Two-photon fluorescence (TPF) and second-harmonic generation (SHG) microscopy are powerful tools for imaging unstained biological tissues [1
]. These label-free techniques are capable of producing high-resolution real-time in vivo
images and have shown great promise for medical diagnostics of various diseases, potentially replacing surgical biopsies [2
]. The maximum imaging depth, however, is limited in most tissues to ~1 mm [8
One strategy to overcome the depth limitation is to develop miniaturized TPF microscopes that could be used as endoscopes in a clinical setting. A number of different endoscopes and techniques have been demonstrated [10
], including in vivo
imaging of unstained tissues [18
]. These devices are typically composed of a miniaturized scanning mechanism and focusing optics in a protective housing. The scanners proposed generally use either a miniaturized fiber scanning mechanism or microelectromechanical systems scanning mirror. The need to encapsulate a scanning mechanism into a housing of suitable size for minimally invasive procedures poses several challenges including: (1) uniformity of scan, (2) sensitivity, durability and reliability of the scanner, and (3) miniaturization of the distal scan mechanism and optics. A different approach has been to use gradient index (GRIN) lenses to relay the excitation light and TPF/SHG emission to and from an external microscope deep into soft tissue [19
]. Since only the GRIN lens penetrates the tissue, the excitation, scanning, and collection optics need not be miniaturized for in vivo
imaging. GRIN lenses have been shown to be biocompatible and previously used in a clinical setting for non-penetrative imaging of chronic leg ulcers with delayed wound healing [23
]. Use of a small diameter lens to penetrate deeply within tissue has been demonstrated with a hypodermic needle GRIN system [24
]. These studies show great promise for GRIN endoscopy to be used as either a guide for or a replacement of traditional surgical biopsies.
Previous studies, however, have been limited to short (<4 cm) GRIN systems primarily intended for small animals and external clinical use [19
]. The eventual adaptation of GRIN lenses in the diagnosis of human diseases would require significantly longer GRIN endoscope systems. Commonly used prostate biopsy needles, for example, are as long as 25 cm. In this study we investigate the effect of GRIN system length on the imaging performance. We characterize the loss in imaging performance with increased GRIN system length and present a compact and portable two-photon microscope that could be used in a clinical environment in combination with various GRIN lens systems. Finally, the capability of the system for in vivo
imaging of unstained tissues is demonstrated in live, anesthetized rats.