High Resolution imaging in Deep Tissue
description
Transcript of High Resolution imaging in Deep Tissue
High Resolution High Resolution imaging in Deep imaging in Deep TissueTissue
Rainer HeintzmannRainer Heintzmann,,
Institute for Photonic Technologies (IPHT),Institute for Photonic Technologies (IPHT),Friedrich Schiller University of JenaFriedrich Schiller University of JenaRandall Division, King‘s College LondonRandall Division, King‘s College London
Biophotonics, 2011
The Problem: 1. The Problem: 1. AbsorbtionAbsorbtion
100 1000 10000
Wellenlänge / nm
Abs
orp
tions
koef
fizie
nt /
cm
-1
100
10
1
1000
10000
UV Vis IR
Aorta
Haut
gesamtes Blut
Melanosom
Epidermis
Wasser
100 1000 10000
Wellenlänge / nm
Abs
orp
tions
koef
fizie
nt /
cm
-1
100
10
1
1000
10000
UV Vis IR
Aorta
Haut
gesamtes Blut
Melanosom
Epidermis
Wasser water
epidermis skin
aorta
blood
melanosom
Wavelength / nm
Abs
orbt
ion
Coe
ffic
ient
/ cm
-1
User requirementsUser requirements• as „lifelike“ as possible:
• the illumination light should not influence the behaviour
• sample-mounting shound not disturbe• fluorphores (vFPs) should behave close to
wildtype• images have to be taken quickly
„temporal sampling“to avoid artefacts, distortions and allow tracking
The Problem: 2. The Problem: 2. ScatteringScattering
Rayleigh scattering:
~ -4
Blue: Bad!Red / Infrared: OK!
Physics of LightPhysics of Light
Light as a Wavefocal distance
plane of focus
Moving Phase Front
The Problem: 3. The Problem: 3. AberrationsAberrations
Distorted Wavefront(Aberrated)
h
ps
psPs0
Ps1
Singletps
psPs0
Ps1
Singlet
h
h
1 Photon 1 Photon absorbedabsorbed
2 Photons 2 Photons absorbedabsorbedProbability ~ Probability ~
IntensityIntensity22
Solution 1: Two Photon Solution 1: Two Photon EffectEffect
Two Photon Excitation
Zipfel, Williams, Webb, Nature Biotechnology 21, 1369 - 1377 (2003)
Solution 1: Two Photon Solution 1: Two Photon EffectEffect
Solution 1: Two Photon Solution 1: Two Photon EffectEffect
emission photons will be multiply scattered
Non descanned detection needed to maximize capture area
DichromaticReflector
Wid
e A
rea
Det
ecto
rat
clo
se d
esta
nce
Solution 1: Two Photon Solution 1: Two Photon EffectEffect
• Much less absorption• Much less scattering• Less aberrations• Less out-of-focus bleach
• Fancy technique: Temporal focussing leads to ultrafast scans
Scattering loss is compensated untilSurface starts „burning“
Solution 1: Two Photon Solution 1: Two Photon EffectEffect
Some techniques (Multifocus, Temporal focussing) require imaging of the emitted light
Problem: haze from emission scattering remains
Solution: 1. Use different technique (e.g. single beam)2. Combination with modulated excitation
e.g. Structured illumination or focal modulation
removes haze, but subtraction noise remains3. temporal gating (may reduce noise but also signal)
Rainer Heintzmann12
UnnessesaryBleaching
SSelectiveelective P Planelane I Illuminationllumination MMicroscopyicroscopy
Illumination
Plane of focus
Solution 2: Solution 2: UltramicroscopyUltramicroscopy
Rainer Heintzmann13
SSelectiveelective P Planelane I Illuminationllumination MMicroscopyicroscopy
Illumination
Light Sheet
Cylinder Lens
Solution 2: Solution 2: UltramicroscopyUltramicroscopy
Rainer Heintzmann14
SSelectiveelective P Planelane I Illuminationllumination MMicroscopyicroscopy
Illumination
Detection
Solution 2: Solution 2: UltramicroscopyUltramicroscopy
Rainer Heintzmann15
3D reconstruction of large 3-dimensional microscopic systems
Material above or below the focal plane are not illuminated no out-of-focus blur & no out-of-focus bleaching
Simple collection optics images illuminated area onto a camera
Rotation different angles of view 3D tomographic image reconstruction
Solution 2: Solution 2: UltramicroscopyUltramicroscopy
Rainer Heintzmann16
3D reconstructionMouse embryo E12.5.
(A) Surface (B) Stained nerve fibers (C) Surface of head(D) Sensory nerve fibers innervating the vibrissae.
Because the mouse embryos are opaque, a special clearing technique is applied:
Index matching:2 parts benzyl benzoate and one part benzyl alcohol).
Solution 2: Solution 2: UltramicroscopyUltramicroscopy
Rainer Heintzmann17
Overview: Direct ImagingOverview: Direct Imaging
Stimulated Emission Depletion Stimulated Emission Depletion MicroscopyMicroscopy
Pointillism, PALM and STORMPointillism, PALM and STORM SSelective elective PPlane lane IIllumination llumination
MMicroscopyicroscopy Structured IlluminationStructured Illumination Circumventing the limit: Circumventing the limit:
NonlinearityNonlinearity Interferometric Resolution Interferometric Resolution
EnhancementEnhancement
Rainer Heintzmann18
Problem: Limited Problem: Limited NNumerical umerical AApertureperture
Immersion Medium
Objective Lense
Cell
Cover Slip
Immersion Medium
x,yz
Slide
NA = n sin
Rainer Heintzmann19
Solution: Axial Solution: Axial TomographyTomography
Glass Fiber
Immersion Medium
Objective Lense
Aperture increaseby rotation of the specimen
Cell
Cover Slip
Immersion Medium
Shaw et al.,Cogswell et al.,Kawata et al.,Heintzmann et al.
x,yz
Rainer Heintzmann20
Combined Combined MLML--DeconvolutionDeconvolution
Compare
Register
View 1View 1 View 2View 2
MiEi
Convolution
Reconstructed Estimate
Simulated
Apply
Back Convolution
,,,,
CC NCNC
Measured
View 1View 1 View 2View 2
Rainer Heintzmann21
Biological SpecimenBiological Specimen
Polytrichum Commune
Fully automatically registered
Moss Spore
R. Heintzmann and C. Cremer., J. Microsc., 206 (1), 7-23, 2002
Rainer Heintzmann22
Biological SpecimenBiological Specimen
Polytrichum Commune
Fully automatically registered
Moss Spore
Rainer Heintzmann23
Solution 2: Solution 2: UltramicroscopyUltramicroscopy
• Much less out-of-focus bleach• complicated sample mount• Absorption/scattering loss can be a problem
(excitation and emisission)
• combinations (2-photon excitation) with structured illumination orswept line illumination are possible
Rainer Heintzmann24
Solution 3: Aberration Solution 3: Aberration CorrectionCorrection
• Quality improvement is often only moderate• Estimation of aberrations is
difficult and time consuming(Selective Aperture, iterative procedure, ..)
• spatially varying aberrations are very hard
Pre-compensation element (SLM)