2. Microscopy Flashcards
What are focal points and focal lengths of convex lenses?
Convex lenses cause parallel light rays from a distant source to converge at a specific point termed the focal point. The focal length is the distance from the centre of the lens to the focal point.
What does the shape of a lens affect?
Smaller rounder lenses tend to have higher magnification and shorter focal lengths
Working distance is the distance from the specimen to the objective lens. High mag have shorter working distances and narrower depth of field (range of focus)
What does the resulting image look like if the object is located more than 2 focal lengths away?
It will be real, smaller, and inverted
What does the resulting image look like if the object is located 2 focal lengths away?
The image will be real, the same size, and inverted
What does the resulting image look like if the object is located between 1 and 2 focal lengths away?
The image will be real, magnified, and inverted
What does the resulting image look like if the object is located exactly 1 focal length away?
The light rays emerge parallel and image can no longer be focused on a screen
What does the resulting image look like if the object is located within 1 focal length away?
The image will be virtual, magnified, and erect (not inverted)
Can only be seen by looking through the lens, the image forms on the same side of the lens as the object itself
What is total magnification?
When you multiply the magnifications of the two lenses (objective and ocular)
E.g. 40x X 10x = 400x
What is a convex lens?
Causes light rays to converge
Magnification is related to the curvature of the lens
Looks like an eye shape
What are two lens aberrations?
Chromatic aberration - shorter wavelengths come to focus closer to the lens than longer wavelengths, causing colour distortion and a fringe of colours around the periphery of the field.
Spherical aberration - light passing through lens does not bend as much as those passing through the periphery, causing a blurred image on the outer edges.
How do you correct spherical aberrations?
Use a combination of convex and concave lenses
Termed “plan-“
What is the amount of refraction dependent on?
Angle of incidence Refractive index (density)
What is the angle of incidence?
The angle at which approaching light strikes the surface
Light entering more dense mediums bends towards normal , air to glass
Light entering less dense mediums bends away from normal , glass to air
What is the refractive index?
Is an expression of the density of a medium and its effect on light rays
Grown glass = 1.52
Immersion oil = 1.52
The denser a medium the higher the refractive index
What is immersion oil? What does it do?
Immersion oil can help control the refraction of light preventing the loss of light rays, maintaining image sharpness and brightness
What is resolution?
Is expressed as the minimum distance two objects must be apart in order to be seen as distinct
What is resolution dependent on?
Wavelength of light used
Numerical aperture of the lens (ability of a lens to gather light)
Resolution = wavelength / 2 NA (numerical aperture)
What is numerical aperture? What does it do?
Ability of a lens to gather light
Higher the numerical aperture, the better resolution
Oil lens NA = 1.25
The wider a cone of light gathered by the lens, the higher the NA
The NA of the condenser should match the NA of the objective lens to obtain the best resolution
What is Useful vs. Empty magnification?
Useful magnification occurs when 1000 X NA is greater than the total magnification, results in loss of resolution
Empty magnification occurs when 1000 X NA is less than the total magnification, results in too much magnification
What is the procedure for setting Kohler?
- Close radiant field diaphragm
- Open condenser aperture
- Raise condenser to highest position
- Lower condenser until diaphragm image is in sharp focus (small circle crisp edges)
- Centre image using condenser centering screws
- Open field diaphragm until field is filled with light (stop when edge is just out of field of view)
- Remove ocular and close condenser aperture until 3/4 of circle is light
- Adjust brightness
What region of the light spectrum is visible?
380nm to 780nm
What can light waves be described as?
Wavelength
Amplitude
Frequency
Velocity
What are the key components of a compound microscope?
Light source Radiant field diaphragm Condenser assembly Aperture diaphragm Stage assembly Nosepiece and objectives Body tube Oculars
What does parfocal mean?
The object stays in focus even when the objective is switched, requires only minimal fine focusing
