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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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(a-c) Tryptophan fluorescence image (a), second harmonic generation image (b) and merged image (c) (red: tryptophan, blue: SHG. scale bar 20 µm). (d-f) Blood vessels at a depth of 50 µm visualized by tryptophan fluorescence image (d), FITC fluorescence image, (e), and merged image in (f) (red: tryptophan, green: FITC. scale bar 20 µm).
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g003: (a-c) Tryptophan fluorescence image (a), second harmonic generation image (b) and merged image (c) (red: tryptophan, blue: SHG. scale bar 20 µm). (d-f) Blood vessels at a depth of 50 µm visualized by tryptophan fluorescence image (d), FITC fluorescence image, (e), and merged image in (f) (red: tryptophan, green: FITC. scale bar 20 µm).

Mentions: Below 30 µm deep we are imaging in the dermis. Features that are prominent in the dermis include hair follicles (round structure in Fig. 2 (d)), dermal cells (Fig. 2 (e)), and vascular structures characterized by low tryptophan signal with occasional bright cells moving inside (Fig. 2 (f)). Notably absent in these images are fibrous structures characteristic of collagen, the main protein of connective tissue that is abundant in the dermis. The amino acid content in collagen measured by the ion-exchange chromatographic method has shown no tryptophan in human bone and tendon collagen [22]. In order to further investigate if mouse skin collagen emits tryptophan fluorescence, we performed both MPM and second harmonic generation (SHG) microscopy on the same mouse skin area for comparison. Example tryptophan fluorescence and SHG images are shown in Fig. 3 (a)-(c)Fig. 3


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)

(a-c) Tryptophan fluorescence image (a), second harmonic generation image (b) and merged image (c) (red: tryptophan, blue: SHG. scale bar 20 µm). (d-f) Blood vessels at a depth of 50 µm visualized by tryptophan fluorescence image (d), FITC fluorescence image, (e), and merged image in (f) (red: tryptophan, green: FITC. scale bar 20 µm).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

g003: (a-c) Tryptophan fluorescence image (a), second harmonic generation image (b) and merged image (c) (red: tryptophan, blue: SHG. scale bar 20 µm). (d-f) Blood vessels at a depth of 50 µm visualized by tryptophan fluorescence image (d), FITC fluorescence image, (e), and merged image in (f) (red: tryptophan, green: FITC. scale bar 20 µm).
Mentions: Below 30 µm deep we are imaging in the dermis. Features that are prominent in the dermis include hair follicles (round structure in Fig. 2 (d)), dermal cells (Fig. 2 (e)), and vascular structures characterized by low tryptophan signal with occasional bright cells moving inside (Fig. 2 (f)). Notably absent in these images are fibrous structures characteristic of collagen, the main protein of connective tissue that is abundant in the dermis. The amino acid content in collagen measured by the ion-exchange chromatographic method has shown no tryptophan in human bone and tendon collagen [22]. In order to further investigate if mouse skin collagen emits tryptophan fluorescence, we performed both MPM and second harmonic generation (SHG) microscopy on the same mouse skin area for comparison. Example tryptophan fluorescence and SHG images are shown in Fig. 3 (a)-(c)Fig. 3

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