Inverted structured illumination microscope (SIM) and single molecule imaging (PALM/STORM) total internal reflection fluorescence (TIRF) instrument

Structured Illumination Microscopy (SIM) provides an approximate 2-fold resolution improvement in XY and Z compared to confocal imaging with a comparable acquisition speed. Resolution = ~120nm XY and 300nm with ~2.5 seconds per frame.

Single molecule imaging (PALM/STORM) can be used to generate images with up to 10-fold resolution improvement. Reconstructed images localise fluorescent markers with a ~20-40nm resolution. For more information on these techniques and to request training  please use the PPMS booking system.

When preparing samples for Structured Illumination Microscope please follow these recommendations.

Features Include:

  • Fully motorised X-Y-Z stage
  • Piezo Z-drive for rapid Z-stacks
  • PCO sCMOS camera for SIM
  • Andor 897 EMCCD for Single Molecule Imaging

Objectives:

  • 10x 0.45 NA / Plan-Apochromat / 520µm working distance / 0.406 μm/pixel / Air
  • 63x 1.2 NA / C-Apochromat / 280µm working distance / 0.064 μm/pixel / Water – For SIM
  • 63x 1.4 NA / Plan-Apochromat / 190µm working distance / 0.064 μm/pixel/ Oil – For SIM
  • 100x 1.46 NA / Alpha Plan-Apochromat / 110µm working distance / 0.041 μm/pixel / Oil – For SIM and PALM

Fluorescent Filter Sets:

Filter Turret #1

(for checking sample with oculars)

  • 77HE – Dual Green/Red filter for locating sample by eye (allows viewing of 488 and 560 emitting fluorophores together)

(for imaging with laser)

  • SR Cube 07 RL – BP 420-480
  • SR Cube 08 RL – BP 495-550
  • SR Cube 09 RL – BP 570-620
  • SR Cube 10 RL – LP 655

Filter Turret #2

(multi-band dichroic and emission filters)

  • SR Cube 03 RL – LBF 561/642
  • SR Cube 04 RL – LBF 488/561
  • SR Cube 05 RL –  BP 420-480 / BP570-640 / LP 740
  • SR Cube 06 RL –  BP 420-480 / BP495-550 / LP 650

Lasers:

  • 405nm
    • DAPI
  • 491nm
    • GFP / YFP / FITC / Alexa488
  • 561nm
    • Cy3 / Alexa 546/555/568 / Alexa 594 / mCherry
  • 642nm
    • Cy5 / Alexa647

Laser Power:

Measurement at BFP

  • 405nm (0.6 mW),
  • 488nm (18.72 mW),
  • 568nm (20.6 mW),
  • 647nm (17.03 mW)

Measurement at 10x

  • 405nm (5.73 mW),
  • 488nm (19.86 mW),
  • 568nm (18.47 mW),
  • 647nm (18.94 mW)

Cameras

User guide to SIM here and PALM/STORM here.