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Unique System features

  • Simultaneous capture of image z-stacks comprised of 9 separate, aberration- and chromatic-corrected focal planes
     

  • Perfect temporal alignment across each z-stack eliminates ambiguity inherent to sequential z-sectioning
     

  • Rapid acquisition of z-stacks, up to 35 stacks per second
     

  • Simultaneous two color imaging (green and red channel)

  • Stage top incubator chamber for temperature, humidity, and CO2 control

System specifications

MFM Gratings

  • 100x/1.45, 515nm emission, 400nm Z-plane separation

  • 100x/1.45, 600nm emission, 400nm Z-plane separation

  • 100x/1.45, 680nm emission, 400nm Z-plane separation

Lasers

  • 405 nm

  • 488 nm

  • 561 nm

  • 635 nm


Dichroics (used with LED white light source, single wavelength illumination) 

  • DAPI-1160B (Semrock)

  • CFP-2432C (Semrock)

  • FITC-3540C (Semrock)

  • TRITC-B (Semrock)

  • Cy5-4040C (Semrock)


Dichroic (used with laser excitation, multi wavelength illumination)  

  • Di01-R405/488/561/635 (Semrock)


Filters

  • FF01-525/45 (Semrock)

  • FF01-593/40 (Semrock)

  • FF01-676/37 (Semrock)

   
Objectives

  • 100x NA 1.45 Oil (Nikon) - for MFM imaging

  • 60x NA 1.4 Oil (Nikon) - for wide field imaging only

  • 60x NA 1.2 Water (Nikon)  - for wide field imaging only

  • 20x NA 0.75 Air (Nikon) - for wide field imaging only

Camera

  • iXon3-DU897E EMCCD (Andor Technologies), one for each color channel

System Limitations

  • The field of view in multi-focal mode is limited to ca. 20x20 microns (100x magnification).  Larger field of view is available in single focal plane mode.
     

  • Sample is illuminated in epi-fluorescent mode (i.e. illuminated along Z axis), which can cause issues with out-of-focus fluorescence beyond desired 2D stack depth.
     

  • Epi-fluorescent illumination may require optimization to further reduce fluorophore photobleaching or phototoxicity.
     

  • Ability to distinguish 9 focal planes relies on the multifocal grating, which causes a 20-25% reduction in detection efficiency compared to typical widefield fluorescent techniques.
     

  • Spatial resolution is diffraction limited, and dependent on microscope configuration (i.e. objective and excitation/emission wavelength

Suggested Reading

  1. Abrahamsson, S., Chen, J., Hajj, B., et al. (2013). Fast and sensitive multi-color 3D imaging using aberration-corrected mutli-focus microscopy. Nat Methods (1). 10, pp 60-63