The present dataset includes data generated as part of the 2PhotonECL, focusing on the imaging limit of shadow electrochemiluminescence (SECL). Content of the folders:• folder "decrease ratio" contains data regarding: - the SECL raw images used to extract the ECL intensity (a.u.) for calculating decrease SECL ratio recorded in the Procell solution contained 30 µM [Ru(bpy)3]2+ on GCE or 0.1 M PBS (pH = 7.4) containing 1 mM [Ru(bpy)3]2+ and 100 mM TPA on ITO. - the decrease SECL ratio of different size of sperichal targets and spores on ITO and GCE.• folder "ITO_FL BF SECL" contains fluorescence (FL) and bright field (BF) and SECL raw images of 1 micron beads, 450 nm silica beads, 200 nm and 100 nm PS beads recording on the ITO setup at 1.2 V vs Ag/AgCl. The solution is 0.1 M PBS (pH = 7.4) containing 1 mM [Ru(bpy)3]2+ and 100 mM TPA. • folder "particle stacking" contains data regarding: - FL and SECL images of tightly stacked cluster of 6 µm particles on glassy carbon electrode (GCE) recorded in the Procell solution contained 30 μM [Ru(bpy)3]2+ at 1.2 V. - The decrease ratio calculated from the center, corner, edge and other single 6 µm particles. • folder "SECL FL intensity curve" contains data regarding:- FL and SECL images of 6 µm, 3 µm and 200 nm particles on GCE and 100 nm particles on ITO. - The normalized FL and SECL intensity curve extracted from the FL and SECL images mentioned above. • folder "spores" contains data regarding: - the FL and the SECL images of spores on ITO and GCE. - The normalized FL and SECL intensity curve extracted from the FL and SECL images of spores mentioned above.