1. 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

2. Contrasting techniques - a reminder…
Brightfield -absorption
Darkfield -scattering
Phase Contrast -phase interference
Polarization Contrast -polarization
Differential Interference Contrast (DIC) -polarization + phase interference
Fluorescence Contrast

3. Fluorescence techniques
Standard techniques: wide-field
confocal
2-photon
Special techniques: FRET
FLIM
FRAP
Photoactivation
TIRF

4. Fluorescence excitation emission shorter wavelength, higher energy
Excited state
Ground state
excitation
shorter wavelength, higher energy
emission
longer wavelength, less energy
 Stoke’s shift

5. Fluorophores (Fluorochromes, chromophores)
Special molecular structure
Aromatic systems (Pi-systems) and metal complexes (with transition metals)
characteristic excitation and emission spectra

6. Excitation / emission Exitation filter Dichroic mirror (beamsplitter)
Excitation/emission spectra always a bit overlapping
filterblock has to separate them
Exitation filter
Dichroic mirror
(beamsplitter)
Emission filter

7. Excitation / emission

8. 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 )
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

9. Excitation / emission  No filter can separate these wavelengths!
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!

10. 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 

11. Standard techniques
wide-field
confocal
2-photon

12. 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

13. 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

14. Wide-field vs confocal
Wide-field image confocal image
Molecular probes test slide Nr 4, mouse intestine

15. 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

16. Confocal
Use:
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)

17. 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

19. 2-photon microscopy Excitation: long wavelength (low energy)
Excited state
Ground state
Excitation: long wavelength (low energy)
Each photon gives ½ the required energy
Emission: shorter wavelength (higher energy) than excitation

20. 2-photon microscopy Advantages:  Use for deep tissue imaging
Use of lower energy light to excite the sample (higher wavelength)
1-photon: 488nm
2-photon: 843nm
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)

21. Special applications:
FRET and FLIM
FRAP and photoactivation
TIRF

22. 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)
FRET situation: Excitation of the donor (GFP) but emission comes from the acceptor (RFP)
No FRET
Exited state
Ground state
Exited state
Ground state
Energy transfer, no emission!
Exited state
Ground state
Donor (GFP)
Acceptor (RFP)

23. FRET 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 bleaching take image of donor, then bleach acceptor (with acceptor excitation wavelength - RFP:580nm), take another image of donor  should be brighter!

24. FRET You need: a suitable FRET pair Disadvantages:
(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

25. 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 concentration
Oxygen concentration pH
Protein-protein interactions
∆t=lifetime

26. FLIM 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

27. Example of FLIM-FRET measurement
GFP expressed in COS 1 cell: average lifetime of ps
fused GFP-RFP expressed in COS 1 cell: average lifetime of 2108 ps
Joan Grindlay, R7

28. FLIM
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

29. Special applications:
FRET and FLIM
FRAP and photoactivation
TIRF

30. 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

31. photoactivation
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

32. Special applications:
FRET and FLIM
FRAP and photoactivation
TIRF

33. 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

34. TIRF micro.magnet.fsu.edu
Result: very thin section at the bottom of the sample  nm
Use: to study membrane dynamics (endocytosis, focal adhesions, receptor binding)
 Nikon TE 2000

35. TIRF vs epi FAK-lasp in epi mode (wide field)
FAK-lasp in tirf mode (wide field)
Heather Spence, R10

36. TIRF vs epi Lasp in confocal sectioning Lasp in TIRF mode
Heather Spence, R10

37. Summary/comparison method excitation detection sectioning use
Wide field
Whole sample
No sectioning
Simple fluorescence samples
confocal
One z-plane nm
High contrast images, optical sectioning
2-Photon nm
Deep tissue imaging, optical sectioning
FLIM/FRET
Protein interactions
FRAP + photoactivation
405 laser (UV)
dynamics/mobility
TIRF
Only bottom plane nm
Membrane dynamics

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

39. BAIR webpage demonstration: