Module 2: Microscopy Flashcards
Convex
Positive
causes light rays to converge
Magnification is related to curvature of lens surface
Concave
Negative (thinner in center)
Causes light rays to Diverge
Not used for magnification
Focal Point
point at which parallel light rays converge after passing through a convex lens
Focal Length (F)
distance from the centre of the lens to the focal point
Focal Plane
vertical plane the focal point lies within
Lens with larger diameter and flatter lens faces
result in longer focal lengths
Smaller lens with rounder lens faces
Result in shorter focal lengths (and more magnification than larger lens)
Working distance
distance from specimen to the objective lens
Higher magnification objective = shorter working distance
Depth of field
range in which an object is in focus
Conjugate foci
for convex lenses
The object and its formed image
When object is greater than 2 focal lengths from the lens
The image will be:
REAL
SMALLER
INVERTED
When object is exactly 2 focal lengths away from the lens
The image will be:
REAL
SAME SIZE
INVERTED
When object is between 1-2 focal lengths from the lens
The image will be:
REAL
MAGNIFIED
INVERTED
When object is exactly 1 focal length from the lens
Light rays emerge from lens in parallel
Image can no longer be focused
When object is less than 1 focal lengths from the lens
The image will be: on the same side as the object (can only be seen by looking through the lens) VIRTUAL MAGNIFIED ERECT
Compound microscope
Primary magnification made by the objective lens
Object on stage is 1-2 focal lengths from the lens (real, magnified, inverted)
Image formed by objective lens is focused inside the microscope tube.
Image and ocular lens are less than 1 FL apart (image we see if virtual, magnified and erect)
Total magnification
multiply magnification of objective lens and ocular lens
OR
Total mag = mag of ocular X (tube length/objective focal length)
Chromatic Aberration
White light passes through lens, shorter wavelengths are refracted more than longer wavelengths
Different wave lengths will have different focal points
Produces distortion in the colors of the image
May produce a fringe of colors around the periphery of the field
Spherical Aberration
Light passing through the centre of the lens does not bend as much as rays passing through edge of lens
Outer edges will be blurred
Corrections for chromatic aberration (3)
Achromatic Lenses $
Semi-apochromats (fluorites) $$
Apochromats $$$
Achromatic Lenses
Corrective lense for chromatic aberration
Least expensive
Corrected for red and blue
Semi-apochromats (fluorites)
Corrective lense for chromatic aberration
Moderately expensive
Incorporate fluorite into lens to correct for red, blue, and SOME green
Apochromats
Corrective lense for chromatic aberration
Most expensive
Correct all 3 colors (red, blue, green)
Corrections for Spherical Aberration
Combined use of convex and concave lenses
Combined lense produces flat fields of view
Termed:
“plan-“
ex. plan-apochromats
Light source
Typically tungsten bulb, but now LED available
LED more expensive but lasts longer
Coloured glass filters
Used with lamps to reduce certain wavelengths of light.
Ex. Blue filter to reduce yellow light
Radiant Field Diaphragm
Controls the diameter of the light bundle directed at the specimen
Condenser Assembly
Lens system located between light source and specimen in the light path (right below stage assembly)
Acts to focus the illuminating light onto the slide on the stage
Aperture Diaphragm
controls the angle of the cone of light reaching the specimen
Slide controls
move stage in a horizontal plane, allowing viewing different areas of the slide
Focus knob
move stage in a vertical plane
Fine and course focus
Vernier scales
used to note particular location of an object on a slide
rulers on stage
Parfocal
nosepiece/objective assembly that allows the different objects to all focus the image at the same time
Mechanical Tube length
in mm
distance from top of ocular to the objective/nosepiece junction
Optical tube length
distance from optical centre of the objective lens to the focal plan of the ocular
Infinity corrected objectives
can be used on newer microscopes
Oculars
can be individually focused
Typically made for 18mm field of view
Hygenian (negative)
Ramsden (positive)
Hygenian (negative) oculars
less expensive
less corrections for aberrations
Ramsden (positive) oculars
more expensive
greater corrections for aberration
Best used for micrometry (measuring)
Refraction
Bending of light due to change in medium
Amount of refraction is based on:
angle of incidence
Refractive index of the mediums
Angle of Incidence (normal line)
normal line is 90 degrees relative to the surface
light will pass straight through the normal line without refracting
Light ray angles are measures from the “normal” pathway
Angle of incidence
the angle that the light strikes the surface
Angle of refraction
angle at which it leaves the surface
Light entering MORE DENSE medium
Bend TOWARDS normal
Light entering LESS DENSE medium
Bend AWAY from normal
Critical angle
Angle of refraction = 90degrees
Emerging ray will be parallel to the surface of the new medium
Total Internal Reflection
increase of angle of refraction past 90 degrees
Refractive index (RI or n)
expression of the density of a medium Denser medium = slower light rays = higher RI Air = 1.00 Crown glass = 1.52 Immersion oil = 1.52
Snell’s Law
RI = Sin(angle of incidence)/sin(angle of refraction)
Immersion oil
helps control refraction of light
prevents loss of light rays between slide and objective
Resolution
Minimum distance 2 objects must be apart in order to be seen as distinct
Depends on wavelength of light used and numerical aperture of the lens
Resolution = λ/2NA
Wavelength
shorter wavelength=greater amount of refraction = greater separation of 2 points
Numerical aperture (NA)
expression of the ability of a lens to gather light
(cone of light from slide to objective)
Wider cone (higher NA) = better resolution
NA = n sin μ
n=refractive index of the medium between the object and the lens
μ = one half the angle of aperture
NA for oil lens (100X) = 1.25
NA of condenser should match NA of objective for best resolution
Disadvantages to high NA
working distance, depth of field and flatness of field are all decreased
Useful magnification
occurs when 1000 X NA is greater than the total magnification
Empty magnification
occurs when 1000 X NA is less than the total magnification
Kohler
aligns microscope components to provide even illumination of specimen
match NA of condenser assembly to NA of objective lens
Collector lens
focuses image of the light source at the focal plane of the condenser lens
Light emerges as parallel rays
Radiant field diaphragm (RFD)
controls amount of light by controlling diameter of the light bundle
Setting Kohler steps
Raise condenser
Open condenser aperture
Close radiant field diaphragm
Lower condenser until image is in sharp focus
Centre using centering screws
Open field diaphragm until field is completely filled with light
Remove ocular, close condenser aperture until light field is 75% open
Infinity Corrective Objectives
Will have infinity symbol instead of tube length
Must be used with a microscope that has a tube lens
Allow for other components to enter light path (ex. filters)
Common objective markings
- oil immersion
- apochromat
- 100x magnification
- 1.25 NA
- 170mm tube length
- Coverslip thickness not important
Oil/Apo
100/1.25
170/-
