ImageXpress Micro Confocal

Both microscopes are equipped with:

  • Widefield imaging options for high-throughput screening
  • High-quantum efficiency sCMOS detection camera with a large field of view

Molecular Devices ImageXpress Micro Confocal

IXMC with stacker

  • An inverted fluorescent microscope with a transmitted light option for label-free imaging 
  • Features a 60 um pinhole single spinning disc for confocal imaging
  • Integrated with a PAA GX robot arm and 45-plate hotel suite

Molecular Devices ImageXpress Micro Confocal Laser

IXMC LZR

  • An inverted fluorescent microscope with a transmitted LED light (red 630nm)
  • Features 2 spinning discs for confocal imaging:
    • 60 um pinhole with a 50 um slit
    • 42 um pinhole 
  • Equipped with a Lumencor Celesta light engine, a solid-state laser light source with up to 1 watt of output power

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Designing an Image-Based Screen

Assay plates:

Black-walled, clear-bottom (plastic or glass) microplates are used for image-based screens. A common 384-well plate type is Corning #3764 (previously #3712).

Seeding density:

Test a variety of cell densities under the conditions you plan to use for the screen. Image analysis is often improved when the cells are sub-confluents. Depending upon cell size and length of assay, a starting range may be 500 to 2,000 cells per well.

Imaging live cells:

This is possible when using the Acumen. Remember that the microscope stage is not heated and your cells will be at room temperature during imaging.

Treating plates after imaging:

After processing cells for imaging, fill your wells with PBS to prevent the cells from drying out and to reduce background noise. Some investigators include an anti-fungal agent (eg sodium azide) for long term storage at 4oC.

Selecting fluorophores:

Please make sure the fluorophores you select do not conflict with the filter sets on the screening scope. See details for each microscope. For high throughput screens, antibodies are frequently used at higher concentrations to decrease acquisition time.

Plate Seals:

Clear and foil microplate seals can significantly increase background fluorescence to the point where images may be unusable. Black opaque plate seals are ideal for fluorescence imaging. Microplates must be sealed prior to imaging in the microscope to protect the equipment. 

ImageXpress C Objective Options

There are 4 objective positions within the IXM-C, the standard instrument configuration has the 4x, 10x, and 20x objectives in the instrument (bold in the table below). If you are interested in using one of the objectives not installed in the instrument, please contact the screening facility with your request before your scheduled usage time. 

  • The 2x objective captures the whole well of a 96-well microplate.
  • The 4x objective captures the full well of a 384-well microplate, or four 1536-microplate wells at a time. 
  • The 10x objective captures a whole well in a 1536-well microplate.  
Magnification Objective Type NA (um) WD (mm)
2X Plan Apo λ 0.10 8.5
4X S Fluor 0.20 15.5
4X Plan Apo λ 0.20 20
10X Plan Fluor 0.30 16
10X Plan Apo 0.45 4
20X S Plan Fluor * 0.45 8.2-6.9
40X Plan Fluor 0.60 3.7-2.7

* This objective has a correction collar

ImageXpress C Laser Objective Options

There are 4 objective positions within the IXM-C laser, the standard instrument configuration has the 4x, 10x, and 20x objectives in the instrument (bold in the table below). If you are interested in using one of the objectives not installed in the instrument, please contact the screening facility with your request before your scheduled usage time.

  • The 4x objective captures the full well of a 384-well microplate, or four 1536-microplate wells at a time
  • The 10x objective captures a whole well in a 1536-well microplate
Magnification Objective Type NA (um) WD (mm)
4X Plan Apo λ 0.20 20
10X Plan Apo λ 0.45 4
20X S Plan Fluor 0.45 8.2-6.9
20X Plan Apo 0.75 1
40X S Plan Fluor 0.60 3.6-2.8
40X Plan Apo λ * 0.95 0.25-0.17
60X Plan Apo λ 0.95 0.20-0.11

* This objective has a correction collar

ImageXpress C Filters

For excitation wavelengths, instead of using traditional excitation filters or lasers, the IXM-C uses a power-optimized solid-state light source. In addition there is a 5 position dichroic wheel, and an 8 position emission filter wheel. 

Fluorophores Excitation (nm) Emission (nm) Dichroic (nm)
DAPI, Hoechst 380-420  417-477 409
FITC, GFP 440-500 516-556 506
CY3, TRITC 490-550 573-613 562
Texas Red, mCherry 530-610 604-644 593
CY5 610-660 672-712  660

ImageXpress C Laser Filters

For excitation wavelengths, the IXM-C laser uses the Lumencor Celesta light engine, a solid-state laser light source with up to 1 watt of output power. High output laser excitation reduces exposure times by up to 75%. In addition, there is an 8-position emission filter wheel that is supported by a 5-position dichroic wheel that features a pentaband dichroic beamsplitter spanning multiple wavelengths indicated in bold.

Fluorophores Excitation (nm) Emission (nm) Dichroic (nm)
DAPI, Hoechst 405 452/45 421
CFP 446 483/32 462
FITC 477 520/28 488
YFP 520 562/40 542
TRITC 546 595/31 564
CY3, Texas Red 546 624/40 593
CY5 638 692/40 656
CY7 749 819/44 774