sentence: The Mlrc2 sequence was then subcloned into the tdEos Gateway vector using the LR clonase ( Invitrogen ) , following the manufacturer ’ s procedure , to produce the Eos-MLC fusion construct.Single-molecule imaging of Eos-MLC was performed using a modified epifluorescence microscope ( Olympus IX81 ; Olympus , Tokyo , Japan ) equipped with 60 × microscope objective ( numerical aperture , 1.45 ; Olympus ) and a thermoelectric-cooled , electron-multiplying CCD camera ( PhotonMax ; Roper Scientific , Trenton , NJ ) .