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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

Signals in the SHG channel for gels of collagen I to V.(Fig. 2a) SHG intensity image of collagen I to V (left to right). (Fig. 2b) SHG intensity images overlapped with the color mask chosen in the phasor plots (Fig. 2c). Red cursor was used to select the phasor points of zero lifetime (SHG) and the green cursor was used to select the fluorescence phasor points (non-zero lifetime). It is evident that the signal in the SHG channel for the collagen III (Fig. 2a) can be identified with fluorescence since the position in the phasor plot is not at the (1,0) position.
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f2: Signals in the SHG channel for gels of collagen I to V.(Fig. 2a) SHG intensity image of collagen I to V (left to right). (Fig. 2b) SHG intensity images overlapped with the color mask chosen in the phasor plots (Fig. 2c). Red cursor was used to select the phasor points of zero lifetime (SHG) and the green cursor was used to select the fluorescence phasor points (non-zero lifetime). It is evident that the signal in the SHG channel for the collagen III (Fig. 2a) can be identified with fluorescence since the position in the phasor plot is not at the (1,0) position.

Mentions: The other important observation is that the relative intensity of SHG and fluorescence are dependent on the type of collagens. Collagen I and II give very strong second harmonic signals. Collagen IV and V do not produce SHG. Collagen III has a weak contribution in the SHG channel, but this signal in our instrument is due to leakage of the very strong fluorescence signal through the filters used for the separation of SHG signals, as collagen III is responsible for the strongest fluorescence amongst the collagens under study. Figure 2a shows the SHG intensity images acquired in the same field of view as that of the fluorescence images of Fig. 1a. The phasor points originating from SHG images appear at the coordinate of s = 0 and g = 1, as the lifetime of SHG is basically zero. Figure 2c shows the phasor plots arising from each of the five collagen SHG images. The phasor plot for collagen III has a non-zero lifetime and is similar to the fluorescence lifetime of collagen III, thus signifying that the origin of the signal for collagen III is not SHG and is actually fluorescence. After selection of the different populations of the phasor plot (Fig. 2c) using colored cursors, the image for collagen I and II the images were masked with red (SHG mask) and the collagen III image was masked with green, the mask for non-zero lifetime. On the contrary, collagens IV and V do not produce any SHG signal. Collagen IV has a non-fibrous structure and hence does not produce second harmonic signals. Collagen V is known to be fibrillar, but only in the presence of collagen I. In a gel formed from the mixture of collagen I and V, increasing fraction of collagen V results in decrease of fibrillar structure and also a decrease in the fibril diameter. An increase of 20% of collagen V in the mixture of collagen I and V decreases the fibril structures by 40%. Thus a gel formed by only collagen V does not produce fibrillar structure and SHG signals21.


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)

Signals in the SHG channel for gels of collagen I to V.(Fig. 2a) SHG intensity image of collagen I to V (left to right). (Fig. 2b) SHG intensity images overlapped with the color mask chosen in the phasor plots (Fig. 2c). Red cursor was used to select the phasor points of zero lifetime (SHG) and the green cursor was used to select the fluorescence phasor points (non-zero lifetime). It is evident that the signal in the SHG channel for the collagen III (Fig. 2a) can be identified with fluorescence since the position in the phasor plot is not at the (1,0) position.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Signals in the SHG channel for gels of collagen I to V.(Fig. 2a) SHG intensity image of collagen I to V (left to right). (Fig. 2b) SHG intensity images overlapped with the color mask chosen in the phasor plots (Fig. 2c). Red cursor was used to select the phasor points of zero lifetime (SHG) and the green cursor was used to select the fluorescence phasor points (non-zero lifetime). It is evident that the signal in the SHG channel for the collagen III (Fig. 2a) can be identified with fluorescence since the position in the phasor plot is not at the (1,0) position.
Mentions: The other important observation is that the relative intensity of SHG and fluorescence are dependent on the type of collagens. Collagen I and II give very strong second harmonic signals. Collagen IV and V do not produce SHG. Collagen III has a weak contribution in the SHG channel, but this signal in our instrument is due to leakage of the very strong fluorescence signal through the filters used for the separation of SHG signals, as collagen III is responsible for the strongest fluorescence amongst the collagens under study. Figure 2a shows the SHG intensity images acquired in the same field of view as that of the fluorescence images of Fig. 1a. The phasor points originating from SHG images appear at the coordinate of s = 0 and g = 1, as the lifetime of SHG is basically zero. Figure 2c shows the phasor plots arising from each of the five collagen SHG images. The phasor plot for collagen III has a non-zero lifetime and is similar to the fluorescence lifetime of collagen III, thus signifying that the origin of the signal for collagen III is not SHG and is actually fluorescence. After selection of the different populations of the phasor plot (Fig. 2c) using colored cursors, the image for collagen I and II the images were masked with red (SHG mask) and the collagen III image was masked with green, the mask for non-zero lifetime. On the contrary, collagens IV and V do not produce any SHG signal. Collagen IV has a non-fibrous structure and hence does not produce second harmonic signals. Collagen V is known to be fibrillar, but only in the presence of collagen I. In a gel formed from the mixture of collagen I and V, increasing fraction of collagen V results in decrease of fibrillar structure and also a decrease in the fibril diameter. An increase of 20% of collagen V in the mixture of collagen I and V decreases the fibril structures by 40%. Thus a gel formed by only collagen V does not produce fibrillar structure and SHG signals21.

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