Bringing Light into the chaos:a general introduction to optics

and light microscopy

Part 2:

Part 1: The root of all evil

Contrasting techniques - a reminder…

• Brightfield -absorption

• Darkfield -scattering

• Phase Contrast -phase interference

• Polarization Contrast -polarization

• Differential Interference Contrast (DIC) -polarization + phase interference

• Fluorescence Contrast

Fluorescence techniques

• Standard techniques: wide-fieldconfocal2-photon

• Special techniques: FRETFLIMFRAPPhotoactivationTIRF

Fluorescence

Excited state

Ground state

excitation

shorter wavelength, higher energy

emission

longer wavelength, less energy

Stoke’s shift

Fluorophores (Fluorochromes, chromophores)

• Special molecular structure

• Aromatic systems (Pi-systems) and metal complexes (with transition metals)

• characteristic excitation and emission spectra

Excitation / emission

Excitation/emission spectra always a bit overlapping

filterblock has to separate them

a) Exitation filterb) Dichroic mirror (beamsplitter)a) Emission filter

Excitation / emission

Filter nomenclature• Excitation filters: x• Emission filters: m• Beamsplitter (dichroic mirror): bs, dc, FT

• 480/30 = the center wavelength is at 480nm; full bandwidth is 30 [ = +/- 15]

• BP = bandpass, light within the given range of wavelengths passes through (BP 450-490)

• LP = indicates a longpass filter which transmits wavelengths longer than the shown number and blocks shorter wavelengths (LP 500)

• SP = indicates a shortpass filter which transmits wavelengths shorter than the shown number,

and blocks longer wavelengths

Excitation / emissionexcitation and emission spectra of EGFP (green) and Cy5 (blue)

excitation and emission spectra of EGFP (green) and Cy2 (blue)

No filter can separate these wavelengths!

You can plot and compare spectra and check spectra compatibility for many fluorophores using the following Spectra Viewers.

Invitrogen Data Base BD Fluorescence Spectrum Viewer University of Arizona Data Base NCI ETI Branch flow Cytometry 

Where to check spectra?

Standard techniques

• wide-field

• confocal

• 2-photon

Wide-field fluorescence

• reflected light method

• Multiple wavelength source (polychromatic, i.e. mercury lamp)

• Illumination of whole sample

upright Zeiss microscopes, fluorescence tissue culture microscopes, timelapse microscopes

PFS timelapse

• New long term timelapse (Nikon)• System adjusts the focus by using IR laser to

measure the distance to the glass of your dish

Wide-field vs confocal

Wide-field image confocal image

Molecular probes test slide Nr 4, mouse intestine

Confocal

• method to get rid of the out of focus light less blur

• whole sample illuminated (by scanning single wavelength laser)

• only light from the focal plane is passing through the pinhole to the detector

ConfocalUse:• to reduce blur in the picture high contrast

fluorescence pictures (low background)• optical sectioning (without cutting);

3D reassembly possible

Careful: increasing image size (more pixels) does not mean that the objective can resolve the same!!! (resolution determined by NA, a property of the objective)

Timelapse with confocal

Two Leica confocals and one Olympus FV 1000

You can do timelapse movies with the confocal.

Mainly for fast processes

Be aware that not all our confocals have incubation chamber and CO2!

Excited state

Ground state

2-photon microscopy

Excitation: long wavelength (low energy)

Each photon gives ½ the required energy

Emission: shorter wavelength (higher energy) than excitation

2-photon microscopy

Advantages: IR light penetrates deeper into the tissue than shorter wavelength

2-photon excitation only occurs at the focal plane less bleaching above and below the section

Use for deep tissue imaging

new La Vision microscope (live mouse imaging, will be installed in the new building)

Use of lower energy light to excite the sample (higher wavelength)

1-photon: 488nm

2-photon: 843nm

Special applications:

• FRET and FLIM• FRAP and photoactivation• TIRF

FRET (Fluorescence Resonance Energy Transfer)

• method to investigate molecular interactions• Principle: a close acceptor molecule can take the excitation energy

from the donor (distance ca 1-10 nm)

Donor (GFP)

FRET situation: Excitation of the donor (GFP) but emission comes from the acceptor (RFP)

Exited state

Ground state

Acceptor (RFP)

Exited state

Ground state

Energy transfer, no emission!

Exited state

Ground state

No FRET

FRET

ways to measure:

• Acceptor emissionDetect the emission of the acceptor after excitation of the donor, e.g. excite GFP with 488 but detect RFP at 610 (GFP emission at 520)

• Donor emission after acceptor bleaching take image of donor, then bleach acceptor (with acceptor excitation wavelength - RFP:580nm), take another image of donor should be brighter!

You need: • a suitable FRET pair (with overlapping excitation/emission curves)

Disadvantages:• Bleed through (because of overlapping spectra)Limitation of techniques (filters etc)• Photobleaching only with fixed samples• Intensity depends on concentrations etc

FRET

FLIM (Fluorescence Lifetime Imaging Microscopy)

• measures the lifetime of the excited state (delay between excitation and emission)

• every fluorophore has a unique natural lifetime

• lifetime can be changed by the environment, such as: Ion concentrationOxygen concentrationpHProtein-protein interactions

∆t=lifetime

FLIM

Excitation of many electrons at the same time count the different times when they are falling back down (i.e. photons are emitted)

lifetime = ½ of all electrons are fallen backdecay curve

Lifetime histogram

Example of FLIM-FRET measurement

GFP expressed in COS 1 cell: average lifetime of 2523 ps

fused GFP-RFP expressed in COS 1 cell: average lifetime of 2108 ps

Joan Grindlay, R7

You still need: a suitable FRET-pair with the right orientation of the π-orbitals

Interaction of proteins is not enough, because fluorophores have to be close enough and in the right orientation!

Use of FLIM: measurements of concentration changes (Ca2+), pH change etc, Protein interactions

FRET: Leica confocal 2 or Olympus FV 1000

FLIM: Leica confocal 1 and soon LIFA system from Lambert Instruments

FLIM

Special applications:

• FRET and FLIM

• FRAP and photoactivation• TIRF

FRAP (Fluorescence Recovery After Photobleaching)

• Intense illumination with 405 laser bleaches the sample within the selected region observation of the recovery

before 0.65 s 0.78 s

Olympus FV 1000

Use: to measure the mobility/dynamics of proteins under different conditions

photoactivation• Fluorophore only becomes active (= fluorescent) if

excited (e.g. with 405 laser) due to structural change

Olympus FV 1000

Pictures taken from a activation movie: activation of a line trough the lamellipodia of the cell, activated GFP_F diffuses quickly

Special applications:

• FRET and FLIM

• FRAP and photoactivation

• TIRF

TIRF (Total Internal Reflection Fluorescence)

You need:

• TIRF objectives with high NA

• TIRF condensor, where you are able to change the angle of illumination

• Glass coverslips

TIRF

micro.magnet.fsu.edu

Result: very thin section at the bottom of the sample 150-200nm

Use: to study membrane dynamics (endocytosis, focal adhesions, receptor binding)

Nikon TE 2000

TIRF vs epi

FAK-lasp in epi mode (wide field)

FAK-lasp in tirf mode (wide field)Heather Spence, R10

TIRF vs epi

Lasp in TIRF mode

Lasp in confocal sectioning

Heather Spence, R10

Summary/comparison

method excitation detection sectioning use

Wide field Whole sample Whole sample No sectioningSimple fluorescence samples

confocal Whole sample One z-plane 350-500nmHigh contrast images, optical sectioning

2-Photon One z-plane One z-plane 500-700nmDeep tissue imaging, optical sectioning

FLIM/FRET Protein interactions

FRAP + photoactivation

405 laser (UV) dynamics/mobility

TIRFOnly bottom plane

Only bottom plane

150-200nm Membrane dynamics

• Please book proper training with Tom or Margaret before using BAIR equipment!

BAIR webpage demonstration: