fig. 3ai) using the top histogram (Fig. 3ci) or for the low intensity (Fig. 3aii) using the bottom histogram in (Fig. 3ci). The fluorescence intensity images were masked using the cursor colors in the phasor plot (Fig. 3cii) and colored accordingly to show the prominently collagen III rich region (Fig. 3bi) and collagen I rich region (Fig. 3bii). The SHG intensity image, phasor masked image and the corresponding phasor plot for SHG generation is shown in Fig. 3aiii–3ciii, respectively.
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f3: Fluorescence and SHG signals of the gel prepared from the mixture of collagen I and III.The fluorescence image was selected either for the high intensity (Fig. 3ai) using the top histogram (Fig. 3ci) or for the low intensity (Fig. 3aii) using the bottom histogram in (Fig. 3ci). The fluorescence intensity images were masked using the cursor colors in the phasor plot (Fig. 3cii) and colored accordingly to show the prominently collagen III rich region (Fig. 3bi) and collagen I rich region (Fig. 3bii). The SHG intensity image, phasor masked image and the corresponding phasor plot for SHG generation is shown in Fig. 3aiii–3ciii, respectively.

Mentions: Collagen I and III are known to coexist in tissues. Both the ratio of the two collagens and the total amount of them has been shown to indicate the extent of different fibrotic diseases. As mentioned earlier, collagen I results in stiffness and tensile strength and large amount of collagen III results in greater elasticity. Thus a change in the relative ratios of these two collagens can determine the behavior of the extra cellular matrix and was shown to be an important factor in cardiac myopathy345. Therefore separating the signals of collagen I and III becomes important for diagnostic purposes. To distinguish if the phasor approach to FLIM can separate the collagen I and III signals, a mixed gel was formed from a 3:1 mixture of these two collagens and then SHG and FLIM images were acquired. Figure 3ai,ii show the fluorescence images selected for regions of high intensity and the low intensity, respectively. This selection was done based on the histogram in Fig. 3ci, where the top shows the selection for the Fig. 3ai and the bottom shows the selection for the Fig. 3aii. Collagen III is much more fluorescent than collagen I and thus to observe collagen I, the lower fluorescence intensity must be selected. Figure 3bii,bi show the masked image of the intensity overlapped with the phasor color in Fig. 3cii. In Fig. 3bi, most of the image is colored cyan and in Fig. 3bii most of the image is colored red, the cursor colors (Fig. 3ci) for the FLIM signature of collagen III and I, respectively. Figure 3aiii–ciii show the SHG intensity image, phasor masked image and the phasor plot of the second harmonic generation, respectively. The lifetime of SHG is zero and thus the phasor points appears at s = 0, g = 1 (Fig. 3ciii). A comparison between Fig. 3bii,biii shows that most of the fiber structures in the SHG image can also be separated by the red fluorescence mask in Fig. 3biii. Collagen I has a very strong SHG signal. In the mixture, the bleed through of collagen III fluorescence in the SHG channel has a much lower intensity than the SHG signal of collagen I and is actually very close to the background. Thus after background correction, the phasor points originating from the bleed through disappears from the phasor plot (Fig. 3ciii). It is also evident that the bright image in Fig. 3ai does not give rise to the signals in the SHG channels and only the dim fluorescent spots, i.e. the ones from collagen I coexist in both SHG and fluorescence images. This proves that at least in gels, collagen I and III can be separated based on lifetime.


Imaging Fibrosis and Separating Collagens using Second Harmonic Generation and Phasor Approach to Fluorescence Lifetime Imaging.

Ranjit S, Dvornikov A, Stakic M, Hong SH, Levi M, Evans RM, Gratton E - Sci Rep (2015)

Fluorescence and SHG signals of the gel prepared from the mixture of collagen I and III.The fluorescence image was selected either for the high intensity (Fig. 3ai) using the top histogram (Fig. 3ci) or for the low intensity (Fig. 3aii) using the bottom histogram in (Fig. 3ci). The fluorescence intensity images were masked using the cursor colors in the phasor plot (Fig. 3cii) and colored accordingly to show the prominently collagen III rich region (Fig. 3bi) and collagen I rich region (Fig. 3bii). The SHG intensity image, phasor masked image and the corresponding phasor plot for SHG generation is shown in Fig. 3aiii–3ciii, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543938&req=5

f3: Fluorescence and SHG signals of the gel prepared from the mixture of collagen I and III.The fluorescence image was selected either for the high intensity (Fig. 3ai) using the top histogram (Fig. 3ci) or for the low intensity (Fig. 3aii) using the bottom histogram in (Fig. 3ci). The fluorescence intensity images were masked using the cursor colors in the phasor plot (Fig. 3cii) and colored accordingly to show the prominently collagen III rich region (Fig. 3bi) and collagen I rich region (Fig. 3bii). The SHG intensity image, phasor masked image and the corresponding phasor plot for SHG generation is shown in Fig. 3aiii–3ciii, respectively.
Mentions: Collagen I and III are known to coexist in tissues. Both the ratio of the two collagens and the total amount of them has been shown to indicate the extent of different fibrotic diseases. As mentioned earlier, collagen I results in stiffness and tensile strength and large amount of collagen III results in greater elasticity. Thus a change in the relative ratios of these two collagens can determine the behavior of the extra cellular matrix and was shown to be an important factor in cardiac myopathy345. Therefore separating the signals of collagen I and III becomes important for diagnostic purposes. To distinguish if the phasor approach to FLIM can separate the collagen I and III signals, a mixed gel was formed from a 3:1 mixture of these two collagens and then SHG and FLIM images were acquired. Figure 3ai,ii show the fluorescence images selected for regions of high intensity and the low intensity, respectively. This selection was done based on the histogram in Fig. 3ci, where the top shows the selection for the Fig. 3ai and the bottom shows the selection for the Fig. 3aii. Collagen III is much more fluorescent than collagen I and thus to observe collagen I, the lower fluorescence intensity must be selected. Figure 3bii,bi show the masked image of the intensity overlapped with the phasor color in Fig. 3cii. In Fig. 3bi, most of the image is colored cyan and in Fig. 3bii most of the image is colored red, the cursor colors (Fig. 3ci) for the FLIM signature of collagen III and I, respectively. Figure 3aiii–ciii show the SHG intensity image, phasor masked image and the phasor plot of the second harmonic generation, respectively. The lifetime of SHG is zero and thus the phasor points appears at s = 0, g = 1 (Fig. 3ciii). A comparison between Fig. 3bii,biii shows that most of the fiber structures in the SHG image can also be separated by the red fluorescence mask in Fig. 3biii. Collagen I has a very strong SHG signal. In the mixture, the bleed through of collagen III fluorescence in the SHG channel has a much lower intensity than the SHG signal of collagen I and is actually very close to the background. Thus after background correction, the phasor points originating from the bleed through disappears from the phasor plot (Fig. 3ciii). It is also evident that the bright image in Fig. 3ai does not give rise to the signals in the SHG channels and only the dim fluorescent spots, i.e. the ones from collagen I coexist in both SHG and fluorescence images. This proves that at least in gels, collagen I and III can be separated based on lifetime.

Bottom Line: In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur.FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy.Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, University of California Irvine, California.

ABSTRACT
In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur. This is a label free approach towards fast automatable detection of fibrosis in tissue samples. FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy. Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated.

No MeSH data available.


Related in: MedlinePlus