Module # 2 - Microscopy Flashcards
Convex lens
Thicker at the centre, causes light rays to converge, used in microscopy.
Concave lens
Thinner at the centre, causes light rays to diverge.
Parallel light rays
Are caused by convex lenses and make distant sources converge at a specific point.
Focal point
The specific point at which light rays converge.
Focal length
The distance from the centre of the lens to the focal point. The more the lens is curved, the shorter the focal length.
Focal plane
The vertical plane the focal point lies within.
Working distance
The distance from the specimen to the objective sense. high magnification objectives have a shorter working distance.
Depth of field
The range at which an object is in focus.
When an object is located a great distance from the lens, greater than two focal lengths, the resulting image will be:
Real, smaller and inverted.
When an object is located exactly two focal lengths from the lens, the resulting image will be:
Real, same size and inverted
When an object is between one and two focal lengths from the lens, the resulting image will be:
Real, magnified and inverted (most microscopes have this property.)
When an object is located exactly one focal length from the lens, the resulting image will be:
Parallel. No image is produced.
When an object is brought within one focal length from the lens, the resulting image will be:
Virtual, magnified and erect not inverted. Can only be visualized by looking through the lens. How oculars work on a microscope.
Total magnification
Is the combination of the ocular and objective lenses magnifying an image (e.g. 40X objective used with a 10X ocular lens is a total magnification of 400X.)
Chromatic Aberration
Produces a distortion in the colours of the image, may also produce a fringe of colour around the periphery of the field of view. Occurs when each wavelength of light has a specific focal point. Alternating convex and concave lenses in the objective can correct an chromatic aberration.
Spherical Aberration
Light passing through the centre of the lens does not bend as much as those rays passing through the periphery, resulting in a blurred image. Happens most often when the magnification is higher.
Achromatic lenses
Used to correct chromatic aberrations. Least expensive option. Corrected for two colours (red and blue.)
Semi-apochromats (fluorites)
Involve incorporating fluorite into the composition of the lenses to correct for red and blue, with some correction for green light in chromatic aberration.
Apochromats
Most expensive corrected lenses. Corrects all three colours; red, green and blue.
Plan-achromats/Plan-apochromats
Correction for spherical aberrations.
Light source
Light source for a microscope. Tungsten or tungsten halogen bulbs are usually used. LED bulbs are more expensive but also have a longer lifespan. An adjustable radiant field diaphragm is located above the light source to control the diameter of the light directed onto the specimen.
Condenser assembly
Acts to focus the illuminating light onto the slide on the stage. An adjustable aperture diaphragm controls the angle of the cone of light reaching the specimen.
Kohler illumination
Used to make sure the condenser provides proper illumination onto the specimen. Results in an even distribution of light to illuminate the slide. Provides a nice even light, angle of cone of light matches the NA of the objective.
Stage assembly
The stage is where the slide is placed to be viewed, and is held in place by a slide holder. Slide controls move the stage in a horizontal plane. Focus knob controls the movement of the stage in a vertical plane. Corner scales can be used to note the particular location of an object on the slide.
Nosepieces and Objectives
Holds three to five objectives and can be rotated to bring the desired objective into the light path. The term par focal is used to describe a objective assembly that allows the different objectives to all focus the image on the same plane.
Body tube
The body of the microscope consists of an enclosed tube that physically separates the objective and ocular lenses. Tube length is an important feature in proper image formation.
Mechanical tube length
Is the distance from the top of the ocular to the objective/nosepiece junction.
Optical tube length
The distance from the optical centre of the objective lense to the focal plane of the ocular.
Oculars
Can be individually focused. The viewing field number is stamped on the ocular itself, and is expressed in mm.
Refraction
When light passes through an optically dense medium, such as glass it slows down. When it passes from a denser medium to a less dense medium, it speeds up.
Refraction is dependant on:
Angle of incidence (angle at which it enters), refractive index (density) of the mediums.
Angle of incidence
Is the angle at which approaching light strikes the surface. Light entering a more dense medium bends towards normal. Light entering a less dense medium bends away from normal.
Angle of refraction
The angle at which it leaves the surface (if able to do so.)
Critical angle
Is the angle of incidence that produces the following effect: increases and at some point the emerging ray of light will have bent far enough away from normal that the emerging ray will be parallel to the surface.
Total internal reflection
Is when the angle of incidence has refracted enough to be reflected back towards the inside of the glass. The light ray will not exit through the glass at all, but bounce back.
Refractive index
Is an expression of the density of a medium and its effect on light rays. The denser the medium, the more it will slow light rays and will have a higher refractive index.
Immersion oil
Light passing from a glass slide or coverslip into air at larger angles of incidence will be refracted away from the objective lens. Immersion oil which has the same refractive index as glass in the lenses will help control the refraction of light, preventing the loss of light rays.
Resolution
Is expressed as the minimum distance two objects must be apart in order to be seen as distinct. The smaller the separation, the better the resolution.
Resolution equation
Resolution = Wavelength/ 2 x numerical aperture
Numerical aperture
Is the mathematical expression of the ability of a lens to gather light. The higher the value, the better the resolution. Disadvantages to a high NA; working distance, depth of field and flatness of field are all decreased. Can be calculated by N.A = n sin u (n = refractive index of the medium, u = one half the angle of aperture.) For best resolution the NA of the condenser should match the NA of the objective.
Total magnification
Can be calculated by mag of ocular x mag of objective or; mag of ocular x tube length/objective focal length
Useful magnification
Magnification to a certain point. Any magnification above this results in a loss of resolution. Occurs when 1000 x NA is greater than the total magnification.
Empty magnification
Magnifying an object too much that it serves no purpose. Occurs when 1000 x NA is less than the total magnification.
Example of markings on an objective lens
Plan40/0.65
160/0.17
Planachromat, 40X Mag, 0.65 NA, 160mm tube length, 0.17 thick coverslip. Some tubes will use an infinity symbol instead of tube length. Infinity corrected microscopes allow for the introduction of other components into the light path.
