Dark field microscopy enhances the contrast of transparent and unstained specimens by illuminating them with light that does not enter the objective lens unless it is scattered by the specimen.
This creates a bright image of the specimen against a dark background, making normally invisible structures visible.
Procedure for Dark Field Microscopy
Preparation of the Microscope:
Dark Field Condenser: Install a dark field condenser that directs light obliquely onto the specimen. High-power objectives may require an oil immersion condenser to achieve optimal results.
Specimen Preparation:
Slide Preparation: Place a drop of the specimen on a clean slide and cover it with a coverslip to create a thin, even layer suitable for observation.
Mounting Media: Use media that do not interfere with the specimen’s refractive properties or add background fluorescence.
Microscope Settings:
Adjusting the Illumination: Close the iris diaphragm to produce a hollow cone of light and align the condenser to ensure that direct light does not enter the objective.
Objective Selection: High numerical aperture objectives are necessary to capture scattered light effectively.
Observation:
Focusing: Initially focus on the specimen under low magnification, then switch to higher magnification and adjust the fine focus.
Image Enhancement: Adjust the light intensity to optimize the brightness of the specimen against the dark background.
Documentation:
Image Capture: Use a sensitive camera to record images, as the light levels in dark field microscopy are generally low.
Applications
Microbiology: Detecting thin organisms like spirochetes (e.g., Treponema pallidum) that are difficult to see under bright field illumination.
Cell Biology: Observing the motility and behavior of live cells and organelles.
Aquatic Biology: Examining plankton and other transparent aquatic organisms.
Advantages
Visualizes specimens that are invisible under bright field microscopy.
No need for staining or complex preparation steps.
Suitable for live specimens, maintaining their natural state.
Limitations
Low light levels necessitate sensitive detection equipment.
Not ideal for quantitative measurements due to varying light scattering properties.
Artifacts, dust, or debris can scatter light, potentially leading to misleading images.