First steps in exploring epi DIC (on a Zeiss Photomicroscope)

by David Walker, UK

The Zeiss documentation states that the Epiplan Pol range of objectives are used for DIC when each are matched with the correct INKO prism.

Right: The Zeiss Epiplan 4/0.10 Pol objective disassembled from its INKO prism and mount. My example is labelled for DIC; I'm not clear whether DIC labelled examples differed in any way to the Pol. This INKO mount is labelled as being also suited for the 8/0.2 objective.

The bar on the prism only translates the prism in the x-y plane to change the amount of so-called bias retardation.

(I believe some makers e.g. Reichert used a single prism for their epi DIC objective range and the prism block also had facilities to adjust the prism vertically to set the correct plane with respect to the back focal plane of each objective.)

Right: The Zeiss INKO Epiplan 40/0.85 DIC disassembled from its prism and mount. On this objective the prism is translated by rotating the knurled ring on the prism mount.

Note that the two objectives shown use different sized threads in the dovetail mount (RMS and M24).

Right: The prism of the 4X objective between crossed polars. The typical central black bar of zero interference and higher order spectra of a Nomarksi modified Wollaston prism are shown. When first installed on the microscope, the correct orientation of the black bar (45° to the E-W axis between crossed polars) should be checked using the Optovar or a phase telescope as described in the manuals. On my example it was not initially 'set' correctly in its dovetail mount. 

Right: A Zeiss Photomicroscope III set up for incident / epi DIC. Mine has the single objective epi head IIC, a turret model was available. The 4X objective is in use studying a cube of iron pyrites. Placing small items for study on a blank microscope slide allows the mechanical stage controls to be retained.

A polariser sits in the lamp optics to right and an analyser is fitted in the upper slot (a variable one show here isn't vital if the polariser can also be rotated manually to set full extinction).

On the Photomicroscope, a lever on limb allows quick change from transmitted to incident light use. Unfortunately the epi condenser for the Photomicroscope shown fitted, seems very hard to source (the Universal uses a different design), it took me five years of searching to find one. Although prior to that, a right angle prism sitting on a card mount within the port worked well. The Zeiss side bar lamp support that fits in epi port may also work but the rear fittings of the epi head need removing to allow clearance for it.

The reflector in the epi head is quickly interchangeable (the drum with knurled nut just above objective and facing to rear. My example has the H-Pl reflector (a half silvered mirror) which Zeiss do state is suitable for DIC but remark that the H-Pl Pol reflector (a double reflecting design with both a half-silvered and fully silvered mirror) is better because the former suffers from some depolarisation.

The theory and general practice of reflected light DIC is very well explained and illustrated on the Molecular Expressions website. Its implementation on Zeiss 160 mm tube length microscopes is described in the Zeiss literature of the time, most of which is available online (see Resources).

Essentially the typical subjects should be reflective, flat and usually opaque. epi DIC can reveal fine changes in surface relief and surface composition. Unlike in transmitted DIC, the 3D imagery if correctly interpreted is an indication of the surface topography.

One of the classic and most illustrated examples of its professional use is in metallurgy of prepared polished, sometimes etched, metals and alloys. I don't have access to these type of samples unfortunately at present.

The technique is also widely used for studying electronic components such as silicon chips during and after manufacture.

Right. Some typically suitable subjects from my own studies. Minerals with essentially flat reflective such as the faces of iron pyrites, electronic components (a B/W and colour sensor from old handheld scanners, the electronic plate from an old inkjet cartridge and an exposed EEPROM chip are shown). Engineered metals such as coins, watch parts etc.

Exposing the chips of integrated circuits can be tricky and needs care especially if mounted in hard, brittle ceramic substrates. Trace toxic elements may also be present.

Examples of subjects that were not suitable are shown right. Paper and textiles have plenty of surface texture, can be essentially flat but unsuited if not reflective. Selected areas of paper or plastic goods which are reflective can work e.g. the hologram areas of credit cards, security features on bank notes etc.

The uncovered coal slide shown had a matte unpolished surface and gave poor images. Polished examples may work better.

Although the right-hand coal section was uncovered and did contain fossil micro structure, it had a none reflective, coarsely ground surface.

Iron pyrites face of cube, 4X objective, horizontal field width (HFW) 1.7 mm. To the eye the faces are essentially flat.

Top left: analyser omitted, i.e. effectively axial epi illumination without DIC. The crystal boundaries are clearly seen but no clear relief information.
Top right: epi DIC with bias retardation minimal, i.e. prism set centrally. Although not as clear on the image, seen visually, there is a distinct three dimensionality with a 'cliff type' boundary across the diagonal where there is a marked change in surface relief.
Bottom right: epi DIC with strong bias retardation set by moving prism laterally. The changes in relief are highlighted in colour.

Right: 4X objective, 2X Optovar. HFW 1.1 mm. epi DIC. Corner detail from the digital sensor on an old monochrome security camera.

The imaging area is top left, supplementary circuitry and what may be alignment marks for manufacture are show on the periphery.

The sensor still has its dust plate on, but the long working distance of the 4X objective could focus through to the sensor. This was not possible with the 40X objective. There was evidence of contrast loss because of the dust plate, but levels adjustment in image editing software corrected for this.

Right: 4X objective, 2X Optovar. HFW 1.1 mm. epi DIC.

Mint Canadian Trade Dollar (Niagara Falls). Area of the highly polished 'silver' coin base and some dimples around the edge. epi DIC is not designed to study gross relief found on subjects like this coin, off-axis incident lighting is adequate for this. epi DIC is more suited for revealing the finer surface relief e.g. in this case on the coin base, which to the eye is near featureless and highly polished.

Right: 4X objective, 1.6X Optovar. HFW 1.35 mm. epi DIC.

Edge detail from the sensor of an old handheld scanner, shown earlier as the thin strip in the collage of suitable subjects. The sensor area is out of shot, the ancillary circuitry and likely alignment / calibration marks are seen.

Right: 40X objective HFW 0.19 mm. epi DIC.

A powerful feature of transmitted DIC is its ability to optically section, which can be very useful e.g. for studying important features of microorganisms such as surface cilia on some protists.

Optical sectioning also applies to epi DIC and can be useful in industry for studying selected complex layers of an integrated circuit.

The image right shows circuit detail of the monochrome handheld scanner sensor. One layer of tracks is picked out in focus, a lower layer is not in focus.

The optical sectioning can be a disadvantage in photography where stacking may be needed to capture all the features of a subject in a single image.

Right: 40X objective HFW 0.19 mm. epi DIC.

Circuit detail of the monochrome sensor from a handheld scanner.

Right: 40X objective HFW 0.19 mm. epi DIC.

Right: An exposed EEPROM chip. The block of programmable memory which is erasable by UV light is seen bottom left.

Experimenting with both the rotation and extend of bias retardation can reveal which combination best shows the features of interest.

Right: 40X objective HFW 0.19 mm. epi DIC.

The ability of epi DIC to reveal the finest surface topography in subjects which seem almost featureless to the unaided eye, is perhaps best illustrated with this familiar household example of unused aluminium kitchen foil cut straight from the roll.

The right-hand image shows the polished side and reveals the marks during manufacture.

Right: 40X objective HFW 0.19 mm. epi DIC.

Aluminium foil on the matte side showing the complex disordered surface relief associated with this finish.

To what extent a surface becomes unsuited for epi DIC because it is either too matte and/or its colour, I'm uncertain as the matte side of the foil has low reflectivity. A matte coal section with microfossils tried showed very little under epi DIC (although of course is much darker as well as having little reflectivity).