Development of a real-time flexible multiphoton microendoscope for label-free imaging in a live animal.

TPME system

The system is fed by a standard MIRA 900 Ti:Sapphire oscillator followed by a Faraday isolator avoiding reinjection back to the oscillator. More than 400 mW of the laser power are injected in the first 0.5 m-long PM-SMF in which pulses are temporally and spectrally broadened. Three different half-wave plates allow controlling the polarization where it is necessary in the setup (see in SI Fig. S2a).

Human Tissue and Animal Preparation

The reported investigation was in accordance with the relevant guidelines for both humans and animals. The rat tail tendon was fresh while all other reported ex vivo samples were fixed in 4% paraformaldehyde and stored in phosphate buffered saline. The healthy human pulmonary tissue samples, prepared at Rouen University Hospital, were obtained from a lobectomy. They were sampled from a healthy area of a pulmonary lobe. According to the French regulation, the use of the human tissue sample for the experiment was approved by scientific committee of the Rouen University Hospital Tumor Tissue Bank, and a written informed consent obtained from the patient. Induction of UUO in mice and the preparation of animals for in vivo imaging sessions were carried out by the staff of the UMR CNRS 7276 in accordance with European regulations, applied in France by Decret No. 2013-118 of 1st February 2013 on the protection of animals used by scientists. The protocol was approved by the ethics committee for animals used by scientists registered under the code C2A2-33 by the French “Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche”. During in vivo imaging sessions, the preparation and observation of the mice were made with a protocol similar to the one described by Brown et al. 12 , the main difference being the anesthesia. In our case, mice received a non-barbiturate anesthetic and a muscle relaxant sedative analgesic (ketamine at 18 mg/mL and xylazine at 0.9 mg/mL) through intraperitoneal injection. The kidney, elevated from the body, was clamped between two tongue depressors and placed on the flank of the mice, beneath the probe (see Fig. 5a ). The TPME probe was manipulated by means of a 3-axis precision motorized micromanipulator (Thorlabs, Inc., MAX 343). The micromanipulator was connected to an articulated arm (see Fig. 5a ) holding the TPME distal tip. The depth resolved imaging results reported in Fig. 4e–h and in Fig. 5d were obtained with this micromanipulator that allows precisely controlling the z coordinate.

Supplementary Material Supplementary Information Supplementary Video S1 Supplementary Video S2 Supplementary Video S3