CBIS LM Core: Live-SR spinning disk confocal with TIRF

This system is capable of super-resolution live-cell imaging at much higher speed than regular confocal imaging and with minimal photodamage, or extremely thin optical sections in total internal reflection mode.

FRAP and STORM are also available.

An opaque enclosure with laser safety interlock makes it accessible to users who do not have a laser licence.

Location: CBIS Light Microscopy Core (S1A #01-04 CBIS lab)
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Confocal spinning disk

Yokogawa

The spinning disk is perforated with tens of thousands of pinholes arranged in an Archimedean spiral. Instead of scanning the field of view point by point with a single pinhole as in regular confocal imaging, a single rotation of the disk scans the entire field of view three times.

The pinhole size of our spinning disk is optimized for higher magnifications and weaker fluorescence signals. Less laser is needed to get the same signal because the camera used for imaging is more sensitive than a confocal detector.

Total internal reflection fluorescence (TIRF)

The Plant Journal

In TIRF, the laser contacts the coverslip at a very shallow angle and gets completely reflected by the glass surface adjacent to the sample before it can hit the sample (right). This generates in the sample a very shallow evanescent field of the same wavelength as the laser, which excites the fluorophores in the sample.

One can also steepen the laser’s angle of incidence so it passes through the coverslip into the sample at a very shallow angle (centre). This essentially gives widefield fluorescence with much less out-of-focus illumination, yielding clearer images with less photodamage.

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Features
  • Nikon Eclipse Ti inverted microscope with Perfect Focus System, Nikon TI-S-EJOY xy stage control and piezo z
  • Objectives (see user manual for detailed specs):
    • 10×/0.30 plan fluor phase contrast
    • 20×/0.95 apo DIC water immersion
    • 40×/0.95 plan apo DIC dry
    • 60×/1.40 plan apo DIC oil
    • 100×/1.45 plan apo DIC oil
    • 100×/1.49 apo DIC TIRF oil
  • Black incubation enclosure with temperature, CO2 and humidity control
  • Sample holders for 35mm petri dish, chamber slide, glass slide and multiple well plate
  • Transmitted light for DIC and/or phase contrast
  • Widefield fluorescence:
    • Lumencor light engine Generation I: Sola SE 6-LCR-SB (see blue curve below)
    • Fluorescence cubes, Quadra Filter

      • Yokogawa CSU-W1 spinning disk with microlens array, 50 μm pinholes and 500 μm pinhole spacing
      • iLas2 laser illuminator for ring TIRF and FRAP
      • Lasers:
        • 405 nm 100 mW
        • 488 nm 150mW DPSS
        • 514 nm 100 mW
        • 561 nm 100 mW DPSS
        • 640 nm 100 mW diode
        • 647 nm 500 mW ytterbium-doped fibre (STORM only)
      • Dichroics:
        • 405/488/561/640 multi-bandpass
        • 445/515/640 multi-bandpass
      • Emission bandpass filters (midpoint/bandwidth):
        • 452/45
        • 525/40
        • 571/72
        • 607/36
        • 692/40
      •  Two air-cooled Photometrics 95B back-illuminated sCMOS cameras (FOV 19 mm)
        • up to 40 fps in 16-bit full frame, 80 fps in 12-bit full frame

      • Software: MetaMorph