Significance: Confocal laser scanning enables optical sectioning in fiber bundle endomicroscopy but limits the frame rate. To be able to better explore tissue morphology, it is useful to stitch sequentially acquired frames into a mosaic. However, low frame rates limit the maximum probe translation speed. Line-scanning (LS) confocal endomicroscopy provides higher frame rates, but residual out-of-focus light degrades images. Subtraction-based approaches can suppress this residue at the expense of introducing motion artifacts.

Aim: To generate high-frame-rate endomicroscopy images with improved optical sectioning, we develop a high-speed subtraction method that only requires the acquisition of a single camera frame.

Approach: The rolling shutter of a CMOS camera acts as both the aligned and offset detector slits required for subtraction-based sectioning enhancement. Two images of the bundle are formed on different regions of the camera, allowing both images to be acquired simultaneously.

Results: We confirm improved optical sectioning compared to conventional LS, particularly far from focus, and show that motion artifacts are not introduced. We demonstrate high-speed mosaicing at frame rates of up to 240 Hz.

Conclusion: High-speed acquisition of optically sectioned images using the new subtraction based-approach leads to improved mosaicing at high frame rates.