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Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence.

Li C, Pastila RK, Pitsillides C, Runnels JM, Puoris'haag M, Côté D, Lin CP - Opt Express (2010)

Bottom Line: We describe a new method for imaging leukocytes in vivo by exciting the endogenous protein fluorescence in the ultraviolet (UV) spectral region where tryptophan is the major fluorophore.Two-photon excitation near 590 nm allows noninvasive optical sectioning through the epidermal cell layers into the dermis of mouse skin, where leukocytes can be observed by video-rate microscopy to interact dynamically with the dermal vascular endothelium.Because the new method alleviates the need to introduce exogenous labels, it is potentially applicable for tracking leukocytes and monitoring inflammatory cellular reactions in humans.

View Article: PubMed Central - PubMed

Affiliation: Wellman Center for Photomedicine and Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. Li.Chunqiang@mgh.harvard.edu

ABSTRACT
We describe a new method for imaging leukocytes in vivo by exciting the endogenous protein fluorescence in the ultraviolet (UV) spectral region where tryptophan is the major fluorophore. Two-photon excitation near 590 nm allows noninvasive optical sectioning through the epidermal cell layers into the dermis of mouse skin, where leukocytes can be observed by video-rate microscopy to interact dynamically with the dermal vascular endothelium. Inflammation significantly enhances leukocyte rolling, adhesion, and tissue infiltration. After exiting the vasculature, leukocytes continue to move actively in tissue as observed by time-lapse microscopy, and are distinguishable from resident autofluorescent cells that are not motile. Because the new method alleviates the need to introduce exogenous labels, it is potentially applicable for tracking leukocytes and monitoring inflammatory cellular reactions in humans.

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Tryptophan fluorescence images of leukocytes. (a) Confocal reflectance (green) and tryptophan fluorescence (red) images of mouse blood smear, (b) Tryptophan fluorescence image of isolated granulocytes, and (c) agranulocytes. (scale bar 20 µm). (d) Two-photon excitation spectrum of tryptophan in leukocytes. (e) Two-photon excitation spectrum of tryptophan (solid line) in phosphate buffer solution (reproduced with permission from [17]). (f) Tryptophan fluorescence intensity change of a dermal cell under continuous illumination.
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g004: Tryptophan fluorescence images of leukocytes. (a) Confocal reflectance (green) and tryptophan fluorescence (red) images of mouse blood smear, (b) Tryptophan fluorescence image of isolated granulocytes, and (c) agranulocytes. (scale bar 20 µm). (d) Two-photon excitation spectrum of tryptophan in leukocytes. (e) Two-photon excitation spectrum of tryptophan (solid line) in phosphate buffer solution (reproduced with permission from [17]). (f) Tryptophan fluorescence intensity change of a dermal cell under continuous illumination.

Mentions: It has been reported that leukocytes contribute to tryptophan fluorescence with one-photon excitation and the spectroscopic signal variation in different subpopulations can be distinguished [15]. Since leukocytes make up about 1% of the blood cells in the peripheral circulation, our observation of vascular structures that are low in tryptophan signal with occasional bright cells moving inside is consistent with these bright cells being leukocytes while the majority of blood cells are nonfluorescent erythrocytes (red blood cells). To verify this, we imaged a small drop of mouse blood on a glass slide. Figure 4 (a)Fig. 4


Imaging leukocyte trafficking in vivo with two-photon-excited endogenous tryptophan fluorescence.

Li C, Pastila RK, Pitsillides C, Runnels JM, Puoris'haag M, Côté D, Lin CP - Opt Express (2010)

Tryptophan fluorescence images of leukocytes. (a) Confocal reflectance (green) and tryptophan fluorescence (red) images of mouse blood smear, (b) Tryptophan fluorescence image of isolated granulocytes, and (c) agranulocytes. (scale bar 20 µm). (d) Two-photon excitation spectrum of tryptophan in leukocytes. (e) Two-photon excitation spectrum of tryptophan (solid line) in phosphate buffer solution (reproduced with permission from [17]). (f) Tryptophan fluorescence intensity change of a dermal cell under continuous illumination.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3369551&req=5

g004: Tryptophan fluorescence images of leukocytes. (a) Confocal reflectance (green) and tryptophan fluorescence (red) images of mouse blood smear, (b) Tryptophan fluorescence image of isolated granulocytes, and (c) agranulocytes. (scale bar 20 µm). (d) Two-photon excitation spectrum of tryptophan in leukocytes. (e) Two-photon excitation spectrum of tryptophan (solid line) in phosphate buffer solution (reproduced with permission from [17]). (f) Tryptophan fluorescence intensity change of a dermal cell under continuous illumination.
Mentions: It has been reported that leukocytes contribute to tryptophan fluorescence with one-photon excitation and the spectroscopic signal variation in different subpopulations can be distinguished [15]. Since leukocytes make up about 1% of the blood cells in the peripheral circulation, our observation of vascular structures that are low in tryptophan signal with occasional bright cells moving inside is consistent with these bright cells being leukocytes while the majority of blood cells are nonfluorescent erythrocytes (red blood cells). To verify this, we imaged a small drop of mouse blood on a glass slide. Figure 4 (a)Fig. 4

Bottom Line: We describe a new method for imaging leukocytes in vivo by exciting the endogenous protein fluorescence in the ultraviolet (UV) spectral region where tryptophan is the major fluorophore.Two-photon excitation near 590 nm allows noninvasive optical sectioning through the epidermal cell layers into the dermis of mouse skin, where leukocytes can be observed by video-rate microscopy to interact dynamically with the dermal vascular endothelium.Because the new method alleviates the need to introduce exogenous labels, it is potentially applicable for tracking leukocytes and monitoring inflammatory cellular reactions in humans.

View Article: PubMed Central - PubMed

Affiliation: Wellman Center for Photomedicine and Center for Systems Biology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA. Li.Chunqiang@mgh.harvard.edu

ABSTRACT
We describe a new method for imaging leukocytes in vivo by exciting the endogenous protein fluorescence in the ultraviolet (UV) spectral region where tryptophan is the major fluorophore. Two-photon excitation near 590 nm allows noninvasive optical sectioning through the epidermal cell layers into the dermis of mouse skin, where leukocytes can be observed by video-rate microscopy to interact dynamically with the dermal vascular endothelium. Inflammation significantly enhances leukocyte rolling, adhesion, and tissue infiltration. After exiting the vasculature, leukocytes continue to move actively in tissue as observed by time-lapse microscopy, and are distinguishable from resident autofluorescent cells that are not motile. Because the new method alleviates the need to introduce exogenous labels, it is potentially applicable for tracking leukocytes and monitoring inflammatory cellular reactions in humans.

Show MeSH
Related in: MedlinePlus