Exam 2 Confocal Microscopy: Lec. 7 Brightfield, Phase Contrast, Polarized light, DIC

what are the four types of transmitted light imaging

1) brightfield

2) phase contrast

3) polarized light

4)differential interference contrast (DIC)

what is transmitted light image dependent on?

how light interacts with the sample

how light interacts with the specimen

how light interacts with the optics

visualizing the types of transmitted light

transmitted light interacting with sample

image formed when the undeviated background light is focused in the same image plane as the deviated light from the sample and interference takes place

amplitutde

wave height

changes in amplitutde…

change in brightness

S and A waves

light passing through the sample is absorbed and therefore we see a decrease in intensity when compared to the brighter surrounding light

phase shifts

constructive intereference

when light waves combine, their intensities are added together

if peaks and troughs of both waves are near the same position constructive interefencce occurs (A+B=C)

the resulting ray (C) will be more intense (brighter) than the individual waves

destructive intereference

if peaks and troughs of both waves are in oppositve positions (out of phase), then deconstructive intereference occurs (A+B=c)

the resulting ray c will be less intense (darker) then the individual waves

D and S Wave definitions

S wave- undeviated or surround wave

D wave- deviated or diffracted wave

P waves

S+ D

When the waves recombine, they undergo interference and generate a resultant Particle wave

only when amplitutde of P is sign. different from S can we visually see the structures causing the interference

phase contrast optics: phase annulus in condenser

restricts illumination light to a ring

phase contrast optics: phase plate in objective

ring of low refractive index material

phase advances S waves only

phase contrast microscopy

optics enhance the phase shift between the undeviated surrounding light (S) and the deviated light (d) that interact with sample

deviated light is slowed (phase retarted) by interaction with the sample

S waves are faster (phase advanced) using optics with low refractive index

lineraly polarized light

when all light waves vibrate in a single plane, this is referred to as linearly polarized light

light from most illuminators - a halogen lamp is randomly polarized

randomly polarized light may also be referred to as unpolarized/non-polarized

polarizing lenses

birefringence

in some crystals (quartz, calcite) atoms are ordered in a precise geometic arrangement

ordered arrangment can result in direction-dependent differences in refractive index within a crysal

birefringence occurs when light entering the crystal lattice is refracted into two polarized rays

birefringence-onlique incident light results in O and E rays that

are linearly polarized

have perpendicular vibration planes to each other

have different trajectories (path) through the crystal

are phase shifted to each other

examples of birefringence in biology

most ordered macromolecular assemblies - lipid bilayers, bundles of microtubules, plant cell walls, starch granules

pretty uncommon in biology - more common for geology to have polarized light microscopy

Differential intereference contrast microscopy (DIC)

uses a birefringent crystal to generate the E and O rays

small difference s in refractive index and thickness of the sample will result in the E and O rays experincing a phase shift relative to each other

DIC picture

compare and contrast enhancing optics

both DIC and phase contast techniques depend on phase differences within the sample

DIC - amplitutde change due to phase shift of two perpendicullary polarized, deviated light rays (E and O waves)

phase contrast- amplitude change after destructive interference of deviated rays (D waves ) and undeviated rays (S wabes)