Ch. 9A: Light microscopy

Visualizing cells

in phase light

bright

out of phase light

dark, waves cancel each other out = dimmer

interaction between light and objects makes phase changes

parallel lines

concentric circles

two objects that are close together

• may have overlapping images

• appear as one object

• can’t tell if same thing or not

objective lens

collects light and performs primary magnification

• makes an image

condenser lens

focuses cone of light onto specimen

• illuminates and shapes the light/beam for the specimen

• controls intensity and angle

resolution

shortest distance at which two separate objects can still be distinguished

resolving power of a microscope

microscope ability to distinguish two very close points as separate, not a blur

• depends on objective lens

0.61lambda/n(sin(theta))

• theta: half the angular width of cones of rays collected by objective lens

• n: refractive index of medium (air or oil)

  • separates specimens from object and condenser lens

• lambda: wavelength of light used

• n(sin(theta)): numerical aperture

  • light entering lens

higher numerical aperture

greater resolution and brighter image

white light wavelength

purple = 400 nm

red = 700 nm

• longer wavelength

• infrared

living cells are seen in

bright field

dark-field

phase-contrast/PIC

bright field

colored stains

• specimen has been fixed, dead, and stained

• staining: allows contrast when there’s light

dark-field

stained at an angle

• specimen observable over time

• living specimen

phase-contrast/PIC

specimen knocking in or out of phase

• specimen observable over time

glutataraldehyde

fixes tissues

• forms covalent bonds between peptide sides chains

• cross-linking, stabilization in position

In-situ hybridization

uses DNA or RNA probe to probe for specific RNA sequences

• determines location of an RNA

fluorescence microscopy

brightness of fluorescent image = fourth power of numerical aperture

• emission wavelength is greater than excitation wavelength

  • excited with yellow light, emits red light (longer than yellow)

excitation wavelength

absorption of photon

emission wavelength

emission of photon

immunohistochemistry

uses antibodies to detect specific proteins (antigens) in tissue

primary antibody

directed against antigen A

• binds to target antigen

• unlabeled

secondary antibody

binds to primary antibodies

• labeled with marker

• amplifies signals

  • each primary antibody can be bound by multiple secondary antibodies

• detects

markers

enzymes, flourophore

biotin-streptavidin method

similar to immunohistochemistry

• amplifies signal for antigen detection

• uses secondary antibody with biotin

fluorescent proteins

gene expression reporters

• promoter part of gene fused with fluorescent protein coding region

• expressed in cells

• fluorescent = readout of gene X expression

inducible cell tracer

• specific wavelength of light used to cause expression of the reporter in cell

• cell can be followed over time

FRET

fluorescent resonance energy transfer

• assesses if two fluorescent proteins co-localize (near each other)

• blue protein: violet light excitation, blue light emission

• green protein: blue light excitation, green light emission

• no protein interaction: blue light emits

• protein interaction: green light emits

• emission wavelength of first pair = excitation wavelength

FRAD

fluorescent recovery after photobleaching

• photo bleach one area of cell/membrane

• bleach destroys fluorescence in small region then watch how quickly fluorescence recover as non-bleached molecules move in

• determines if a protein or lipid diffuses freely around membrane or can be replaced in sub-cellular compartment

fluorescent sensors

engineered molecules/systems that change their fluorescence in response o a signal

• ion-sensitive fluorophores

  • fluorescent in presence of Ca2+ and H+

• biosensors

  • fluorescence structure change when bound to H2O2

  • can

• red = low concentration

• yellow = intermediate concentration

• blue = high concentration

confocal microscope

focuses on a single plane of a specimen

• sharp image of fluorescent

  • illumination path: focuses light onto a single point

  • emission path: fluorophores at focal point are excited and emit fluorescence

  • confocal pinhole: makes a blur disk

TIRF

total internal reflection fluorescence microscope

• light shined at an angle

• makes a thin sheet that only excites molecules at cell surface

  • makes everything else invisible

multi photon imaging

allows penetration of deep light in tissues

• less risk of tissue damage bc lower energy

• two photon excitement allows use of lower energy (infrared) light

expansion microscopy

differs higher resolution

• pokes holes in membranes

• injects gel in cell and expands it

SIM

structured illumination microscopy

• patterned light onto sample

• fast imaging

STED

stimulated emission depletion

• instead of blurry spot, makes spot smaller

• excite: laser makes molecules in spot glow

• deplete: doughnut shape laser turns off

• result: only molecules in center remaining glowing

SMLM

single-molecule locationalize microscopy

• switching individuals fluorescent molecules on and off

  • take many pics

  • pinpoints molecules location

• AKA PALM or STORM