Fluorescence Nanoscopy

Microscopy

Lasers

Optics 高甫仁高甫仁高甫仁高甫仁& Collaborators陽明大學生醫光電工程研究所陽明大學生醫光電工程研究所陽明大學生醫光電工程研究所陽明大學生醫光電工程研究所July 23, 2010

Outline

�A brief overview on optical microscopy

�Toward optical super resolution __ Abbe’s diffraction limit and nanoscopy

�Fundamentals and opportunities of FLIM/FRET

�Visualizing cell metabolism with autofluorescence lifetime imaging

�Future prospects

Imagining imaging’s future

NATURE SS20 | SEPTEMBER 2003

Imagining imaging’s future

NATURE SS20 | SEPTEMBER 2003MEG: Magnetoencephalography AOH: Acoustic Optics Hybrid

Starting in the 16th century from a magnifier

The 19th century saw dramatic progress in the development of the microscope, thanks to the contributions of such great minds as Carl Zeiss, who devoted significant effort to the manufacture of microscopes, Ernst Abbe, who carried out a theoretical study of optical principles, and Otto Schott, who conducted research on optical glass.

Diffraction Limits

M. Minsky, US Patent 3013467, Dec. 19, 1961

T. Wilson and C.J.R. Sheppard, Theory and Practice of Scanning Optical Microscopy, (Academic, London, 1984)

Peter Török, P. Varga, Z. Laczik and G. R. Booker, Electromagnetic diffraction of light focused through a planar interface

between materials of mismatched refractive indices:

an integral representation, J. Opt. Soc. Am. A, 12, 325( 1995)

Confocal Microscopy

Marvin Minsky 1957

Confocal Microscopy

Z Koana 1943

Petrán 1968

16ms 20ms 24ms 28ms

Post-electrostimulation changes in Ca²+ in mouse ventricular cardiac muscle cell labeled with Fluo-3 , images taken at 4 ms intervals

Oxford microscope, 1975

Amar Choudhury, Colin Sheppard, Pete Hale and Rudi Kompfner

Oxford, Summer 1976

Marvin Minsky 1957

Confocal Microscopy

Z Koana 1943

One of the Microscopy Challenges:Elucidating Molecular Dynamics in Biology

without Destroying the MoleculesDiffract Limit: A barrier originated from the Uncertainty Principle

Difficult for life science to be alive at λ λ λ λ < 450 nm

Diffraction Limit: ∆∆∆∆R~λλλλ/NA

Ernst Abbe memorial at

Jena, Germany:

Nanoscopy:Seeing Beyond the Diffraction Limit

1. Single Fluorophore Localization microscopy

(a) Stochastic optical reconstruction microscopy

(STORM), 2006, Xiaowei Zhuang

(b) Photoactivated localization microscopy

(PALM), 2006, by Eric Betzig, Harald Hess, and

Samuel Hess.

(c) SPDM C. Cremer, 2008.

2. Saturated structured illumination microscopy

(SSIM) by Mats Gustafsson.

3. Stimulated emission depletion (STED) by Stefan

Hell.

From micro to nano

200 nm �100 nm � 30 nm (� 10 nm)

Optical Localization Microscopy

1. Single molecule (or fluorophore)

activation

2. Single molecule detection

3. Centralizing

4. Image reconstruction

Courtesy of Eric Betzig

A moiré pattern and structured illumination

Mats Gustaffsonhttp://www.bioengineering.ucsf.edu/faculty-

mats_gustafsson.vp.html

Courtesy of Mats Gustaffson

NATURE BIOTECHNOLOGY V21 # 11 NOVEMBER 2003

Courtesy of Stefan Hell

4Pi Microscopy

http://www.mpibpc.gwdg.de/abteilungen/200/4Pi.htmhttp://www.mpibpc.gwdg.de/abteilungen/200/mmm.html

http://www.uchsc.edu/lightmicroscopy/sted4pi.htm

Courtesy of Stefan Hell & Alex Egner

STED-4Pi Microscopy

http://www.mpibpc.gwdg.de/abteilungen/200/STED.htm

http://www.mpibpc.gwdg.de/groups/hell/

UV microscope

(Near field microscope)

Structured illumination

STED

PALM & STORM

Range of Molecular Interaction

The gap of optical microscopy

http://www.mindfully.org/Technology/2004/Buckyball-Football1jul04.jpg

Nanoscopy:

Seeing Beyond the Diffraction LimitFrom micro to nano

200 nm �100 nm � 30 nm (� 10 nm)

Limits:1. Note that the critical distance for bio-interaction is ~10 nm.

2. The photon statistics

3.The “brute force” dimension

the “free” dimension

PSF Volume ( )4

1

NA∝

The solution may lie in the temporal domain.

Starting with Molecular States __Franck-Condon and Jablonski Energy Diagram

Dipole-dipole interaction1. Van der Waal force

2. FRET (Förster/Fluorescence Resonant Energy Transfer)

2 23

κ ≈

p2A→→→→

p2B→→→→p2

C→→→→

E1→→→→

E1→→→→

E1→→→→r̂

θθθθ1111 →→→→2222

^

r→→→→

θ1

p2^

E1→→→→

⊥⊥⊥⊥∧∧∧∧

E1→→→→

p1→→→→

⊥⊥⊥⊥∧∧∧∧⊥⊥⊥⊥

∧∧∧∧ kT =1

τrD

R0

R

6

Criteria of FRET

• Overlapping of the emission spectrum of D and the absorption spectrum of A. (> 30%).

• Close proximity of two fluorophores (within 1 to 10 nm).

• Favorable mutual orientation.

• High quantum yield of the donor.

R

κ κ κ κ : orientation factor

D A

Fluorescence

photon

Laser pulseFluorescence

photon

Many idling cycles (for

avoiding piling up)

Start-stop-time 1 Start-stop-time 2

Histogram of correlated interval

Fluorescence Lifetime Measurement

1. The concept of a very fast stop watch2. Naturally compatible with confocal microscope using pulsed lasers.

A Time-domain Technique: Time Correlated Single Photon Counting (TCSPC)

2-p Time-correlated single photon counting microscopy

FLIM = Confocal+ Pulsed laser + Time-resolved module

The Advantages of Photon Counting

• High temporal resolution (~0.1 ns)

• Great signal-to-noise ratio (the SMD capacity)• Intensity fluctuations in light source is not critical• Intuitive data interpretation

The Significance of FLIM/FRET

• To distinguish “co-localization” and “binding” with nanometer (1~10 nm) sensitivity

• in vivo and in situ observation

FLIM is far more quantitative than ratio imaging (intensity FRET), not affected by photobleaching, dilution, ..etc.

Scope of FLIM/FRET:Detection of Bio-interactions and Micro-environment

changes that may be revealed by dipole-dipole interaction

1. Cell-to-Cell Signaling: ligand/receptor

2. Immune System: antigen-antibody interactions

3. Expression of Genes : DNA/regulator protein interactions

4. Metabolism: Formation of enzyme complex

5. Cell Division: DNA hybridization

6. Protein Folding

7. Changes of micro-environment surrounding the reporting fluorophores

Cell Vitality and Metabolism

through

Auto-fluorescence

Autofluorescence_first demonstrated in the 50s of the 20th century

1. No labeling

2. Generally ignored due to its weakness

3. Very weak signal: photon statistics is important

4. Short excitation wavelength: 2-p excitation is

recommended

5. Broad spectral distribution: spectrum shift does not

provide much useful information

6. Lifetime may exhibit interesting information

FluorophoreMain exc. peaks

(nm)

Main emission peaks

(nm)

Fluorescence lifetime

(ns)

Tryptophan 275 350 2.8, 1.5

Collagen 340, 270, 285 295, 395, 310 9.9, 5.0, 0.8

Elastin 460, 360, 425, 260 520, 410, 490, 410 6.7, 1.4, 7.8, 2.6, 0.5

NADH 350 460 0.6, 0.2, 2.5, 6

FAD, Flavins 450 535 ~4

FMN 440 520 4.7

β-carotene 520 9.6, 2.0, 0.3

Porphyrins 400 610, 675 9.6, 2.0, 0.3

Lipofuscin 340-395 540, 430-460 -

The The autofluorescenceautofluorescence

300 700500 1100900 15001300 2100 3000 nm

Visible light spectrum

2-photon excitation Most staining dyesSi or GaAs semiconductorSHG

Spectral range of a small frame Ti:Sapphire laser

SHG of Ti:Sa

Kr-Ar

568

Nd:YVO4

532

GaN

403

Ti:Sapphire

He-Ne

633

Light source

λλλλ

Applications

Ar ion

514

He-Ne

543

Ar ion

488

Wide bandgap material Small moleculesGas phase sample

2-p UVB excitation Aromatic amino acidsWide bandgap semiconductor

Bio-window Low IR absorption3-photon processes (THG,EFISH) Interfaces

SHG signal idler

2100 3000

idler1600 2100

OPO signal1350

1300

OPOsignal

1050650525

pump pump

920460 700350

Ar ion

365

Light Sources for Confocal Microscopy

Solid State Laser

The Promises of Ultrafast laser

Nonlinear Optics (SHG, THG, 2-p)Spectral and Time-resolved StudiesFrequency Comb (Nobel 2005)…..

Coming Opportunities on

Optical Microscopy

1. Stimulated Emission_ Stefan Hell & Sunny Xie

2. Fiber laser and Photonics Crystal Fiber_ Nonlinear

optics, Spectroscopy

3. Hybrid detectors__ high QE, large area, and fast

4. Polarization resolving

5. Photon Statistics_ Single Molecule Detection,

Physics at work

Retro-

Reflector

Retro-

Reflector

Long

pass

½ wave

plate

+Polarizer

½ wave

plate

800

NIR

50/50

ultrafast

beam splitter

½ wave

plate

+Polarizer800

NIR800

NIR

800

NIR

800

NIR

800

NIR

800

NIR

800

NIR

Long

passObjectiv

e

20X

Objectiv

e

40X

PCF

800 nm

RELP

FLIM CARS Fluorescence Lifetime Imaging

Live HuH (human hepatoma) liver cells

FLIM

Hoescht:

long

lifetime

DiO: short

lifetime

CARS

0 ns 1 ns 3 ns2 ns

Albert Stolow: This worked

extremely well and, within days, the

team was able to demonstrate - for

the first time - a new capability: a

FLIM-CARS microscope system…..

137mm

105mm

39mm

Mechanical Base Design Mechanical Base Design

50

22--Axis VCM and Pickup ModuleAxis VCM and Pickup Module

150mm

130mm

130mm

X-axis

Y-axis

Z-axis

AdjustStage

X-Y Stage for NRC Project：：：：Resolution of X axis: 0.5 um

Resolution of Y axis: 0.5 um

Thank You for Your Attention !