Bringing Light into the chaos:
a general introduction to optics
and light microscopy
Part 1: The root of all evil
Part 2:
Contrasting techniques a reminder…
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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-field
confocal
2-photon
• Special techniques:
FRET
FLIM
FRAP
Photoactivation
TIRF
Fluorescence
Excited state
excitation
emission
shorter wavelength,
higher energy
longer wavelength,
less energy
Ground state
 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 filter
b) 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 / emission
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!
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
• 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
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)
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
2-photon microscopy
Excited state
Excitation:
long wavelength
(low energy)
Emission: shorter
wavelength (higher
energy) than excitation
Each photon
gives ½ the
required energy
Ground state
2-photon microscopy
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)
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)
No FRET
Exited state
FRET situation: Excitation of the donor (GFP)
but emission comes from the acceptor (RFP)
Exited state
Energy transfer,
no emission!
Exited state
Donor
(GFP)
Acceptor
(RFP)
Ground state
Ground state
Ground state
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!
FRET
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
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
∆t=lifetime
 pH
 Protein-protein interactions
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)
decay curve
lifetime = ½ of all
electrons are fallen
back
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
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
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
Use: to measure the mobility/dynamics of proteins under different conditions
 Olympus FV 1000
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
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 confocal sectioning
Lasp in TIRF mode
Heather Spence, R10
Summary/comparison
method
excitation
detection
sectioning
Wide field
Whole sample
Whole sample
No sectioning
Simple fluorescence
samples
confocal
Whole sample
One z-plane
350-500nm
High contrast images,
optical sectioning
2-Photon
One z-plane
One z-plane
500-700nm
Deep tissue imaging,
optical sectioning
FLIM/FRET
FRAP +
photoactivation
TIRF
use
Protein interactions
dynamics/mobility
405 laser (UV)
Only bottom
plane
Only bottom
plane
150-200nm
Membrane dynamics
• Please book proper training with Tom or
Margaret before using BAIR equipment!
BAIR webpage demonstration: