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• WHEN A TRANSPARENT SPECIMEN IS PLACED ON THE GLASS MICROSCOPE STAGE AND OBSERVED UNDER
DARKFIELD ILLUMINATION, THE OBLIQUE LIGHT RAYS CROSS THE SPECIMEN AND ARE DIFFRACTED, REFLECTED,
AND/OR REFRACTED BY OPTICAL DISCONTINUITIES (SUCH AS THE CELL MEMBRANE, NUCLEUS, AND INTERNAL
ORGANELLES) ALLOWING THESE FAINT RAYS TO ENTER THE OBJECTIVE. THE SPECIMEN THEN APPEARS BRIGHT
ON AN OTHERWISE BLACK BACKGROUND. IN TERMS OF FOURIER OPTICS, DARKFIELD ILLUMINATION REMOVES
THE ZEROTH ORDER (UNSCATTERED LIGHT) FROM THE DIFFRACTION PATTERN FORMED AT THE REAR FOCAL
PLANE OF THE OBJECTIVE. THIS RESULTS IN AN IMAGE FORMED EXCLUSIVELY FROM HIGHER ORDER
DIFFRACTION INTENSITIES SCATTERED BY THE SPECIMEN, AND IS ALSO RESPONSIBLE FOR THE MAIN LIMITATION
OF DARKFIELD OBSERVATION. BECAUSE THE IMAGE IS COMPOSED ENTIRELY FROM SCATTERED LIGHT FROM THE
SPECIMEN, IT IS RICH IN GLARE AND CAN EVEN BE DISTORTED TO VARYING DEGREES, SO IT CANNOT BE
CONSIDERED A FAITHFUL GEOMETRICAL REPRODUCTION OF THE SPECIMEN.
DARKFIELD-BRIGHTFIELD
SAMPLE OF DARKFIELD MICROSCOPY
REFERENCE
• HTTPS://WWW.MICROSCOPYU.COM/TECHNIQUES/STEREOMICROSCOPY/DARKFIELD-
ILLUMINATION