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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 collagen III in mouse femur bones.Picture of the bone, two photon auto-fluorescence intensity image and the SHG images are shown in Fig. 4a–c, respectively. The FLIM and SHG phasor masked images are shown in Fig. 4d,e where the masking color indicates the cursor color of the phasor plot (Fig. 4f). Most of the parts chosen by cyan (Fig. 4d) are not present in Fig. 4e and mostly the red part of the masked image correlates with the SHG image.
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f4: Separation of collagen I and collagen III in mouse femur bones.Picture of the bone, two photon auto-fluorescence intensity image and the SHG images are shown in Fig. 4a–c, respectively. The FLIM and SHG phasor masked images are shown in Fig. 4d,e where the masking color indicates the cursor color of the phasor plot (Fig. 4f). Most of the parts chosen by cyan (Fig. 4d) are not present in Fig. 4e and mostly the red part of the masked image correlates with the SHG image.

Mentions: A mouse model that spontaneously develops myelofibrosis is the SMRTmRID mouse. In these mice, two receptor interaction domains of epigenetic repressor silencing mediator of retinoid and thyroid hormone receptors are targeted and these mice develops spontaneous myelofibrosis, characterized by the bone marrow fibrosis and increasing collagen content in the bone marrow32. The FLIM and SHG measurements were further extended to study these mouse femur slices, obtained from Dr. Ronald Evans’ lab at Salk Institute, San Diego, CA. Each individual image was taken with 925 μm field of view and with 256 × 256 pixels. Both FLIM and SHG images were then tiled and are shown in Fig. 4b,c, respectively. Figure 4a shows the image of the bone taken with a camera. Red, cyan and orange colored cursors were chosen in the phasor plot (Fig. 4f) to select collagen I fluorescence, collagen III fluorescence and the SHG signals, respectively. Fig. 4d,e represent the phasor masked image of the corresponding intensity images, Fig. 4b,c. A comparison between the masked FLIM (Fig. 4d) and masked SHG (Fig. 4e) shows that the part of the image covered in SHG is mostly covered by red in the fluorescence image, red being the cursor used for collagen I FLIM signature, signifying the inability of collagen III to produce SHG. This result shows that in the mouse femur tissue, collagen I and III can be separated by the FLIM. The strong correlation between the pixels measured by SHG and the red mask (the mask for collagen I) in the FLIM images shows that collagen I can be identified by both SHG and FLIM (Supplementary Fig. S2).


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 collagen III in mouse femur bones.Picture of the bone, two photon auto-fluorescence intensity image and the SHG images are shown in Fig. 4a–c, respectively. The FLIM and SHG phasor masked images are shown in Fig. 4d,e where the masking color indicates the cursor color of the phasor plot (Fig. 4f). Most of the parts chosen by cyan (Fig. 4d) are not present in Fig. 4e and mostly the red part of the masked image correlates with the SHG image.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: Separation of collagen I and collagen III in mouse femur bones.Picture of the bone, two photon auto-fluorescence intensity image and the SHG images are shown in Fig. 4a–c, respectively. The FLIM and SHG phasor masked images are shown in Fig. 4d,e where the masking color indicates the cursor color of the phasor plot (Fig. 4f). Most of the parts chosen by cyan (Fig. 4d) are not present in Fig. 4e and mostly the red part of the masked image correlates with the SHG image.
Mentions: A mouse model that spontaneously develops myelofibrosis is the SMRTmRID mouse. In these mice, two receptor interaction domains of epigenetic repressor silencing mediator of retinoid and thyroid hormone receptors are targeted and these mice develops spontaneous myelofibrosis, characterized by the bone marrow fibrosis and increasing collagen content in the bone marrow32. The FLIM and SHG measurements were further extended to study these mouse femur slices, obtained from Dr. Ronald Evans’ lab at Salk Institute, San Diego, CA. Each individual image was taken with 925 μm field of view and with 256 × 256 pixels. Both FLIM and SHG images were then tiled and are shown in Fig. 4b,c, respectively. Figure 4a shows the image of the bone taken with a camera. Red, cyan and orange colored cursors were chosen in the phasor plot (Fig. 4f) to select collagen I fluorescence, collagen III fluorescence and the SHG signals, respectively. Fig. 4d,e represent the phasor masked image of the corresponding intensity images, Fig. 4b,c. A comparison between the masked FLIM (Fig. 4d) and masked SHG (Fig. 4e) shows that the part of the image covered in SHG is mostly covered by red in the fluorescence image, red being the cursor used for collagen I FLIM signature, signifying the inability of collagen III to produce SHG. This result shows that in the mouse femur tissue, collagen I and III can be separated by the FLIM. The strong correlation between the pixels measured by SHG and the red mask (the mask for collagen I) in the FLIM images shows that collagen I can be identified by both SHG and FLIM (Supplementary Fig. S2).

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