Needle-based fluorescence endomicroscopy via structured illumination with a plastic, achromatic objective

Abstract

In order to diagnose cancer, a sample must be removed, prepared, and examined under a microscope, which is expensive, invasive, and time consuming. Fiber optic fluorescence endomicroscopy, where an image guide is used to obtain high-resolution images of tissue in vivo, has shown promise as an alternative to conventional biopsies. However, the resolution of standard endomicroscopy is limited by the fiber bundle sampling frequency and out-of-focus light. A system is presented which incorporates a plastic, achromatic objective to increase the sampling and which provides optical sectioning via structured illumination to reject background light. An image is relayed from the sample by a fiber bundle with the custom 2.1-mm outer diameter objective lens integrated to the distal tip. The objective is corrected for the excitation and the emission wavelengths of proflavine (452 and 515 nm). It magnifies the object onto the fiber bundle to improve the system's lateral resolution by increasing the sampling. The plastic lenses were fabricated via single-point diamond turning and assembled using a zero alignment technique. Ex vivo images of normal and neoplastic murine mammary tissues stained with proflavine are captured. The system achieves higher contrast and resolves smaller features than standard fluorescence endomicroscopy.

Fig. 1

Custom achromatic objective design.

Fig. 2

(a) Nominal MTF of the objective. (b) Nominal chromatic focal shift of the objective.

Fig. 3

Structured illumination high-resolution microendoscope (SI-HRME) design layout.

Fig. 4

(a) SolidWorks cutaway of the achromatic objective with built-in alignment features. (b) Inset of (a). The alignment features at the edges define the position of the lenses. (c) SolidWorks model of the achromatic objective and the fiber coupler used to attach the objective to the fiber bundle.

Fig. 5

(a) Sample doublet undergoing tilt measurements with a Zygo white light interferometer. (b) Sample doublet undergoing decenter measurements with a Zeiss microscope. (c) Set of lenses ready for assembly, fully assembled achromatic objective, and a commercial objective, next to a ruler for scale.

Fig. 6

(a) Achromatic objective integrated to a fiber bundle. (b) Assembled SI-HRME. Col collection lens; Ex excitation filter; DM dichroic mirror; Com commercial objective, FB fiber bundle; AO achromatic objective; Em emission filter; Con condenser lens; FM folding mirror.

Fig. 7

(a) Commercial Zeiss microscope objective relaying images from the custom achromatic objective. (b) Fluorescence image of a 1951 USAF resolution target imaged through the achromatic objective.

Fig. 8

The diffraction limited depth of field for a $2\times$, NA0.55 objective represented by the gray rectangle is 6.0 μm. The nominal shift range plotted by the solid line is 4.0 μm. The measured shift range plotted by the small dashed line is 6.9 μm. The nominal shift range of an NA0.55 monochromatic objective plotted by the large dashed line is 96.7 μm.

Fig. 9

Fluorescence images of a 1951 USAF resolution target through the HRME (a) before integrating the achromatic objective to the distal tip of the fiber bundle and (b) after integrating the objective. The lateral resolution limit improves from 7.8 μm (group 7, element 1) to 4.4 μm (group 7, element 6).

Fig. 10

Ex vivo mouse tissue images through the endomicroscope and a benchtop confocal microscope.