Abstract
Graded Index (GRIN) rod microlenses are increasingly employed in the assembly of optical probes for microendoscopy applications. Confocal, two–photon and optical coherence tomography (OCT) based on GRIN optical probes permit in–vivo imaging with penetration depths into tissue up to the centimeter range. However, insertion of the probe can be complicated by the need of several alignment and focusing mechanisms along the optical path. Furthermore, resolution values are generally not limited by diffraction, but rather by optical aberrations within the endoscope probe and feeding optics. Here we describe a multiphoton confocal fluorescence imaging system equipped with a compact objective that incorporates a GRIN probe and requires no adjustment mechanisms. We minimized the effects of aberrations with optical compensation provided by a low–order electrostatic membrane mirror (EMM) inserted in the optical path of the confocal architecture, resulting in greatly enhanced image quality.
Highlights
Optical microscopy for in vivo analysis deep within tissues requires the application of a series of well known observational techniques supported by small and non–invasive optical probes
The devices inserted on the microscope optical path, shown as grey boxes in Figure 1, comprised: 1. an adaptive optics (AO) module based on an electrostatic membrane mirror (EMM) 2. a Graded Index (GRIN) fiber objective mounted on the microscope standard objective receptacle 3. a calibration system based on an imaging camera illuminated by an optical relay reproducing a magnified view of the field covered by the GRIN objective
One could recreate a suitable field invariant pupil after the confocal scanning head using custom designed optics. This proved impossible in our configuration, due to constraints imposed by the underlying commercial architecture
Summary
Optical microscopy for in vivo analysis deep within tissues requires the application of a series of well known observational techniques (confocal, two–photon fluorescence or OCT) supported by small and non–invasive optical probes. GRIN rod lenses guide light using internal variations in the refractive index rather than the curved refractive surfaces employed by conventional lenses [1]. They can be assembled in a sequence of typically 1–3 elements to form a microendoscope probe that acts essentially as an optical relay (http://www.grintech.de/gradient-index-optics.html). GRIN probes for microendoscopy are typically combined with more classic optical elements such lenses [2]. Approximate ranges of typical values for microendoscopy probes based on GRIN microlenses are: 0.5– 3 cm for physical lengths; 150–800 mm for optical working distances; 0.4–0.75 for numerical apertures (NAs) and 100– 1000 mm for fields of view [3]. A probe with an external diameter of 360 mm and carefully engineered fiber structure was able to provide optical manipulation and analysis of microscale specimens [5]
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