Fff555 646 di01

The SPEED microscope setup has been extensively described in previous publications [27] , [30] . Briefly, it consists of an Olympus IX81 with a 1.4 NA 100× oil-immersion apochromatic objective (UPLSAPO 100X, Olympus), a 35 mW 633 nm He-Ne laser (Melles Griot), a 120 mW ArKr tunable ion laser (Melles Griot), an on-chip multiplication gain charge-coupled device camera (Cascade 128+, Roper Scientific) and the Slidebook software package (Intelligent Imaging Innovations) for data acquisition and processing. Cascade 128+ (128×128 pixels) was used to monitor the fast nuclear import process.
An optical chopper (Newport) was used to generate an on-off mode of laser excitation. GFP and Alexa Fluor 647 fluorescence were excited by 488 nm and 633 nm lasers, respectively. The two lasers were combined by an optical filter (FFF555/646 Di01, Semrock), collimated and focused into an overlapped illumination volume in the focal plane. The green and red fluorescence emissions were collected by the same objective, filtered by a dichroic filter (Di01- R405/488/561/635-25×36, Semrock) and an emission filter (NF01- 405/488/561/635-25×5.0, Semrock) and imaged by an identical CCD camera. The system error of alignment between red and green fluorescence channels is 3.0±0.1 nm, determined by measuring 230 immobile Alexa Fluor 647-labeled GFP fluorescent molecules on the surface of a cover-slip.