Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of...

53
Dr. Markus Kohler Page 1 Introduction to Multiphoton Laser Scanning Microscopy Dr. Markus Kohler Carl Zeiss AG Feldbachstrasse 81 8714 Feldbach www.zeiss.ch

Transcript of Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of...

Page 1: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 1

Introduction to Multiphoton Laser Scanning Microscopy

Dr. Markus Kohler

Carl Zeiss AG

Feldbachstrasse 81

8714 Feldbach

www.zeiss.ch

Page 2: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 2

- Fluorescence Microscopy

- Confocal Laser Scanning Microscopy

- Multiphoton Microscopy

Topics

Page 3: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 3

Confocal Laser Scanning Microscopy

Today, it„s about more than pretty pictures….

Page 4: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 4

Conventional fluorescence microscopy

Detection

ZEISS

Plan-NEOFLUAR

40x /1,3 Oil

ZEISS

Plan-NEOFLUAR

40x /1,3 Oil

Excitation

Rat Brain, Double labelling:

Green: Neurons, Blue: Nuclei

Problem:Detecting in-focus information together with out-of-focus signals in wide-field microscopy

Page 5: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 5

Optical sectioning with confocal microscopy

ZEISS

Plan-NEOFLUAR

40x /1,3 Oil

ZEISS

Plan-NEOFLUAR

40x /1,3 Oil

Excitation

Goal:To detect only the fluorescence information from the plane of focus

Detection

Rat Brain, Double labelling:

Green: Neurons, Blue: Nuclei

Page 6: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 6

Fluorescence Microscopy

- Absorption of light rises a fluorochrome molecule to an excited state

of higher energy content

- The molecule remains in the excited state only for a very short period

of time (nsec range)

- The way back to the basic energy level is accompanied by the emission

of light (fluorescence)

- Due to internal energy dissipation the emitted light has a longer wavelength

(=lower energy) than the exciting light (Stokes shift)

- The quantity of emitted light is very small compared to the quantity of

excitation light

Fluorescence Microscopy

Page 7: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 7

E

n

e

r

g

y

W a v e l e n g t h

Internal energy

dissipation

Stokes shift

Excitation light

Fluorescence

Fluorescence Microscopy

Page 8: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 8

FITC Fluorescein-5-isothiocyanat (Isomer I)

Rhodamine, TRITC Tetramethylrhodamine -5 isothiocyanate(5-TRITC; G isomer)

Page 9: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 9

Alexa Fluor™ 350 hydrazide,

sodium salt

TOTO®-3 iodide (642/660)

Page 10: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 10

Comparison of different burners

Ab

so

lute

in

ten

sit

y in

AU

Wavelength (nm)

Page 11: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 11

Excitation filter

Beamsplitter

(dichroic mirror)

Emission filter

HB

O

XB

O

Sample

Page 12: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 12

laser

Emission filter

Monochromatic light,

no excitation filter necessary

Beamsplitter

(dichroic mirror)

Sample

Page 13: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 13

Page 14: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 14

excitation I

emission I emission II

excitation II

Page 15: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 15

Fluorescence Microscopy

www.zeiss.de

Page 16: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 16

Amber fossil (Chironomide)

thickness app. 300 µm

conventional fluorescence

Confocal Microscopy

Page 17: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 17

Amber fossil (Chironomide)

thickness app. 300 µm

confocal imaging

3D reconstruction

Amber fossil (Chironomide)

thickness app. 300 µm

confocal imaging

3D reconstruction

Confocal Microscopy

Page 18: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 18

Laser

Sample

The confocal principle (non multiphoton)

Page 19: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 19

Laser

Sample

PMT

Pinhole

Page 20: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 20

Laser

Sample

PMT

Pinhole

A minute diaphragm, situated in

a conjugated focal plane,

prevents out of focus light to be

detected.

Page 21: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 21

Confocal: Point Scanning

Point scanning confocal systems

XY scanning

Page 22: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 22

X

Y

Sequentially illuminated

sample

Sequentially generated

image

Page 23: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 23

XZ

Y

Optical slicing

Page 24: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 24

Page 25: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 25

Advantages of Confocal Laser Scanning Microscopy

- Efficient excitation by highly focussed laser light

- Adjustable pinhole for the best compromise between resolution and signal

detection

- “Optical slices” for sharp three dimensional reconstructions

- Line scans, spline scans, free definable scan fields, changeable resolution…..

Page 26: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 26

Limitations of Confocal Laser Scanning Microscopy

- The excitation wavelengths are limited by the available laser lines

- The sequential scanning is time consuming

- Direct confocal observation of the sample is not possible

- Excitation and bleaching occurs -as in conventional fluorescence- also in

out of focus planes

Page 27: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 27

Multiphoton Laser Scanning Microscopy

- Confocal Microscopy without pinhole

- The excitation light has a longer wavelength than the emitted fluorescence

- Two -or more- near infrared photons have to be absorbed simultaneously

by a fluorochrome to emit one visible photon

- Only in the focal spot of the laser the excitation energy is high enough to

generate fluorescence

- As the relationship between excitation and emission is no longer linear,

multiphoton microscopy belongs to non linear optics (NLO)

Page 28: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 28

Multiphoton processes - the principle

Multiphoton processes described for the first time in 1931 by Maria Goeppert-

Mayer (Nobel price in physics 1963)

Über Elementarakte mit zwei Quantensprüngen; Göttinger Dissertation: Ann.

Phys. 9: 273-294

Page 29: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 29

Single Photon Excitation: linear process; proportional to the intensity of the

excitation light

Multi Photon Excitation: non linear process; proportional to the square of the

excitation light intensity per excited area

Multiphoton processes - the principle

Pavg

A

2

Page 30: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 30

Multiphoton processes - the principle

Prerequisite for the excitation of fluorochromes with more than one photon of

lower energy

– very high intensity of the excitation light

– strong focus of the light

-> use of pulsed near infrared lasers with high average power

-> use of objectives with high numerical aperture

Page 31: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 31

Multiphoton processes - the principle

Characteristics of Titan:Sapphire Lasers:

• Emission Wavelength: < 700 up to >1000 nm

• Frequency of pulses: 76-90 MHz (F)

• Length of pulses: 100-200 fs (10 –15 s)

Pavg

F Ap

=Peak pulse energy

Page 32: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 32

E

n

e

r

g

y

Internal

energy

dissipation

Visible

fluorescence

Virtual

excitation

state

Excitation light

Page 33: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 33

2-Photon 1-Photon

Focal

Region

Page 34: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 34

2 photon 1 photon

Page 35: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 35

CW laser (continuous wave) for microscopy:

Continuous emission with low average

power

Pulsed laser:

Very short pulses with a very high peak

power

(0,5 W average power with 100 fs pulses

correspond to a peak power of 65.8 kW)

Page 36: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 36

Peak power (kW)

Repetition frequency (Mhz)

Pulse length (fs)

time

Page 37: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 37

Multiphoton Microscopy – practical issues

System components of a Multiphoton Microscope

Laser Scanning Confocal Microscope

Tunable Ultrafast Laser preferably to be tuned via software

Direct coupling of ultrafast lasers

Integration of laser control into the software of the LSM

Page 38: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 38

Multiphoton Microscopy – practical issues

LSM 710 NLO

attached to the

AxioExaminer (fixed

stage) Microscope

with direct coupled

Multiphoton Laser

and AOM Box for

Laser attenuation

Page 39: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 39

Bleaching of fluorescence gels in Z

Single photon excitation

Multi photon excitation

Page 40: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 40

One-Photon – Multiphoton: a comparison

Focal Excitation:

Due to the quadratic dependence on the light intensity only at the focal spot

the excitation of the fluorochrome occurs

Page 41: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 41

Deep tissue penetration of the excitation light due to less absorption and

scattering of NIR light in tissue

One-Photon – Multiphoton: a comparison

Absorption Spectrum of waterAbsorption Spectrum of Oxy-

and Deoxyhaemoglobin

Page 42: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 42

One-Photon – Multiphoton: a comparison

Imaging of fluorescent structures in deep tissue regions

One-Photon excitation Multiphoton excitation

Mouse brain, GFP expressing astrocyte, fixed brain slice, 160 µm deep in the tissue

Page 43: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 43

Bleaching within a Drosophila embryo using multi photon excitation

Page 44: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 44

Examples of Dyes excited with Multiphoton Technique

Fluorochrome Absorption Emission

Alexa Fluor 488 720-800 515

Alexa Fluor 568 720-840 596

Alexa Fluor 633 720-900 647

CY2 780-800 506

CY3 780 565, 615

CY5 780-820 670

DAPI, Hoechst 700-820 455, 478

eCFP 800-900 476

eGFP 820-950 509

eYFP 860-950 532

Fluorescein 780 – 820 519

Lucifer Yellow 860-890 533

Mito Tracker red 750-840 600

Propidium Iodide 820-850 617

Rhodamine 123 780-860 550

Sytox Green 740-760 or 880-940 524

TRITC 800-840 572

Page 45: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 45

5 - Uncompromised Multiphoton Imaging

LSM 710 NLO

Page 46: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 46

5 - Uncompromised Multiphoton Imaging

LSM 710 NLO with new Axio Examiner

Light path and objective to the front

-> no obstruction of view to the sample

-> best accessibility to sample

TFT display as control panel

All control and focus knobs in the front area of the stand

Choice of objective holders for 1, 2 or 4 objectives

Motorized reflector turret

Focus steps of 25 nm

Sample space up to 11 cm for whole animal imaging

-> table and condensor carrier, and transmission NDD port can be mounted and dismounted by user

Page 47: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 47

Up to 5 NDDs in reflection

Up to 5 NDDs in transmission

W Plan Apo 20x 1,0 NA detects 5.6

times more light than IR Achroplan

40x 0.8

AND

Higher NA = tighter spot -> less

laser power necessary to achieve

comparable signal strength

5 - Uncompromised Multiphoton Imaging

LSM 710 NLO with new Axio Examiner

Page 48: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 48

Objective holder with GaAsP NDD available for Axio Examiner

5 - Uncompromised Multiphoton Imaging

LSM 710 NLO with new Axio Examiner

Unique NDD with GaAsP detector inside the objective holder

2x more sensitive than other NDD detectors

Page 49: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 49

Deep Tissue Imaging

GaAsP increases sensitivity over PMT-NDD by factor ~3

Sample: Mouse brain (fixed)

Provided by Steve Turney, Harvard

Labels: YFP

Mode: Multiphoton

LSM 710 (GaAsP-NDD) LSM 710 (PMT-NDD) – Line Average 16

5 - Uncompromised Multiphoton Imaging

LSM 710 NLO with new Axio Examiner

Page 50: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 50

Advantages of Multiphoton Laser Scanning Microscopy

- Lower cytotoxicity of infrared light, especially in comparison to UV light

- Deeper penetration depth into living tissue

- No excitation/bleaching of fluorochromes outside of the focal spot

- With LSM 710 NLO combination of cw and pulsed lasers simultaneous

- LSM 7 MP dedicated multiphoton system

Page 51: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 51

Limitations of Multiphoton Laser Scanning Microscopy

- Pulsed laser is usable just with one tuned wavelength at the time

- Needs some time (seconds) to change the wavelength

- Price for a complete system approx. 800‟000 to 1‟000‟000 Fr

Page 52: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 52

One-Photon – Multiphoton: a comparison

One-Photon Excitation Multiphoton Excitation

Excitation occurs in the whole light

path of the laser beam

Excitation only at the focal spot of the lens

Confocal aperture necessary, reduces

signal gain

No confocal aperture required, higher signal

gain

Imaging limited in thick specimens:

70 - 100 µm are feasible

Visualization of fluorescent dyes up to

several 100 µm deep in the tissue sample

Several lasers to cover a large number

of fluorescent dyes

One laser excites a variety of fluorescent

dyes

Photo toxic events Photo toxicity is reduced

High resolution Slightly lower resolution compared to One-

Photon excitation

Page 53: Introduction to Multiphoton Laser Scanning Microscopy · Fluorescence Microscopy - Absorption of light rises a fluorochrome molecule to an excited state of higher energy content -

Dr. Markus Kohler Page 53

End

Thank you very much for your attention!