Di01 r405 488 561 635 25 36

The SPEED microscope included an Olympus IX81 equipped with a 1.4-NA 100×oil-immersion apochromatic objective (UPLSAPO 100×, Olympus), a 35 mW 633 nm He-Ne laser (Melles Griot), 50 mW solid state 488-nm and 561-nm lasers (Coherent), 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. For individual channel imaging, GFP (or mCitrine), mCherry, and Alexa Fluor 647 were excited by 488 nm, 561 nm, and 633 nm lasers, respectively. The 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 with the above CCD camera operating at 500 Hz.

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.