‫מיקרוסקופיה פלואורסנטית‬
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
‫חוב מהשיעור הקודם‬
‫שיטות להגברת ניגודיות‬
Bright-field
Phase-contrast
DIC
Dark-field
Fluorescence techniques
• Standard techniques: wide-field
confocal
2-photon
• Special applications:
FRET
FLIM
FRAP
Photoactivation
TIRF
Fluorescence
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
Fluorophores
Filters
How can we separate light with specific wavelength from the rest of the light?
Filters
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
Filter nomenclature
Filters
Multiple Band-Pass Filters
Basic idea
Basic design of epi fluorescence
Objective acts as condenser; excitation light reflected away from eyes
The cube
Excitation/emission
spectra always a
bit overlapping
 filterblock has to
separate them
a) Exitation filter
b) Dichroic mirror
(beamsplitter)
a) Emission filter
The cube
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
Fluorescent Probe Excitation Efficiency (Olympus jave tutorial)
Choosing Fluorophore Combinations for Confocal Microscopy (Olympus
java tutorial)
Photobleaching
• Photobleaching - When a fluorophore permanently loses the ability
to fluoresce due to photon-induced chemical damage and covalent
modification.
Photobleaching
• At low excitation intensities, pb occurs but at lower rate.
• Bleaching is often photodynamic - involves light and oxygen.
• Singlet oxygen has a lifetime of ~1 µs and a diffusion coefficient
~10-5 cm2/s. Therefore, potential photodamage radius is ~50 nm.
Standard techniques
•
•
•
•
wide-field
Confocal
Spinning disk confocal
2-photon
Wide-field fluorescence
• reflected light
method
• Multiple wavelength
source
(polychromatic, i.e.
mercury lamp)
• Illumination of whole
sample
Wide-field vs confocal
Wide-field image
confocal image
Molecular probes test slide Nr 4, mouse intestine
Point illumination
Widefield Illumination
Point Illumination
Light sources for point illumination
Excitation light must be focused to a
diffraction limited spot
Could be done with an arc lamp
and pinhole – but very inefficient
Enter the laser:
Perfectly collimated and
high power
Excitation light
Objective lens
Sample
Point illumination
Fluorescence Illumination of a single point
Camera
Tube lens
Excitation light
Emission light
Objective lens
Sample
Problem – fluorescence is emitted along
entire illuminated cone, not just at focus
The confocal microscope
Detector
Pinhole
Tube lens
Excitation light
Emission light
Objective lens
Sample
The confocal microscope
• 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
Scanning
Changing entrance
angle of illumination
moves illumination spot
on sample
Objective lens
Sample
The emission spot
moves, so we have to
make sure pinhole is
coincident with it
Improved PSF and pinhole size
Why can’t it be as small as possible?
• Reduced number of photons that arrive at the
detector from the specimen may lead to a reduced
signal-to-noise ratio.
• Raising the intensity of the excitation light can
damage the specimen.
• Optical sectioning does not improve considerably
with the pinhole size below a limit that approximates
the radius of the first zero of the Airy disk.
PSF for the focal plane and planes parallel
to it: (a) conventional diffraction pattern
(b) Confocal case.
How big should your pinhole be?
• Width of point spread function at pinhole =
Airy disk diameter × magnification of lens = 1 Airy unit
= resolution of lens × magnification of lens × 2
– 100x / 1.4 NA: resolution = 220nm, so 1 Airy unit = 44 mm
– 40x / 1.3 NA: resolution = 235nm, so 1 Airy unit = 19 mm
– 20x / 0.75 NA: resolution = 407nm, so 1 Airy unit = 16 mm
– 10x / 0.45 NA: resolution = 678nm, so 1 Airy unit = 14 mm
How big should your pinhole be?
• A pinhole of 1 airy unit (AU) gives the best signal/noise.
• A pinhole of 0.5 airy units (AU) will often improve resolution
IF THE SIGNAL IS STRONG.
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)
Spinning Disk Confocal
Spinning Disk
• Fast – multiple points are illuminated at once
• Photon efficient – high QE of CCD
• Gentler on live samples – usually lower laser
power
• Fixed pinhole
• Small field of view (usually)
• Crosstalk through adjacent pinholes limits
sample thickness
Relative Sensitivity
• Widefield
100
• Spinning-Disk Confocal 25
• Laser-scanning Confocal 1
• See Murray JM et al, J. Microscopy 2007 vol.
228 p390-405
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
Special applications:
• FRET and FLIM
• FRAP/FLIP 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 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
• Both Acceptor and Donor are fluorescent
• The Donor is excited and its emission excites the Acceptor
Ex(D)
Em(D)
Em(A)
Ex(A)
FRET
• FRET is a competing process for
the disposition of the energy of a
photo excited electron.
• Donor emission decreases
• Donor lifetime decreases
• Acceptor emission increases
FRET
Energy transfer efficiency
• Depends on: Donor emission and acceptor absorption spectra,
relative orientation of D and A
FRET
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
Advantages
Disadvantages
Cheap implementation
Free fluorophors can
mask energy transfer
high resolution (1-10nm) pH sensitive
Living cells
Weak effect
Real time
Location of fluorophors
is critical
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 - advantages
In this method we measure the lifetime of the excited
state and not the fluorescence intensity, therefore:
• We can separate fluorophores with similar spectra.
• We minimize the effect of photon scattering in thick
layers of sample.
lifetime = ½ of all electrons are fallen back
1/e
1 2
FLIM - Measurement approaches
• Frequency domain
• Modulated excitation
• Lock-in detect emission phase
• Time domain (pulsed exc.)
• Gated intensifier
Photon inefficient
• Time-correlated
single photon counting
Very efficient
 one photon per pulse  slow
Time gates
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
Hepatocyte membrane-stained
with NBD, which has a
hydrophobicity-dependent lifetime
(TCSPC, 3 minutes for 300x300 pixels )
FLIM
For FLIM-FRET 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
(Ca+2), pH change etc, Protein interactions
Special applications:
• FRET and FLIM
• FRAP/FLIP and photoactivation
• TIRF
FRAP
(Fluorescence Recovery After Photobleaching)
Need: to probe transport
Idea: bleach in one area,
watch recovery by transport from other areas
FRAP
Measuring Cdc42 diffusion constant in yeast
Result: df = (0.036 ± 0.017) μm2/s
Marco et al. 2007 Cell 129:411-422
FRAP
• 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
FLIP
(Fluorescence Loss in Photo-bleaching)
Need: probe connectivity
Idea: bleach in one compartment,
watch loss in connected compartments by exchange
Bleach one area repeatedly. Entire ER dims.
 ER is contiguous
Photoactivation
(Better?) FRAP/FLIP alternative
Some fluorophores can be activated by light
• Photo-uncagable dyes
• GFP-family proteins
Look for weak light
against dark background
Instead of slight dimming
of bright background
Activate a small area
Watch fluorescence spread
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
Photoactivation - Proteins
Off-On
• PA-GFP, PS-CFP
Color change
• Kaede, KikGR, Eos,
• Dendra (activatable by blue)
Reversibly Switchable
• asCP, KFP (tetrameric)
• Dronpa
Dendra2 demo
green
Activate
before
after
red
photoactivation
Dronpa – photoswitchable on and off
Ando et al. 2004, Science 306: 1370-1373
photoactivation
Tracking actin flow with Dronpa
Kiuchi, T. et al. J. Cell Biol. 2007;177:465-476
Special applications:
• FRET and FLIM
• FRAP/FLIP 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
use
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
FRET
Protein interactions,
small distances
FLIM
Environmental changes,
protein interactions
FRAP/FLIP +
dynamics/mobility
photoactivation
TIRF
Only bottom
plane
Only bottom
plane
150-200nm
Membrane dynamics
Light source for fluorescence microscopy
Arc lamps
Xenon
Mercury
Laser types
UV
Argon
Blue diode
IR
351 364
457 477 488
405 440
Helium-Cadmium 354
Krypton-Argon
Green Helium-Neon
Yellow Helium-Neon
Orange Helium-Neon
514
442
488
569
647
543
594
612
Red Helium-Neon
633
Red diode
635 650
Ti:Sapphire
720-980