New techniques for fluorescence background rejection in microscopy and endoscopy

Confocal microscopy is a popular technique in the bioimaging community, mainly because it provides optical sectioning. However, its standard implementation requires 3-dimensional scanning of focused illumination throughout the sample. Efficient non-scanning alternatives have been implemented, among which the simple and well-established incoherent structured illumination microscopy (SIM) [1]. We recently proposed a similar technique, called Dynamic Speckle Illumination (DSI) microscopy, wherein the incoherent grid illumination pattern is replaced with a coherent speckle illumination pattern from a laser, taking advantage of the fact that speckle contrast is highly maintained in a scattering media, making the technique well adapted to tissue imaging [2]. DSI microscopy relies on the illumination of a sample with a sequence of dynamic speckle patterns and an image processing algorithm based only on an a priori knowledge of speckle statistics. The choice of this post-processing algorithm is crucial to obtain a good sectioning strength: in particular, we developed a novel post-processing algorithm based one wavelet pre-filtering of the raw images and obtained near-confocal fluorescence sectioning in a mouse brain labeled with GFP, with a good image quality maintained throughout a depth of $\sim $100 $\mu $m [3]. In the purpose of imaging fluorescent tissue at higher depth, we recently applied structured illumination to endoscopy. We used a similar set-up wherein the illumination pattern (a one-dimensional grid) is transported to the sample with an imaging fiber bundle with miniaturized objective and the fluorescence image is collected through the same bundle. Using a post-processing algorithm similar to the one previously described [3], we obtained high-quality images of a fluorescein-labeled rat colonic mucosa [4], establishing the potential of our endomicroscope for bioimaging applications. \\[4pt] \underline {Ref:} \\[0pt] [1] M. A. A. Neil \textit{et al}, Opt. Lett. 22, 1905 (1997) \\[0pt] [2] C. Ventalon \textit{et al}, Opt. Lett. \textbf{30}, 3350 (2005) \\[0pt] [3] C. Ventalon \textit{et al}, Opt. Lett. \textbf{32,} 1417 (2007) \\[0pt] [4] N. Bozinovic \textit{et al}, Opt. Express \textbf{16}, 8016 (2008)