Fluorescence-Based Microendoscopic Sensing System for Minimally Invasive In Vivo Bladder Cancer Diagnosis

Abstract

Bladder cancer is commonly diagnosed by evaluating the tissue morphology through cystoscopy, and tumor resection is used as the primary treatment approach. However, these methods are limited by lesion site specificity and resection margin, and can thereby fail to detect cancer lesions at early stages. Nevertheless, rapid diagnosis without biopsy may be possible through fluorescence sensing. Herein, we describe a minimally invasive imaging system capable of sensing even small tumors through a 1.2 mm diameter flexible fiber bundle microprobe. We demonstrate that this new device can be used for the early diagnosis of bladder cancer in rats. Bladder cancer was induced in rats using the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), and a togglable filter capable of PpIX fluorescence sensing was installed in the microendoscopic system. Following 5-aminolevulinic acid administration, tissue in the early stages of bladder cancer was successfully identified with fluorescence detection and confirmed with hematoxylin/eosin and ferrochelatase staining. Although the time required for BBN to induce bladder cancer varied between 3 and 4 weeks among the rats, the microendoscopic system allowed the minimally invasive follow-up on cancer development.

Keywords: 5-aminolevulinic acid; bladder cancer; ferrochelatase staining; microendoscopy; minimally invasive diagnosis; protoporphyrin IX.

Figure 1

Schematic diagram and images of the experimental minimally invasive monitoring procedure and fluorescence imaging of BBN-induced bladder cancer using a flexible fluorescence imaging microprobe. 5-ALA, 5-aminolevulinic acid; BBN, N-butyl-N-(4-hydroxybutyl)nitrosamine; H/E, hematoxylin/eosin staining; FECH, ferrochelatase staining.

Figure 2

Optical structure and dimensions of the microendoscopy-based fluorescence sensing device.

Figure 3

Minimally invasive in vivo brightfield and fluorescence images of bladder in rats. (a–c) The inside of a healthy bladder, imaged with (a) brightfield illumination, (b) excitation filter only, and (c) with an excitation filter and emission filter both inserted. (d–f) Images of the inside of a bladder with cancerous lesions taken with (d) brightfield illumination, (e) excitation-filtered illumination, and (f) both excitation and emission filters at week 13 following BBN treatment (Case 1). (g,h) Cases 2 and 3 show additional cases of bladder cancer in other rats.

Figure 4

Tracking and monitoring of bladder cancer development. Brightfield and fluorescence images were obtained from the same bladder tissue locations at weeks (a,d) 4, (b,e) 10, and (c,f) 16 of BBN exposure. Orange circles in (a–c) brightfield images denote the approximate corresponding positions in (d–f) fluorescence identification based on the vascular patterns. The green arrow indicates the location of a cancer lesion. The shape of the major blood vessels is highlighted in the small lower circles in (a–c). The fluorescent signal (red) of the cancer tissue is highlighted in the small lower circle in (f).

Figure 5

Biopsy process and H/E staining results of bladder cancer tissue at week 10 observed with brightfield and fluorescence imaging. (a,c) Two neighboring cancer lesions observed with fluorescence imaging and (b) corresponding brightfield image. (d,f) H/E staining results of the excised bladder cancer tissues. (e) Image of the excised bladder with the cancer lesions (highlighted in green dotted circles).

Figure 6

(a) H/E and (b) FECH staining of healthy bladder tissue, and (c) magnified image of the green dotted line in (b). (d) H/E and (e) FECH staining of a bladder cancer lesion, and (f) magnified image of the square dotted line in (e).