OMX V4 Structured Illumination Super-resolution Scope

mouse embryonic stem cell image, top view
mouse embryonic stem cell image, side view
Structured illumination reconstructed image of a mouse embryonic stem cell stained for DAPI (blue), APC (green), alpha tubulin (red). Top view (above) and side view (below) of a 5.6 μm z-stack. Scale bar = 5 μm. Structured illumination microscopy (SIM) provides a two-fold increase in resolution beyond the diffraction limit in both the image and axial planes. Credit: Dr. Shukry Habib, Nusse lab

The OMX V4 is a structured illumination super-resolution microscope from the Applied Precision division of GE Healthcare. This inverted microscope uses a three-dimensional, laser-generated structured illumination pattern; high-speed imaging with three scientific CMOS cameras; and sophisticated computational algorithms to create three-dimensional reconstructions of fluorescent markers. The resulting image stacks offer two-fold improvement in resolution beyond the optical diffraction limit in both the image (xy) and axial (z) planes. Sample preparation is quite similar to standard methods used for laser scanning confocal microscopes. The OMX microscope is also equipped for fast widefield imaging (using solid state illumination) and for TIRF imaging.

exploded-view diagram of OMX microscope modules
OMX exploded view diagram. Image © 2012 GE Healthcare

Specifications
3D structured illumination imaging system High spatial resolution beyond the diffraction limit:
  90–130 nm lateral (depending on wavelength and optics)
  250–350 nm axial (depending on wavelengths and optics).
SIM imaging acquisition time: 5–10 sec for 2 um stack (512 × 512 pixels)
Single mode fiber light launch for speckle free imaging
Diffraction grating stage closed-loop drive controller for imaging 5 phases and 3 rotations of grating per z-plane.
laser illumination Solid state lasers at 405, 488 and 568 nm provide illumination for structured illumination and TIRF imaging modes. There is a six-position neutral density filter wheel to set the power output to the scope for each laser line. The three laser lines are combined into a single optical path using dichroic mirrors. A beam splitter sends the combined beams to two fiber optics: one for the structured illumination module, the other for the TIRF module.
solid state illumination Custom 6-color InsightSSI™ widefield illumination module. Nominal excitation wavelengths: 405, 445, 488, 514, 568, and 642 nm. Capable of simultaneous operation of 4 user-selectable wavelengths.
X, Y, Z Nanomotion stage Absolute accuracy: ±0.02%
Repeatability: ±150 nm (x, y), ±10 nm (z)
Step resolution: 10 nm (x, y), 5 nm (z)
Maximum travel: limited to 25 mm (x, y, and z). Note: to insure stage access for imaging your sample, center your 22 × 22 mm coverslip on the slide!
Laser autofocus UltimateFocus module provides Focus Assist (automated search for focal plane) and Focus Maintenance.
Transmitted light Custom Applied Precision trans-illuminator with ultra-white LED
Cameras Three custom liquid-cooled sCMOS cameras. Full specifications below.
Workstations Windows7 64-bit/Pro OS acquisition workstation.
CentOS 5.5 image processing workstation for reconstructions
Software API DeltaVision OMX Master acquisition software.
API DeltaVision OMX softWoRx image processing software.
Objectives
mag. type NA working distance
(mm)
60× (oil) Olympus U-PLAN APO 1.42 0.15
60× (oil) Olympus APO TIRFM 1.49 0.1
fluorescence filter sets for SIM and TIRF modes (laser illumination)
description excitation
peak
(nm)
emission
bandpass
(nm)
DAPI 405 420–470
AlexaFluor 488/GFP 488 500–550
AlexaFluor 568/mCherry 568 570–610
fluorescence filter sets for widefield mode (solid state illumination)
description excitation
peak
(nm)
emission
bandpass
(nm)
DAPI 405 420–470
CFP 445 455–500
AlexaFluor 488/GFP 488 500–550
YFP 514 525–570
AlexaFluor 568/mCherry 568 570–610
AlexaFluor 647/Cy5 642 650–700
Camera Specifications: pco.edge scientific CMOS camera
(custom version for Applied Precision)
Sensor CIS2051 scientific CMOS (sCMOS)
Maximum resolution 1024 × 1024 pixels (full sensor area is 2560 × 2160 pixels)
Pixel size 6.5 μm (h) × 6.5 μm (v)
Sensor size/diagonal 16.6 mm × 14.0 mm / 21.8 mm
Spectral range 370 nm–1100 nm
Dynamic range 22,000:1 (86.9 dB)
Full well 30,000 e-