Optical microscopy

Investigation using a light-based optical microscope. Resolution, specimen preparation and interpretation constrain what can be established from an image.

Polarized light microscopy uses controlled polarization of illumination and detection to reveal optical properties of a specimen. A polarizer before the specimen sets the incident polarization; a second polarizer, called the analyzer, selects which outgoing component reaches the image. With the two pass axes perpendicular, an ideal empty optical path is dark. A specimen can alter this result through birefringence or polarization-dependent absorption.^[ref]

For an ideal nonabsorbing birefringent plate between crossed polarizers, the transmitted light intensity iswhere $I_0$ is the intensity after the first polarizer, $\theta$ is the angle of a plate axis to its pass axis, $d$ is thickness, $\Delta n$ the birefringence, and $\lambda$ the vacuum wavelength. The phase retardation $\delta$ comes from different optical path lengths. With $\delta =\pi$, the plate is bright at $\theta =45^{\circ }$ and dark at $\theta =0$: rotating a specimen changes contrast without removing material.^[ref]

This sensitivity is useful for oriented biological materials. A2018 ivory study combined polarized-light observations with X-ray scattering and scanning electron microscopy to investigate mineralized collagen architecture. Brightness alone does not uniquely identify a substance: orientation, thickness, wavelength and optical properties all contribute.^[ref]

Aligned two-dimensional sections constrain the three-dimensional structure of a specimen. For parallel sections of known spacing $\Delta z$, the Riemann sum ${\sum }_i{A}_i\Delta z$ approximates volume. Distortion, missing sections and alignment errors constrain the reconstruction; the volume estimate alone does not determine the identity or developmental fate of each visible structure.