Stereo Microscope Imaging: Exploring Advanced Illumination Techniques
In compound microscopes, an illumination parallel to the optical axis is the standard. A lot of hardware is implemented to ensure this axial illumination: Center-adjustable Field and Aperture diaphragm, centerable light rings for Phase contrast and Darkfield for transmitted light, center-adjustable objectives and/or centerable rotatable stages in Polarization microscopes. With all these microscopes we are working on 2-dimensional samples.
In stereo microscopy, a 3D image from a 3-dimensional sample is our ambition. Different viewing angles for both eyes create different images on the retina of the left and the right eye. Our brain is going to process both images. This concept is best comprehensible in the Greenough construction of a traditional stereo microscope: two complete separate beam paths from objective to the eyes, mounted in a relative angle of 11°-16°. But even in the modern, more flexible CMO (Central Main Objective) concept a different viewing angle is realized.
Greenough type / CMO (Galilean) type
OBLIQUE ILLUMINATION:
In stereo microscopy, this is the standard illumination. The following setup is well-known. This setup delivers a flexible illumination for three-dimensional opaque samples. The upright (erect) image helps in cases where manipulation with tweezers or preparation needles is necessary. Depending on the illumination direction and angle, a shadowing is intended to emphasize the topography of the sample surface.
Cold light source with double light guide
RING LIGHT:
A ring light also has a defined illumination angle differing from the optical axis, although the illumination mostly is symmetrical for a shadow-free image of the sample.
Ring light
If we compare both illuminations in the following pictures, note how the outline of the continents and the lines of longitude on the coin surface are emphasized by an oblique illumination
Oblique illumination (8X visual mag.) | Ring light illumination (8X visual mag.)
To work out the tiniest topography phenomena a slit-shaped illumination opening at the end of a round light guide (cross-section converter) supplied by a cold light source is a recommendation. An almost horizontal illumination angle is necessary for best results. A 20 Euro bank note with its embossed printing for blind people (braille!) gives a traceable example.
20€ bill with Ring light, 10X mag. | 20€ bill Horizontal illumination, 10X mag
TRANSMITTED LIGHT APPLICATIONS:
Here the stereo microscope of course requires an at least partly transparent sample. In case of a built-in illumination, a shadowing, underlining the surface topography needs a clever concept. A necessary oblique illumination may be realized by a tiltable reflector in the microscope base.
Base of SMZ171TLED with tiltable reflector, cover of frosted glass removed
A small and effective piece of hardware for almost no money. The glued bottom of a transparent plastic bag is shown below.
| Brightfield illumination | Oblique illumination |
Protein crystals in a nutritive fluid, transparent multilayer foils (packaging films from the kitchen), scratches on a watch glass during the revision procedure, a native cell culture in a petri dish: samples worth to be evaluated by an oblique illumination.
ACT (Advanced Contrast Transmitted):
The most sophisticated and flexible transmitted illumination for the microscopes worldwide is only available from a specialist of light. The ACT (Advanced Contrast Transmitted) base offers Brightfield, Darkfield, single controllable segments and a variable, movable (right - left) slit for relief contrast. Perfect flexibility, unfortunately for the price of an international mid-class microscope.
Light exit of ACT base. Note the movable slit (relief contrast), the ring of LEDs (Darkfield and segments!) and the diffusing lens on top of the central Bright field illumination.
A RECOMMENDATION:
Drive down the general light in your lab if you like to work out the effect of a dedicated illumination. You’ll be surprised.
CONCLUSION:
Illumination always is the key issue in stereo microscopy. Make up your mind about what you need to see and adapt your illumination: Incident, transmitted, oblique or +/- axial illumination. Play around, find your personal solution for your samples.