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

Abstract

Blood vessel endothelium forms a semi-permeable barrier and its permeability controls the traffics of plasma contents. Here we report an intravital evaluation system for vascular permeability in mice using two-photon microscopy. We used various sizes of fluorescein-conjugated dextran as a tracer and its efflux was quantified by measuring the changes of fluorescent intensity both on the blood vessel area and the interstitial space. Using this system, we demonstrated that skin blood vessels limited the passage of dextran larger than 70 kDa under homeostatic conditions. We evaluated the kinetics of vascular permeability in histamine- or IgE-induced type I allergic models and a hapten-induced type IV allergic model. In such inflammatory conditions, the hyperpermeability was selectively induced in the postcapillary venules and dextran as large as 2000-kDa leaked from the bloods. Taken together, our study provides a convenient method to characterize the skin blood vessels as a traffic barrier in physiological conditions.

Imaging protective mast cells in living mice during severe contact hypersensitivity

“…injection of FITC-labeled dextran; see refs. 15,20) and hair follicle areas (corresponding to regions of low collagen content and identified by a second harmonic generation signal; see ref. 21) ( Figure 1A).…”

Label-free optical-resolution photoacoustic endomicroscopy in vivo

“…57 and 58 demonstrated 19.6 µm and 15.7 µm transverse resolutions, respectively), and an SNR of 29 dB from a 3-µm diameter microsphere illuminated by a pulse energy of 500-nJ in a 9.2 µm beam diameter. As shown in the in vivo and ex vivo image demonstrations, the major benefit of the OR-PAEM over existing IVM techniques [44][45][46][47][48][49][50][51][52][53][54][55][56] lies in its label-free angiographic imaging capability, which provides critical image in experimental biology and clinical medicine. Although other groups [57,58] developed endoscopic devices with optical focusing and achieved an even smaller probe diameter (i.e., ~1.1 mm in the case of ref.…”

“…OR-PAM realizes the key benefits of PAT in biological experimentation through its high-resolution imaging capability; further, its unique optical absorption-based contrast mechanism enables it to complement conventional highresolution microscopy tools, such as confocal microscopy [44][45][46][47][48][49], two-photon microscopy [44][45][46][50][51][52][53], and optical coherence tomography [44][45][46][53][54][55][56]. So far, considerable efforts have been made to improve the spatial resolution [15], scanning speed [7,12], and functional imaging capability [4][5][6][7][8][9] of OR-PAM.…”

Optical-resolution photoacoustic endomicroscopy in vivo

“…Intravital microscopy (IVM) techniques [1][2][3][4][5][6][7][8][9][10][11][12][13] have been opening new research and clinical avenues by providing unprecedented image information on biological processes in vivo. In addition to earlier approaches that imaged the body surfaces of small animals [1][2][3][4][7][8][9], technical advances in miniaturized imaging devices have now enabled extending IVM to endomicroscopy [1,5,6,10,11,13], which enables the imaging of internal organs.…”

“…In addition to earlier approaches that imaged the body surfaces of small animals [1][2][3][4][7][8][9], technical advances in miniaturized imaging devices have now enabled extending IVM to endomicroscopy [1,5,6,10,11,13], which enables the imaging of internal organs. So far, two-photon microscopy [1][2][3][7][8][9][10] and confocal microscopy [1][2][3][11][12][13] have been the major technical platforms for this approach. However, in most cases, these techniques are based on complex labeling with fluorescent molecules, which could disturb delicate biological processes.…”