Differential Interference Contrast (DIC)

General remarks

DIC allows observation of unstained specimens by generation of image contrast
Structures of interest must have different refractive indices (r.i.).

Beam path for DIC microscopy

Keep in mind: there are dedicated DIC prism / objective combinations. Check appropriate combinations from the Objective list of the manual or the Facility web site.

1. Upright microscope in laser scanning mode (from top to bottom)

Light source (laser)
No Polarizer (laser has to provide correctly polarized light)
DIC prism at objective
Objective
Sample
Condenser front lens
DIC prism (condenser)
Analyzer (polarizing filter)
Transmission detector (PMT)

2. Inverted microscope in halogen transillumination mode (from top to bottom)

light source (halogen lamp)
Polarizer (polarizing filter)
DIC prism (condenser)
Condenser front lens
Sample
Objective
DIC prism at objective
Analyzer (polarizing filter)
Detector (camera, eye)

3. Upright microscope in halogen transillumination mode (from top to bottom)

Detector (camera, eye)
Analyzer (polarizing filter)
DIC prism at objective
Objective
Sample
Condenser front lens
DIC prism (condenser)
Polarizer (polarizing filter)
light source (halogen lamp)

4. Inverted microscope in laser scanning mode (from top to bottom)

Transmission detector (PMT)
Analyzer (polarizing filter)
DIC prism (condenser)
Condenser front lens
Sample
Objective
DIC prism at objective
no Polarizer (laser has to provide correctly polarized light)
light source (laser)

Prerequisites for DIC observation / recording

1.Koehler alignment of illumination path (transmitted light mode)

If objective iris aperture is present: check if open)
Focus a sample in transmitted light mode
Close field aperture of illumination (first aperture after lamp)
Focus field aperture (sharp contrast)
Center field aperture
Open field aperture (until aperture is no more visible)

2. DIC alignment

Remove both DIC prisms from the illumination path
Insert polarizer and analyzer into the illumination path
Rotate polarizer or analyzer to minimize brightness
Insert appropriate objective and condenser DIC prisms
Adjust the condenser aperture roughly to the objective's aperture

DIC observation / recording

1. Visual inspection / CCD recording

Fine tune objective DIC prism angle (using the dedicated screw or rotation facility at the objective's DIC prism)
Switch the slider (tube slider at the eyepiece) to the appropriate beam path setting for CCD recording
Adjust camera gain / brightness etc.

2. Laser scanning

After DIC alignment (above), remove the polarizer (the laser illumination beam path should provide the correct laser polarization direction at the objective DIC prism. (In case of questions, ask your administrator)
Set the beam path to the transmission photomultiplier tube (PMT) recording mode (for details, see the respective microscope's manual)
Adjust PMT offset / gain etc.

Note: with some microscopes, there may be problems in case of simultaneous DIC / fluorescence recording. Check the respective microscope's manual). It is important to match the immersion medium between sample and condenser to the immersion medium between lens and sample.