Di01 r405 488 561 635 25x36

Images of BS-C-1 cells were acquired using a standard inverted epifluorescence microscope (Observer A1, Zeiss) fitted with a 63× (1.4 NA) Plan-Apochromat oil-immersion objective (Carl Zeiss). The sample was illuminated using 635 nm and 450 nm diode lasers (OptoEngine) for the excitation of Alexa 647 and for photoswitching respectively. The illumination light was directed towards the microscope using custom laser excitation optics, reflected into the sample using a dichroic filter (Di01-R405/488/561/635-25x36, Semrock), and focused onto the back focal plane of the objective lens. Signal from the sample was filtered using a bandpass filter (FF01-676/29-25, Semrock) and acquired by an electron multiplying charge coupled device (EMCCD) camera (iXon DU897-BV, Andor) set to conventional readout mode. Axial drift during the course of the acquisition was corrected in real time using a custom focus stabilization system comprising an 850 nm diode laser (PI, USA), a quadrant position detector (Thorlabs), and an XYZ piezo positioner (PI, USA). All devices including lasers, shutters, and cameras were controlled and synchronized using custom-written software in the C programming language.

For FRAP experiments, 488 nm solid-state and 630 nm diode lasers were focused to the back focal plane of the objective through an appropriate dichroic mirror (Di01-R405/488/561/635-25x36, Semrock) through the objective lens to illuminate a ~5 or ~10 μm (for 488 nm or 630 nm, respectively) diameter spot in the center of the sample area. The laser power was adjusted for ~80% bleaching with 5 s exposure for the eGFP or Alexa 647 signals, and four cycles of bleaching/recovery were recorded for each sample and averaged.

Live yeast expressing GFP-tagged nucleoporins were first observed using a wide-field fluorescence microscope equipped with a charge-coupled device camera (Coolsnap HQ2, Photometrics) to locate the nucleus equator. Then the selected area was observed under the SPEED microscope, which includes an Olympus IX81 instrument equipped with a 1.4 NA 100× oil-immersion apochromatic objective (UPLSAPO 100X, Olympus), 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. Excitation was provided by a 488 nm laser. Green fluorescence emissions were collected through the same objective, filtered by a dichroic filter (Di01-R405/488/561/635-25x36, Semrock) and an emission filter (NF01-405/488/561/635-25X5.0, Semrock), and imaged by the CCD camera. Images were captured continuously; the exposure time of each image was 10–50 ms.