2-intro to light microscopy

purpose of microscopy

magnification; resolution, contrast

magnification

makes fine details more visible = increase resolution

resolution

minimum difference between 2 points that can be observed as separate distinct points

empty magnification

magnification with no increased resolution

introducing contrast to samples

staining, special optics to enhance small amount of existing contrast in unstained specimens

light microscope 2 systems

light and glass lens system

properties that change how light travels

RI of a medium, wavelength

diffraction

light scatters as it passes by and contacts objects

shorter wavelengths diffract less

diffraction leads to

coloured halos = decreased resolution

refraction

bend light as it passes through one medium to another

refractive index

measure of how much medium affects speed of light

amount of refraction through curved glass lens is affected by

lens shape, thickness, wavelength of light

focal point

point at which refracted rays converge on other side of lens

focal length

distance from midpoint of lens to focal point

convex lens

positive lens

light rays converge to focal point

concave lens

negative lens

upon exit, rays diverge/no focal point

optical aberrations

deviation from what is expected

chromatic aberration

due to different wavelengths refracting to different degrees as they pass through lens → image won’t be in complete focus

spherical aberration

light rays refract different depending on where in the lens they hit → image not focused

field curvature

image appears curved → image field can’t be focused simultaneously

correcting for aberrations

combine positive and negative lenses → cancels out aberrations

use different lens material

virtual image

image seen relative to specimen on slide

will be: enlarged, inverted, reversed

ocular

further magnifies real image of specimen

objective

creates magnified real image of specimen, primary determinant of image resolution, does most of image correction

types of microscopes

brightfield, phase contrast, inverted, polarized, fluorescent

plan corrects for

field curvature

highest level of objective lens correction

Plan apochromat

increasing correction

Numerical Aperture and resolution increases

field lens

directs column of light to substage condenser

field diaphragm

atop field lens; controls amount of light

sub stage condenser

gathers light from field lens and focuses it onto specimen; contains aperture diaphragm

numerical aperture (NA)

measure of light gathering capacity of objective lens

10x objective AD setting

0.2

20X objective ad setting

0.4

40X objective ad setting

0.5

100X objective ad setting

1

if ad diameter is too wide

light goes past objective → information is lost

kohler illumination

technique to align illumination and optic systems

sets correct amount of light entering objective = maximized resolution; provides sufficient and even illumination of FOV

minimizes artifact and misinformation

3 main components involved in kohler

AD, sub stage condenser, field diaphragm

when magification increases

NA, AD, and resolution increases

FOV, depth of field, working distance decreases

field of view

sample area diameter → how much of sample is seen

depth of field

number of specimen layers simultaneously in focus

shallower DOF = less layers focused at one time

working distance

distance between specimen and objective lens

cover glass

protects slide mounted specimen; needs to match objective

microscope slide

thin flat piece of glass used to support specimen

medium

what is between the cover glass and objective

immersion oil

RI similar to glass → minimizes light loss due to internal refraction

for higher magnification objectives to maximize resolving power