by Lin Yangchen

The "Large Micron Collider", a state-of-the-art Nikon Eclipse Ni-E almost 100cm high, bombarding a small sample of fibres with high-energy photons traveling at the speed of light. At the top of the superstructure is a Nikon DS-Ri2 35-mm-format full-frame camera. The brown bottle contains high-viscosity, low-autofluorescence immersion oil of refractive index 1.515 (23° C), specifically formulated for Nikon objectives. The conical flask dispenses 100% ethanol for dissolving the oil after use.

The computerized turret with the 20× Plan Apochromatic objective selected.

The mythical 100× Plan Apochromatic VC (violet-corrected) oil-immersion objective, which corrects for violet aberration on top of the red, green and blue correction of apochromats. Only this objective is capable of eliminating purple fringing.

Köhler illumination was used to ensure even lighting and optimal resolution. For each objective, the specimen was first brought into focus. Then the field diaphragm was stopped down and the condenser was focused to get a sharp image of the field diaphragm in the ocular together with the specimen. The condenser was centered using two fine-bore hex wrenches controlling the x- and y-axes until the field diaphragm appeared centered in the field of view. The field diaphragm was then opened until its blades lay just beyond the image circle. Then one of the oculars was removed to view the condenser diaphragm on the back focal plane of the objective. The condenser aperture was adjusted to limit the light cone to about 80% of the objective aperture.

For fibre analysis, the microscope was configured for Nomarski interference contrast, which extracts high-resolution detail from transparent unstained biological material. The optical wizardry starts with the polarization of light rays into two orthogonal directions by a Wollaston prism, a compound prism made up of two birefringent crystals of materials such as quartz or calcite. The differently polarized rays are deliberately passed through the specimen at a slight offset from each other. On exit from the specimen they are rotated to the same direction of polarization, and aligned so that there is no more offset. At this moment, rays that passed through adjacent points in the specimen undergo interference. If the adjacent points were of different density or topography, the destructive or constructive interference will darken or brighten that location in the image relative to cases where adjacent points did not differ.

Photomicrographs were taken with a Nikon DS-Ri2, which has a 35-mm-format full-frame complementary metal-oxide-semiconductor sensor with a higher resolution than the microscope objectives. The sensor was colour-corrected by “zeroing” it to record the halogen light source through a blank microscope slide as pure white. Photomicrographs may however appear yellower than expected if the transmitted light passes through browned gum or hinge remnants. Where necessary, the extended-depth-of-field algorithm in NIS-Elements software was used for motorized focus stacking at sub-micrometer scale.

I am grateful to Benedict Sim, Ernest Cheah, Clement Khaw and Goh Wah Ing for discussions and technical assistance, and to the Nikon Imaging Centre at the Singapore Bioimaging Consortium for the facilities that made this research possible.


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