Multiphoton microscopy is a cutting edge imaging modality leading to increasing advances in biology and also in the clinical field. To use it at its full potential and at the very heart of clinical practice, there have been several developments of fiber-based multiphoton microendoscopes. The application for those probes is now limited by few major restrictions, such as the difficulty to collect autofluorescence signals from tissues and cells theses being inherently weak (e.g. the ones from intracellular NADH or FAD metabolites). This limitation reduces the usefulness of microendoscopy in general, effectively restraining it to morphological imaging modality requiring staining of the tissues. Our aim is to go beyond this limitation, showing for the first time label-free cellular metabolism monitoring, in vivo in situ in real time.
The experimental setup is an upgrade of a recently published one (Ducourthial et.al, Scientific Reports, 2016) where femtosecond pulse fiber delivery is further optimized thank’s to a new transmissive-GRISM-based pulse stretcher permitting high energy throughput and wide bandwidth. This device allows fast sequential operation with two different excitation wavelengths for efficient two-photon excited NADH and FAD autofluorescence endoscopic detection (i.e. 860 nm for FAD and 760 nm for NADH), enabling cellular optical redox ratio quantification at 8 frames/s.
The obtained results on cell models in vitro and also on animal models in vivo (e.g. neurons of a living mouse) prove that we accurately assess the level of NADH and FAD at subcellular resolution through a 3-meters-long fiber with our miniaturized probe (O.D. =2.2 mm).
Pierre Leclerc, Charles-Henri Hage, Marc Fabert, Julien Brevier, Rodney P. O'Connor, Sylvia M. Bardet-Coste, Rémi Habert, Flavie Braud, Alexandre Kudlinski, and Frederic Louradour, "Label-free in vivo in situ diagnostic imaging by cellular metabolism quantification with a flexible multiphoton endomicroscope (Conference Presentation)," Proc. SPIE 10069, Multiphoton Microscopy in the Biomedical Sciences XVII, 100691N (Presented at SPIE BiOS: January 31, 2017; Published: 24 April 2017); https://doi.org/10.1117/12.2251843.5380018911001.
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Study of self-shadowing effect as a simple means to realize nanostructured thin films and layers with special attentions to birefringent obliquely deposited thin films and photo-luminescent porous silicon