Rapid mesoscale multiphoton microscopy of human skin.

Mihaela Balu,Bruce J Tromberg,Eric O Potma,Hideharu Mikami,Jue Hou

doi:10.1364/boe.7.004375

Abstract

We present a multiphoton microscope designed for mesoscale imaging of human skin. The system is based on two-photon excited fluorescence and second-harmonic generation, and images areas of ~0.8x0.8 mm2 at speeds of 0.8 fps (800x800 pixels; 12 frame averages) for high signal-to-noise ratio, with lateral and axial resolutions of 0.5µm and 3.3µm, respectively. The main novelty of this instrument is the design of the scan head, which includes a fast galvanometric scanner, optimized relay optics, a beam expander and high NA objective lens. Computed aberrations in focus are below the Marechal criterion of 0.07λ rms for diffraction-limited performance. We demonstrate the practical utility of this microscope by ex-vivo imaging of wide areas in normal human skin.

Highlights

In vivo multiphoton microscopy (MPM) holds promise as an important research and clinical tool for label-free imaging in human skin
The work described in this manuscript addresses two main technical challenges related to MPM skin imaging: limited field of view and slow acquisition rate of large skin areas
The simulation of the optical performance for the relay and beam expander systems showed that computed aberrations in focus were below the Marechal criterion of 0.07λ rms for diffraction-limited performance

Summary

Introduction

In vivo multiphoton microscopy (MPM) holds promise as an important research and clinical tool for label-free imaging in human skin. Large FOV and automatic translation of the imaging area is complemented by fast image acquisition with high detection sensitivity in order for such a system to be of clinical use for rapid assessment of skin lesions. Presents NLOM images with FOV of 800x600 μm acquired in vivo in mouse skin, with high theoretical spatial resolution, at fast scan rates. All other studies advanced the NLOM systems to improve either the scanning speed or the field of view as required by the application of interest Optimization of both parameters was achieved at the expense of spatial resolution [16]. We describe below in detail the main components of the system

Implementation of fast imaging acquisition

Optical design considerations

Optical design of the relay lens system

MPM imaging system performance

Discussion and conclusion