No abstract

Traditional imaging with one-photon confocal microscopy and organic fluorophores poses several challenges for the visualization of vascular tissue, including autofluorescence, fluorophore crosstalk, and photobleaching. We studied human coronary arteries (HCAs) and mouse aortas with a modified immunohistochemical (IHC) "en face" method using quantum dot (Qdot) bioconjugates and two-photon excitation laser scanning microscopy (TPELSM). We demonstrated the feasibility of multilabeling intimal structures by exciting multicolored Qdots with only one laser wavelength (750 nm). Detailed cell structures, such as the granular appearance of von Willebrand factor (VWF) and the subcellular distribution of endothelial nitric oxide synthase, were visualized using green dots (525 nm), even when the emission maximum of these Qdots overlapped that of tissue autofluorescence (510-520 nm). In addition, sensitive fluorescence quantification of vascular cell adhesion molecule 1 expression at areas of varying hemodynamics (intercostal branches vs. nonbranching areas) was performed in normal C57Bl/6 mice. Finally, we took advantage of the photostability of Qdots and the inherent three-dimensional (3D) resolution of TPELSM to obtain large z-stack series without photobleaching. This innovative en face method allowed simple multicolor profiling, highly sensitive fluorescence quantitation, and 3D visualization of the vascular endothelium with excellent spatial resolution. This is a promising technique to define the spatial and temporal interactions of endothelial inflammatory markers and quantify the effects of different interventions on the endothelium.

No abstract

We present the design and feasibility testing of a multimodal co-registered endoscope based on a dual-path optical system integrated with a scanning piezo. This endoscope incorporates three different imaging modalities. A large field-of-view (FOV) reflectance imaging system enables visualization of objects several millimeters in front of the endoscope, while optical coherence microscopy (OCM) and multiphoton microscopy (MPM) are employed in contact with tissue to further analyze suspicious areas. The optical system allows multiple different imaging modalities by employing a dual optical path. One path features a low numerical aperture (NA) and wide FOV to allow reflectance imaging of distant objects. The other path features a high NA and short working distance to allow microscopy techniques such as OCM and MPM. Images of test targets were obtained with each imaging modality to verify and characterize the imaging capabilities of the endoscope. The reflectance modality was demonstrated with a 561 nm laser to allow high contrast with blood vessels. It achieved a lateral resolution of 24.8 μm at 5 mm and a working distance from 5 to 30 mm. OCM was performed with a 1300 nm super-luminescent diode since this wavelength experiences low relative scattering to allow for deeper tissue imaging. Measured OCM lateral and axial resolution was 4.0 and 14.2 μm, respectively. MPM was performed with a custom 1400 nm femtosecond fiber laser, a wavelength suitable for exciting multiple exogenous, and some endogenous fluorophores, as well as providing information on tissue composition through harmonic generation processes. A 4.0 × μm MPM lateral resolution was measured.