bringing light into the chaos a general introduction to optics and light microscopy
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Bringing Light into the chaos: a general introduction to optics and light microscopy. Part 1: The root of all evil. Part 2:. Fluorescence microscopy and special applications. Contrasting techniques - a reminder…. Brightfield -absorption Darkfield -scattering

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bringing light into the chaos a general introduction to optics and light microscopy

Bringing Light into the chaos:a general introduction to optics and light microscopy

Part 1: The root of all evil

Part 2:

Fluorescence microscopy

and special applications

contrasting techniques a reminder
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
Fluorescence techniques
  • Standard techniques: wide-field



  • Special techniques: FRET





Excited state

Ground state



shorter wavelength, higher energy


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
  • Excitation/emission spectra always a bit overlapping
  • filterblock has to separate them
  • Exitation filter
  • Dichroic mirror
  • (beamsplitter)
  • Emission filter
filter nomenclature
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 emission2
excitation 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!

Excitation / emission
Where to check spectra?

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

standard techniques
Standard techniques
  • wide-field
  • confocal
  • 2-photon
wide field fluorescence
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
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 vs confocal

Wide-field image confocal image

Molecular probes test slide Nr 4, mouse intestine

  • 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


  • 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
Timelapse with confocal

You can do timelapse movies with the confocal.

Mainly for fast processes

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

 Two Leica confocals and one Olympus FV 1000

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

  • 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)

2-photon microscopy

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

1-photon: 488nm

2-photon: 843nm

special applications
Special applications:
  • FRET and FLIM
  • FRAP and photoactivation
  • TIRF
fret f luorescence r esonance e nergy t ransfer
Exited state

Exited state

Exited state

Ground state

Ground state

Ground state

  • method to investigate molecular interactions
  • Principle: a close acceptor molecule can take the excitation energy from the donor (distance ca 1-10 nm)

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


Energy transfer, no emission!

Donor (GFP)

Acceptor (RFP)


ways to measure:

  • Acceptor emission

Detect 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 bleachingtake 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



  • Bleed through (because of overlapping spectra)
  • Limitation of techniques (filters etc)
  • Photobleaching only with fixed samples
  • Intensity depends on concentrations etc
flim f luorescence l ifetime i maging m icroscopy
∆t=lifetimeFLIM(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 concentration
    • Oxygen concentration
    • pH
    • Protein-protein interactions

Lifetime histogram

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 back

decay curve

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

Example of FLIM-FRET measurement

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

special applications1
Special applications:
  • FRET and FLIM
  • FRAP and photoactivation
  • TIRF
frap f luorescence r ecovery a fter p hotobleaching
Use: to measure the mobility/dynamics of proteins under different conditions


0.65 s

0.78 s

 Olympus FV 1000

FRAP (Fluorescence Recovery After Photobleaching)
  • Intense illumination with 405 laser bleaches the sample within the selected region  observation of the recovery
  • Fluorophore only becomes active (= fluorescent) if excited (e.g. with 405 laser) due to structural change

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

 Olympus FV 1000

special applications2
Special applications:
  • FRET and FLIM
  • FRAP and photoactivation
  • TIRF
tirf t otal i nternal r eflection f luorescence
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

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)

TIRF vs epi

Heather Spence, R10

tirf vs epi1
Lasp in confocal sectioning

Lasp in TIRF mode

TIRF vs epi

Heather Spence, R10