2) Stain A substance that binds tightly to only specific components, thereby causing those components to absorb or scatter light differently than other components of the cell. Contrast
Understanding the Condenser A condenser is a lens that serves to concentrate light from the illumination source that is in turn focused through the object and magnified by the objective lens. The condenser has two iris diaphragms that are used to achieve uniform illumination, and improve contrast and resolution . This process is called “ setting Köhler ”: Field Diaphragm Controls the positions within the specimen plane that are illuminated. Aperture Diaphragm Controls the angles of light passing through the specimen plane, thus affecting contrast and the resolution . August Köhler (1866-1948)
What’s the highest magnification we can get with the microscopes in our lab? • Ocular = 10X • Objective = 100X • Total Magnification = ? • A. 10X B. 100X C.1000X
Resolution .. • Eye can’t distinguish between objects 0.1mm apart. • Two points cannot be seen separately if the wavelength of the viewing light is greater than 2X the distance between the points. • e.g., wavelength 600 nm, maximum resolution can’t be less than 300 nm
Maximum Resolution? • What is the maximum resolution of a light microscope if we use blue light (400 nm) to illuminate the subject? • Can’t resolve detail finer than about 2μm (200 nm).
Abbe’s Equation defines resolving power of a microscope d = 0.612 λ n sin θ
Values for Abbe’s Equation • 0.612 = constant • λ = wavelength of visible light (400-700 nm) • n = refractive index of medium between objective and specimen (air = 1.0, oil = 1.4) • 2 θ = 180 o • θ = 90 o • Numerical aperture (NA) = n sin θ
So… d = 0.612 λ n sin θ • Becomes d = 0.612 λ N A
d =(1.22 ) λ / ( N A objective + NA condenser ) *Manipulation of condenser aperture changes the angle of the cone of light entering specimen. Closing aperture lowers the NA of the condenser *If NA of condenser is low, it may limit the resolution of the objective lens.
Manipulation of aperture influences the resolving power and contrast.
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- Spring '08
- Optical microscope, Telescope, Compound Light Microscope