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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 collagens based on the fluorescence lifetime using the clusters in the phasor plot.Figure 1a, from left to right, shows the fluorescence intensity signals originating from gels of collagen I to V. Figure 1b shows the same intensity image masked with the cursor color chosen in the phasor plot (Fig.1c). Red, green, cyan, yellow and magenta colors were chosen to select the phasor clusters in Fig. 1c and the intensity images were painted correspondingly. Figure 1d shows the calculated phase lifetimes and shows the separation in phasor plot (Fig. 1c) is more significant. The field of view in these images is 38 μm.
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f1: Separation of collagens based on the fluorescence lifetime using the clusters in the phasor plot.Figure 1a, from left to right, shows the fluorescence intensity signals originating from gels of collagen I to V. Figure 1b shows the same intensity image masked with the cursor color chosen in the phasor plot (Fig.1c). Red, green, cyan, yellow and magenta colors were chosen to select the phasor clusters in Fig. 1c and the intensity images were painted correspondingly. Figure 1d shows the calculated phase lifetimes and shows the separation in phasor plot (Fig. 1c) is more significant. The field of view in these images is 38 μm.

Mentions: The objective of this paper was to identify locations in the phasor plot that can be used to separate different collagen types in an image. Collagen gels consisting of purified collagen I through V were prepared and imaged with a 32 μs pixel dwell time for 20 repeat images. The images were acquired with a 38 μm field of view and a resolution of 256 × 256 pixels. Each individual fluorescence image was first corrected for the background. The position of the phasor points in the phasor plot originating from that particular image were selected with a colored cursor and the image was painted accordingly. Figure 1a shows the intensity images after background correction (from left to right are collagen I through V). The corresponding FLIM images (Fig. 1b) were colored according to the chosen cursors in Fig. 1c. In the phasor plot (Fig. 1c) each type of collagen forms a different cluster of phasor points. Each cluster is indicated by a circular colored cursor. The points in the images are colored according to the cluster they belong. The red, green, cyan, yellow and magenta represent the selected phasor points for collagen I, II, III, IV and V, respectively. Figure 1b, showing the intensity image painted with the chosen cursor colors, proves that the clusters in the phasor plot are completely separate and can be used to identify the type of collagen.


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 collagens based on the fluorescence lifetime using the clusters in the phasor plot.Figure 1a, from left to right, shows the fluorescence intensity signals originating from gels of collagen I to V. Figure 1b shows the same intensity image masked with the cursor color chosen in the phasor plot (Fig.1c). Red, green, cyan, yellow and magenta colors were chosen to select the phasor clusters in Fig. 1c and the intensity images were painted correspondingly. Figure 1d shows the calculated phase lifetimes and shows the separation in phasor plot (Fig. 1c) is more significant. The field of view in these images is 38 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Separation of collagens based on the fluorescence lifetime using the clusters in the phasor plot.Figure 1a, from left to right, shows the fluorescence intensity signals originating from gels of collagen I to V. Figure 1b shows the same intensity image masked with the cursor color chosen in the phasor plot (Fig.1c). Red, green, cyan, yellow and magenta colors were chosen to select the phasor clusters in Fig. 1c and the intensity images were painted correspondingly. Figure 1d shows the calculated phase lifetimes and shows the separation in phasor plot (Fig. 1c) is more significant. The field of view in these images is 38 μm.
Mentions: The objective of this paper was to identify locations in the phasor plot that can be used to separate different collagen types in an image. Collagen gels consisting of purified collagen I through V were prepared and imaged with a 32 μs pixel dwell time for 20 repeat images. The images were acquired with a 38 μm field of view and a resolution of 256 × 256 pixels. Each individual fluorescence image was first corrected for the background. The position of the phasor points in the phasor plot originating from that particular image were selected with a colored cursor and the image was painted accordingly. Figure 1a shows the intensity images after background correction (from left to right are collagen I through V). The corresponding FLIM images (Fig. 1b) were colored according to the chosen cursors in Fig. 1c. In the phasor plot (Fig. 1c) each type of collagen forms a different cluster of phasor points. Each cluster is indicated by a circular colored cursor. The points in the images are colored according to the cluster they belong. The red, green, cyan, yellow and magenta represent the selected phasor points for collagen I, II, III, IV and V, respectively. Figure 1b, showing the intensity image painted with the chosen cursor colors, proves that the clusters in the phasor plot are completely separate and can be used to identify the type of collagen.

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