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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

Separation of collagen I and III in normal and fibrotic bone marrows.(Fig. 5a) and (Fig. 5b) show the phasor masked FLIM images of the non-fibrotic wild type mice and fibrotic SMRTmRID mice, respectively. The more violet color in the Fig. 5b is representative of higher contribution from collagen III. Figure 5c shows the phasor plot and the continuous cursor used for the analysis.
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f5: Separation of collagen I and III in normal and fibrotic bone marrows.(Fig. 5a) and (Fig. 5b) show the phasor masked FLIM images of the non-fibrotic wild type mice and fibrotic SMRTmRID mice, respectively. The more violet color in the Fig. 5b is representative of higher contribution from collagen III. Figure 5c shows the phasor plot and the continuous cursor used for the analysis.

Mentions: The femur slices from two different mice; one wild type control mouse and one from SMRTmRID mouse were imaged using the phasor approach to the fluorescent lifetime imaging. The different areas of these two samples imaged using the FLIM technique are shown in Supplementary Figure S3. These images were then analyzed using the continuous cursor analysis in the phasor plot. As mentioned in materials and methods section, one of the key unique features of the phasor approach is that, in this approach a continuous color scheme can be used to show differential contribution of two separate species in any individual pixel. Figure 5c shows the continuous color scheme used to show the differential contribution of collagen I and collagen III in these images. The more red color is representative of the more collagen I rich areas and the more violet color is representative of more collagen III rich areas. A comparison between the phasor masked images of the wild type mice in Fig. 5a and the SMRTmRID mouse femurs slices in Fig. 5b shows that while the periphery in both cases is made of mostly collagen I, the bone marrow of the SMRTmRID mouse is more violet in color and hence have more contribution from collagen III. This is similar to the results of staining shown before32. Thus Fig. 5 demonstrates that phasor approach to FLIM can indeed be used image fibrosis in tissues.


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)

Separation of collagen I and III in normal and fibrotic bone marrows.(Fig. 5a) and (Fig. 5b) show the phasor masked FLIM images of the non-fibrotic wild type mice and fibrotic SMRTmRID mice, respectively. The more violet color in the Fig. 5b is representative of higher contribution from collagen III. Figure 5c shows the phasor plot and the continuous cursor used for the analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Separation of collagen I and III in normal and fibrotic bone marrows.(Fig. 5a) and (Fig. 5b) show the phasor masked FLIM images of the non-fibrotic wild type mice and fibrotic SMRTmRID mice, respectively. The more violet color in the Fig. 5b is representative of higher contribution from collagen III. Figure 5c shows the phasor plot and the continuous cursor used for the analysis.
Mentions: The femur slices from two different mice; one wild type control mouse and one from SMRTmRID mouse were imaged using the phasor approach to the fluorescent lifetime imaging. The different areas of these two samples imaged using the FLIM technique are shown in Supplementary Figure S3. These images were then analyzed using the continuous cursor analysis in the phasor plot. As mentioned in materials and methods section, one of the key unique features of the phasor approach is that, in this approach a continuous color scheme can be used to show differential contribution of two separate species in any individual pixel. Figure 5c shows the continuous color scheme used to show the differential contribution of collagen I and collagen III in these images. The more red color is representative of the more collagen I rich areas and the more violet color is representative of more collagen III rich areas. A comparison between the phasor masked images of the wild type mice in Fig. 5a and the SMRTmRID mouse femurs slices in Fig. 5b shows that while the periphery in both cases is made of mostly collagen I, the bone marrow of the SMRTmRID mouse is more violet in color and hence have more contribution from collagen III. This is similar to the results of staining shown before32. Thus Fig. 5 demonstrates that phasor approach to FLIM can indeed be used image fibrosis in tissues.

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