Fluorescence microscopy II Advanced approaches Martin Hof, Radek Macháň CZECH TECHNICAL UNIVERSITY IN PRAGUE FACULTY OF BIOMEDICAL ENGINEERING.

Fluorescence microscopy IIAdvanced approaches

Martin Hof, Radek Macháň

CZECH TECHNICAL UNIVERSITY IN PRAGUE

FACULTY OF BIOMEDICAL ENGINEERING

Microscope resolution:

The lateral resolution of an optical microscope d:

25,0

NA

d

The axial resolution (in the direction of optical axis) dz:

Sufficient contrast is necessary for full utilization of the available resolution

However fluorescence from planes below and above focus also contributes to signal

blurred image, decreased contrast

2

4,1NA

ndz

Total internal reflection fluorescence - TIRF:

When total reflection appears, only an exponentially decaying evanescent wave crosses the interface only fluorophores close to the interface are excited

~ 3 – 300 nm

)/exp()0()( dzIzI

Total internal reflection fluorescence - TIRF:When total reflection appears, only an exponentially decaying evanescent wave

crosses the interface only fluorophores close to the interface are excited

prism-based objective-based

Confocal microscopy – Basic principle:A pinhole in the back focal plane rejects the light coming from outside the focal

plane. The pinhole size is a trade-off between good rejecting ability and sufficient light throughput (typically ~ 30 – 150 m)

focal plane

objective

tube lens

detection

pinhole

wide field confocal

Confocal microscopy – Basic principle:The pinhole restricts the observed volume of the sample to a single point (the size of which is restricted by the pinhole size). Excitation by a collimated beam (point source

optically conjugated to the pinhole) focused to a diffraction limited spot

wide field confocal

whole image at once

dichroic

image is scanned point by pointCCD

PMT MPD …

Confocal microscopy – Scanning systems:

Collimated laser beam focus is scanned through the sample:

sample scanning by a piezo crystalo slowpossible combination with scanning

probe microscopy (AFM, STM, …)

spinning disk laser scanning microscope (LSM)

M. Petráň and M. Hadravský (1967) Wide-filed illumination passes

through pinholes in Nipkow disk (arranged in Archimedean spiral)

either a single pinhole for excitation and emission or 2 tandem disks

beam scanning by a mirrors mounted on galvanometers

optical path for excitation and emission formed by the same mirrors

o low excitation efficiency – only a small fraction of light passes pinhole

nowadays enhanced by microlens arrays on another Nipkow disk

more points in parallel possible – faster imaging X

Y

Axial scanning (Z) usually by a piezo or stepper motor actuator

Wide-field:

Confocal:

Elimination of out-of-focus light improves contrast and, thus, resolution

Confocal vs. Wide field microscopy:

Focusing only in one plane axial sectioning of the sample to ~ m slices

Confocal vs. Wide field microscopy:

Resolution in confocal microscopy:

collimated laser beam is focused by the objective into a diffraction limited spot

PSF (point spread function) = focus profile × collection efficiency of the objective. Those two are approximately the same diffraction limited spot.

x ~ 200 nm

z ~ 1 m

~ 3D Gaussian profile

Slightly higher resolution than in wide field microscopy (improvement ~ 1.4)

The image is a convolution of the object and the PSF

single-photon excitation

h

h

Ab

sorp

tio

n

Em

issi

on

two-photon excitation

hh*

h*

Ab

sorp

tio

n

Em

issi

on

Two photons at the same time and at the same place with doubled wavelength

E ~ 1 / E = h

c = E* ~ 1 / 2E* = 1/2 E

high photon density (6 – 7 orders of magnitude higher than in single photon confocal microscopy)

photons from the infra red spectrum (> 750 nm) – typically Ti:Sa laser

Two-photon microscopy – Basic idea:

excitation probability proportional to I2 reduced detection volume, higher resolution (improvement mainly in axial direction, in lateral it can be negligible due to larger )

laser pulse

focal plane

the required photon density for two-photon

excitationcan be established only in the focal plane no

out-of focus fluorescence no pinhole neededphoton

non-exciteddye molecule

2p-exciteddye molecule

Two-photon microscopy – Focus profile:

1p-excitation 2p-excitation

Two-photon microscopy:

Advantages improved axial resolution reduced bleaching out of focus higher light collection efficiency (no

pinhole) higher depth of light penetration broader excitation spectra –

simultaneous excitation of more dyes

Limitations

o more costly and complicated

instrumental setup

o higher bleaching in the focus

o broader excitation spectra –

decreased selectivity of excitation

o scanning technique like confocal

microscopy

Advantages improved contrast optical sectioning ability possibility to perform fluorescence

measurements in individual points

(lifetime, spectra, FCS, …)

Limitations

o more complicated and costly setup

o limited speed of image acquisition

o longer imaging more photobleaching

General features of scanning microscopy:

Fluorescence lifetime imaging (FLIM)

Below the diffraction limit:

Going to near-field, where the diffraction limit does not hold – Near-field Scanning Optical Microscope (NSOM)

Effectively increasing the numerical aperture (does not really break the limit, but increases resolution) – Structured (Patterned) Illumination Microscopy (SIM), …

Localization of individual fluorophores and fitting their PSFs, typically combined with switching between dark and fluorescent state (PALM, STORM, …); or utilizing intensity fluctuations of individual fluorophores (Superresolution Optical Fluctuation Imaging – SOFI)

Employing nonlinear optical effects:

• Multi-photon excitation

• Optical saturation – nonlinear dependence of fluorescence on excitation intensity, happens at high excitation intensities when large fraction of fluorophores resides in excited state and cannot be excited

• Other saturation phenomena:Dynamic saturation optical microscopy (DSOM) – kinetics of transition to triplet state,Stimulated emission excited state depletion (STED)

Near-field scanning optical microscopy (NSOM):

Diffraction limit is valid in the far-filed, where spherical wave-fronts exiting from an aperture can be regarded locally as plane waves – coming close to the sample changes the situation – scanning probe approach

The probe – usually a metal coated tapered optical fibre moved by a piezo scanner

various operation modes – purely near-field or combining near-/far-field excitation/emission or vice versa

• resolution ~ 20 nm in lateral (determined by tip size) and ~ 2-5 nm in axial direction

o limited only to surfaces

Effective increasing of numerical aperture:

A wide-field approach – faster then scanning

structured illumination

Several images with shifted illumination patterns are recorded and the final image is reconstructed by Fourier transform analysis optical sectioning

Additional spatial frequency increases the resolution power by factor 2

4Pi microscopy

2 opposing objectives – PSF closer to spherical symmetry – 3-7 times improved axial resolution (depends on type) combination with nonlinear image restoration – improvement in 3D

a confocal approach - scanning

Sample is illuminated by a periodically modulated light. Interference of structures in the sample and illumination results in Moiré fringes

Localization of individual molecules:Single fluorophores have dimensions much smaller than the PSF. A single

fluorophore is seen in the image as the PSF

Dtrtrt 4)0()()(MSD 2

By fitting the PSF in the image with a Gaussian profile, fluorophore location can be determined with a few nm accuracy

precise determination of distances, single particle tracking (SPT)

Schmidt et al. (1996) PNAS 93:2926-2629

Localization of individual molecules:At higher densities of fluorophores, the PSFs overlap – impossible to

distinguish the centers of peaks. Nevertheless, fluorophores need to be densely located in the sample to be cover to all structural details

STORM – Stochastic optical reconstruction microscopy

PALM – Photoactivated localization microscopy the same principle with switching of dyes between on and off states

Uses photoswitchable dyes (special organic dyes, GFP mutants):

a strong red laser pulse switches off all fluorophores (to a nonfluorescent state)

a green laser pulse switches on a small fraction of fluorophores, which emit fluorescence when excited with red laser until switched off, cycle repeated …

A wide field technique, but imaging slow because many imaging cycles needed

Rust et al. (2006) Nature Meth 3:793-795

Resolution ~ 20-30 nm

Optical saturation and resolution enhancement:

0 200 4000.0

0.5

1.0

Flu

ores

cenc

e [a

.u.]

Excitation rate [MHz]0 400 800

0.0

0.5

1.0

PS

F(x

)

x (nm)

Optical saturation results in nonlinear relation between excitation and fluorescence intensities broadening of the PSF

We apply a ramp of excitation intensity and the dependence of fluorescence intensity in each pixel on excitation intensity can be fitted with a polynomial expansion

Ifl(x,y) = Iex - Iex2 + Iex

3 - Iex4...

0 20 30 40

Theoretically unlimited resolution, but practically limited by noise and poor stability of polynomial fits (~ 30%)

Saturated excitation microscopy (SAX) – harmonically modulated excitation, Saturated structured illumination (SSIM) – SIM combined with nonlinearity

~

Excitation spot

x

Stimulated emission excited state depletion (STED): Developed by Stefan Hell (http://www.mpibpc.mpg.de/abteilungen/200/STED.htm)• A confocal approach• Fluorophores in the detection volume are excited by an excitation pulse.

• A doughnut-shaped STED pulse is applied, which suppresses the fluorescence completely (by inducing stimulated emission) everywhere except the center of the detection volume

• Photons in STED pulse have lower energy to avoid excitation

• STED pulse duration should be much shorter then S1 lifetime = 1/kfluor

saturation parameter: = I max/ Isaturation

Fluorescence

STED pulse

Imax>> Isaturation

x

kIC >kSE >> kfluor

• Saturation of the stimulated emission in the STED pulse is essential for breaking the diffraction limit

STED:

Theoretically unlimited resolution, usually ~ 3 times in lateral and ~ 6 times in axial direction is achieved

Selective plane illumination microscopy:

http://www.lmg.embl.de/home.html

Based on microscopy (uses excitation and detection optics at 90˚ instead of epi-fluorescence to generate isotropic PSF) – combination with light sheet illumination

faster imaging of 3D objects

Acknowledgement

The course was inspired by courses of:

Prof. David M. Jameson, Ph.D.

Prof. RNDr. Jaromír Plášek, Csc.

Prof. William Reusch

Financial support from the grant:

FRVŠ 33/119970