OMX-SR 3D-SIM Super-Resolution

Offering resolution at twice the diffraction limit of traditional microscopes, the DeltaVision OMX system is the practical solution for three dimensional super resolution imaging that does not depend on special fluorophores or fluorescent proteins. The OMX is also set-up for TIRF and fast conventional imaging of live cells.

Startup

  • Turn on Key and laser power switch (door under microscope). Everything else should be on.
  • Sign-in to iLab. Use kiosk in LMIC or your own phone or computer.
  • If necessary (usually is not), user = worx, password = OMX120SI.
  • Sign in to LMIC software (iCenter Signin, apple icon) to transfer images (IU username).
  • Start AcquireSR software.
  • Click on File/Settings - make sure proper objective is selected (60X/60X TIRF or 60x SIL).
    Objective setting should match objective installed on scope.
  • Make sure focus offsets box is checked for TIRF, UNCHECKED for everything else.
  • Start softWoRx software.

Shutdown

  • Make sure necessary image processing is complete.
  • Quit AcquireSR software (File/Quit/Yes).
  • Quit softWoRx.
  • Transfer images - LMIC Sign out form. Select Data Directory, add title, sign out.
  • At microscope:
    o turn off key and laser power switch.
  • Clean objective with Lens paper, then wipe 1X with Chloroform on a clean cotton tipped applicator.
  • Close all doors.
  • Sign out of iLab

Configuration

  • Structured Illumination mode for high-resolution imaging. X,Y resolution to ~ 100nm, Z resolution to ~ 300nm.
  • Conventional Illumination mode for fast live cell imaging. Up to 65 frames/second in three colors.
  • Lasers lines (405, 488, 561, 642) and and emission filters (419-465, 500-550, 609-654, 665-705) for imaging DAPI, green, red and far red fluorophores.
  • Objectives:
    CorrectionMagnificationImmersionN.A.W.D.CollarItem No.
    UPlanApo60xSIL1.3N/AManual 0.15-0.19UPLSAPO60X2
    TIRF Apo N60xOIL1.49N/AManual 0.13-0.19APON60XOTIRF
    Plan Apo N60xOIL1.42N/ANoPLAPON60XOPFS
  • Ring TIRF.
  • Three PCO Edge 5.5 sCMOS cameras allow simultaneous three color imaging.
  • Environmental control for live imaging.
  • Controlled by AquireSR software, image processing by SoftWorx imaging software

Technical Notes

Everything matters when trying to achieve the best resolution. Care should be taken to optimize sample preparation. The best fixation technique or choice of the best fluorophore for your sample is often an experiment in itself and will be specific to your sample. We are happy to offer advice. Below are a few general guidelines for OMX sample preparation.

  • Check the list of excitation lasers and emission filters above and pick fluorophores that will work well with the microscope.
  • The specimen should be touching or attached to the coverslip. If possible, fix and stain specimens that are already attached to the coverslip that will be used for the imaging experiment.
  • USE ONLY #1.5 COVERSLIPS! This is true for almost all light microscopes but is especially important for the OMX. For best results, the LMIC can supply you with coverslips of the same lot number that we used to calibrate the microscope.
  • Mount only one coverslip in the center of the usual (3in x 1in) glass slide. Stage travel is limited to 22mm X 22mm.
  • Mounting media matters a lot for this system. We recommend the SlowFade (Molecular Probes) mounting medium because it will create a more uniform optical path and prevent bleaching of the fluorescent probes. The LMIC can supply you with an aliquot of SlowFade. We also have ProLong Gold mountant for long term storage of slides. ProLong Gold hardens over time and as it hardens its refractive index changes. It may take 7-8 days for slides to become fully cured. It is too early to tell how this will effect resolution on the OMX.
  • You should seal your coverslips with nail polish. Please make sure it is very dry before you put slides on the microscope. Part of the process of high-resolution imaging is finding the immersion oil that best matches the refractive index of your sample. This may require changing oil several times to find the right one. Cleaning oil off of the coverslip is a lot easier if the coverslip is sealed and fixed in place with nail polish.

OMX Quick reference guides:

Immersion oil optimalization OMX image aquisition SI image reconstruction

 SIM sample preparation OMX image reconstruction

 OMX applications:

OMX Photokinetics  OMX Ring TIRF OMX SIM