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Research Updates

Yoshiki Miyachi, MD, PhD

Live Imaging of the Skin by Two-photon Microscopy

Yoshiki Miyachi

Thursday, January 16, 2014

Introduction

Historically, dermatologists dreamt of observing the fine structure of living skin, because conventional methods were based on histological analysis that allowed us to evaluate only a two-dimensional slice of a certain time point without time or space components. Furthermore, the morphology of skin components may be distorted as a result of the multiple fixation and staining steps. Now, dreams have come true using a novel imaging tool, two-photon microscopy (TPM), with which real-time imaging studies of both anatomic structures and cutaneous reactions are possible. In this article, the fine structures of living skin components, as well as some inflammatory reactions, will be shown as movies.

 

What is two-photon microscopy?

In TPM, fluorescent materials are excited by "two-photon" laser. As the wavelength is longer than conventional "one-photon" laser microscopy, this technique allows the imaging of living tissues up to a very high depth (about 0.5 mm) with low phototoxicity. The mechanics of the workings of this microscope may be beyond the scope of this article!

Image 1

Figure 1. Two-photon microscope

 Image 2

Figure 2. Real-time imaging of living mouse skin under anesthesia

Three-dimensional imaging of keratinocytes using TPM1

We have applied TPM to the ex vivo observation of human skin specimens. Although the collagen and elastin fibers are detectable by auto-excited fluorescent signals, other components in the skin are rarely identified in unprocessed samples. In mice, various kinds of intravital labeling strategies have been developed - such as fluorescein isothiocyanate (FITC) painting, lipid staining and intravenous injection of fluorescein-conjugated dextran - to visualize blood flows. The following movie clips demonstrate the three-dimensional view of mouse ear skin by FITC painting and visualization of sebaceous glands via lipid staining.

Movie 1. FITC-painted mouse ear skin

Movie 2. Three-dimensional view of sebaceous gland visualized by lipid staining

 

We succeeded in visualizing epidermal keratinocytes in human skin biopsy samples using alexa594-conjugated isolectin B4 (IB4; Invitrogen, Carlsbad, CA). Since we found that a keratinocyte-labeling strategy with IB4 was also applicable to mice, we eventually succeeded in live visualization of the cell divisions of keratinocytes in vivo. As shown in movie clip 3, a large, round, IB4-circumscribed keratinocyte was divided into two in a sequential movie (arrow).

Movie 3. In vivo observation of keratinocyte cell division

 

Bone marrow-derived cells moving randomly in the skin

In order to observe the movement of bone marrow (BM)-derived cells in the skin, we transfused BM cells from CAG-eGFP (green fluorescent protein) transgenic mice (a transgenic mouse line with an 'enhanced' GFP) to naïve mice, in which all of the BM-derived cells were labeled green with GFP. Blood vessels were labeled red with dextran. As shown in movie clip 4, many BM-derived cells were randomly moving in the skin. This technique can be used to observe immune reactions in the skin, which will be presented in the next article in this series.

Movie 4. Bone marrow-derived cells are moving randomly in the skin

 

Intravital analysis of vascular permeability in mice using two-photon microscopy2

We have established an intravital evaluation system for vascular permeability in mice using TPM and we have evaluated the kinetics of vascular permeability in a histamine-induced inflammatory model. To mimic vascular hyperpermeability induced by inflammation, we used intravenous injections of histamine and visualized the bloodstream with FITC-dextran. In such inflammatory conditions, the hyperpermeability was selectively induced in the post-capillary venules and dextran as large as 2000-kDa leaked from the blood. This leakage of tracer was completely blocked by a histamine antagonist, confirming the clinical effects of antihistamines.

Movie 5. Histamine antagonist blocked extravasation of the tracer in histamine-induced vascular hyperpermeability (1)

Movie 6. Histamine antagonist blocked extravasation of the tracer in histamine-induced vascular hyperpermeability (2)

 

Conclusions

TPM provides convenient live images of not only the anatomic structure of the skin, but also various scenes occurring in the skin (both in inflammatory and immunologic events), which will improve our understanding of the pathogenesis of various skin diseases in the future.

 

References

  1. Egawa G, Natsuaki Y, Miyachi Y, Kabashima K. Three-dimensional imaging of epidermal keratinocytes and dermal vasculatures using two-photon microscopy. J Dermatol Sci 2013;70:143-145.
  2. Egawa G, Nakamizo S, Natsuaki Y, Doi H, Miyachi Y, Kabashima K. Intravital analysis of vascular permeability in mice using two-photon microscopy. Sci Rep 2013;3:1932. 
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