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DARKFIELD MICROSCOPY

MICROBIOLOGY AND PARASITOLOGY


DR.MYLENE CU
BY
MICHAEL C. AGUILAR BSPH3
DARKFIELD MICROSCOPY

• DARKFIELD MICROSCOPY IS A SIMPLE AND POPULAR METHOD FOR RENDERING UNSTAINED


AND TRANSPARENT SPECIMENS CLEARLY VISIBLE. GOOD CANDIDATES FOR DARKFIELD
OBSERVATION OFTEN HAVE REFRACTIVE INDICES VERY CLOSE IN VALUE TO THAT OF THEIR
SURROUNDINGS AND ARE DIFFICULT TO IMAGE WITH CONVENTIONAL BRIGHTFIELD
TECHNIQUES. AS AN EXAMPLE, SMALL AQUATIC ORGANISMS, OOCYTES, AND CELLS IN TISSUE
CULTURE HAVE A REFRACTIVE INDEX RANGING FROM 1.2 TO 1.4, RESULTING IN A NEGLIGIBLE
OPTICAL DIFFERENCE FROM THE SURROUNDING AQUEOUS MEDIUM (REFRACTIVE INDEX OF
1.3). THESE AND SIMILAR SPECIMENS ARE IDEAL CANDIDATES FOR OBSERVATION WITH
DARKFIELD ILLUMINATION TECHNIQUES.
DARKFIELD MICROSCOPY

• ILLUMINATION OF SPECIMENS BY DARKFIELD REQUIRES BLOCKING OUT OF THE CENTRAL LIGHT


RAYS ALONG THE OPTICAL AXIS OF THE MICROSCOPE, WHICH ORDINARILY PASS THROUGH AND
AROUND (SURROUNDING) THE SPECIMEN. BLOCKING THESE LIGHT RAYS ALLOWS ONLY THOSE
OBLIQUE RAYS ORIGINATING AT LARGE ANGLES TO STRIKE THE SPECIMEN POSITIONED ON THE
MICROSCOPE STAGE. IN A COMPOUND MICROSCOPE EQUIPPED WITH A SIMPLE CONDENSER
SYSTEM, THE CONDENSER (ABBE-STYLE) TOP LENS IS SPHERICALLY CONCAVE, ENABLING LIGHT RAYS
EMERGING FROM THE SURFACE IN ALL AZIMUTHS TO FORM AN INVERTED HOLLOW CONE OF
ILLUMINATION HAVING AN APEX CENTERED IN THE SPECIMEN PLANE. IF NO SPECIMEN IS PRESENT
ON THE STAGE, AND THE NUMERICAL APERTURE OF THE CONDENSER IS GREATER THAN THAT OF
THE OBJECTIVE, THE OBLIQUE RAYS CROSS AND MISS ENTERING THE OBJECTIVE FRONT LENS
BECAUSE OF THEIR OBLIQUITY. THE FIELD OF VIEW WILL APPEAR DARK.
DARKFIELD MICROSCOPY

• 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