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

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SSelectiveelective P Planelane I Illuminationllumination MMicroscopyicroscopy

Illumination

Light Sheet

Cylinder Lens

Solution 2: Solution 2: UltramicroscopyUltramicroscopy

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SSelectiveelective P Planelane I Illuminationllumination MMicroscopyicroscopy

Illumination

Detection

Solution 2: Solution 2: UltramicroscopyUltramicroscopy

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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

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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

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Problem: Limited Problem: Limited NNumerical umerical AApertureperture

Immersion Medium

Objective Lense

Cell

Cover Slip

Immersion Medium

x,yz

Slide

NA = n sin

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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

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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)