Deep vascular imaging in wounds by two-photon fluorescence microscopy

Abstract

Deep imaging within tissue (over 300 μm) at micrometer resolution has become possible with the advent of two-photon fluorescence microscopy (2PFM). The advantages of 2PFM have been used to interrogate endogenous and exogenous fluorophores in the skin. Herein, we employed the integrin (cell-adhesion proteins expressed by invading angiogenic blood vessels) targeting characteristics of a two-photon absorbing fluorescent probe to image new vasculature and fibroblasts up to ≈ 1600 μm within wound (neodermis)/granulation tissue in lesions made on the skin of mice. Reconstruction revealed three dimensional (3D) architecture of the vascular plexus forming at the regenerating wound tissue and the presence of a fibroblast bed surrounding the capillaries. Biologically crucial events, such as angiogenesis for wound healing, may be illustrated and analyzed in 3D on the whole organ level, providing novel tools for biomedical applications.

Figure 1. Two-photon fluorescence microscopy of the “whole-mounted” wounds was performed 800–825 nm to excite probe 1.

3-D reconstruction of invading capillaries from successive 1 μm optical sections. Vascular plexus extends from 0 to approximately 1100 μm and integrin-expressing cells from 1100 to 1600 μm (a). Integrin-expressing cells in optical section (1200–1336 μm). Probe 1 colocalized positively with some granulocytes that were costained with a Gr-1 antibody. Fibroblasts were stained with anti-fibroblast activation protein (FAP) antibody. Many fibroblasts within the fibrin clot colocalized positively with RGD probe-positive cells. Thus, the RGDfK moiety on probe 1 targeted endothelial cells and endothelial cell precursors. (b); capillaries in optical section (200–400 μm, c). Excitation (825 nm, 70 fs, 80 MHz, ≈ 5–7 mW) and collection was done perpendicularly to red-green arrow (excitation: from red to green, collection: from green to red). Objective: Leica 20×, 1.0 N. A., water. Depiction of a wound during the granulation tissue formation (d). During the proliferative phase fibroblast invade the fibrin clot, generating the granulation tissue. Angiogenesis accompanies the invading fibroblasts. Integrins are overregulated in fibroblasts (α₅β₁, α_vβ₆) and in endothelial cells of capillaries (α_vβ_3, inset), which allows for the RGD-containing probe to interact with these cells. 2PA RGD-probe to target integrins (e).

Figure 2. Excised wound healing sample (“whole-mounted”) as imaged and analyzed by 2PFM once it was excised from the mouse.

The image shows four 7-day-old excision wounds (6 mm) with the scab tissue on top of the wounds in a petri capsule prior to imaging.

Figure 3. Segmentation analysis from 2PFM images of a wound healing specimen and immunohystostaining of tissue sections.

Fluorescent pixels were used to account for the vasculature. Probe 1 enabled excellent contrast beyond 1 mm within tissue. Analysis was accomplished with Amira 5.4. Z-axis (blue), green lines delimit optical sections. Immunostaining of horizontal tissue sections of the wounds were used to screen for macrophages (Mac-3), integrin-expressing endothelial cells (CD31), endothelial cell precursors (CD34), granulocytes (Gr1), and fibroblasts (FAP). Fluorescence resulting from the two-photon excitation of probe 1 is shown in green while one-photon fluorescence is shown in red. Lower right is the muscle area and upper left is the area closest to the scab on all sections.

Figure 4. Two-photon fluorescence micrograph of the “whole-mounted” wounds without probe 1.

Excitation at 825 nm, autofluorescence from connective tissue in muscle is observed.