We extended this approach to in vivo imaging by analyzing TSFG signals in 2 dpf zebrafish embryos submitted to artificial hypoxia . Embryos were mounted as described previously except that the observation chamber was sealed ( Fig . 4b ) . The embryo and agarose were deposited in a ≈ 100 µL well at the bottom of the microscope slide , and covered with a coverslip sealed with silicone ( twinsil speed , Picodent , Germany ) while the agarose was still liquid . Within 10 min after mounting , the imaging session started and consisted in recording a time series of 30 images every 5 min during several 2 - 5 hours . After that session , the top cover glass of the chamber was removed to recover normoxia , the dish was filled with embryo medium and placed back under the microscope with the embryo still embedded in agarose . The same region was then imaged . The time between the last hypoxia image and the first one after opening chamber was < 15 min . From the recorded images , we segmented the RBCs and extracted the average oxygenation parameter R = S 401 / S 433 at each time point . Figure 4b shows the time evolution of R in the case of one embryo . The values of R before , during , and after hypoxia for nine embryos is presented in Fig . 4c . We consistently observed a decrease of the R parameter during the hypoxia assay ( typically-40 % after 3 h ) , suggesting that the limited oxygen supply inside the small chamber volume was being consumed by the embryo . After chamber opening , the R parameter returned to its original value , indicating that RBCs recovered their oxygenation state within minutes after hypoxia . This fast recovery is consistent with 58 . Together , these data provide strong evidence for the possibility of label - free functional TSFG microscopy . Importantly , our implementation can probe RBCs in vivo with single - cell resolution and microsecond pixel dwell times .