Pseudodiaptomus marinus copepods are small crustaceans living in estuarine areas endowed with exceptional swimming and adaptative performances. Since the external cuticle acts as an impermeable barrier for most dyes and molecular tools for labeling copepod proteins with fluorescent tags are not available, imaging cellular organelles in these organisms requires label free microscopy. Complementary nonlinear microscopy techniques have been used to investigate the structure and the response of their myofibrils to abrupt changes of temperature or/and salinity. In contrast with previous observations in vertebrates and invertebrates, the flavin autofluorescence which is a signature of mitochondria activity and the Coherent Anti-Stokes Raman Scattering (CARS) pattern assigned to T-tubules overlapped along myofibrils with the second harmonic generation (SHG) striated pattern generated by myosin tails in sarcomeric A bands. Temperature jumps from 18 to 4 degrees C or salinity jumps from 30 to 15 psu mostly affected flavin autofluorescence. Severe salinity jumps from 30 to 0 psu dismantled myofibril organization with major changes both in the SHG and CARS patterns. After a double stress (from 18 degrees C/30 psu to 4 degrees C/0 psu) condensed and distended regions appeared within single myofibrils, with flavin autofluorescence bands located between sarcomeric A bands. These results shed light on the interactions between the different functional compartments which provide fast acting excitation-contraction coupling and adequate power supply in copepods muscles. (C) 2015 Elsevier Inc. All rights reserved.