Introduction to Advanced Instruments

Multiphoton and Confocal Microscope System

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國立交通大學生物科技學院分子醫學與生物工程研究所Phone: 03-5712121 ext 56968 (office) 56969 (Lab)

E-mail: hwangeric@mail.nctu.edu.tw

黃兆祺

Fluorescence Microscopy

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Why Fluorescence?• High contrast

• High specificity

• Quantitative

• Live cell

imaging

Fluorescence

Quinine

Excitation

Emission

http://en.wikipedia.org/wiki/Quinine

Photoluminescence

When specimens, living or non-living, organic or

inorganic, absorb and subsequently re-radiate light,

the process is described as photoluminescence.

Phosphorescence

Emission of light persists after the excitation light is

discontinued.

Fluorescence

Emission of light only during the excitation light is

present.

Fluorescence vs. Phosphorescence

10-9 sec 1~2 sec 10-3 sec

Bioluminescence

Bioluminescent waves

Loghan Call and Jonathan Keena, 2001

Fluorescence in Cell Biology

We can label cells (or subcellular structures) with a fluorescence marker and use a

specific wavelength of light to “excite” it. The excited fluorescence marker can

produce light with a specific wavelength.

Fluorescence Spectra

Caused by the loss of vibrational energy.

Emission spectrum is shifted to longer,

lower energy wavelengths.

Spectral overlap:

This demonstrates why we need the

appropriate excitation filter, dichroic

mirror, and emission filter. This is because

the much brighter excitation light

overwhelms the weaker emitted

fluorescence light.

Changing the wavelength of excitation

does not change the wavelength of the

emission – based on molecular properties.

Stokes Shift

Fluorescence Filters

Excitation Filter

Allows the transmission of a specific portion of excitation light.

Dichromatic Mirror (or beamsplitter)

Reflects shorter wavelengths,and transmits longer wavelengths.

Emission Filter (or barrier filter)

Allows the transmission of a specific portion of emitted light.

Fluorescence Microscope Configuration

Fluorescent Molecules

Extinction coefficient – the ability of a fluorochrome to absorb light.

Quantum yield – ratio of photons emitted relative to photons absorbed, the value is between 0 and 1. It is influenced by molecular characteristics and environment (pH, ions).

Properties that Affect Fluorescence

Traditional Fluorescent Dyes

Notice all of them are cyclic molecules!

Cy Dyes

Enhanced water solubility, higher quantum yield, pH insensitivity, and photostability

Alexa Fluor Dyes

Enhanced water solubility, higher quantum yield, pH insensitivity, and extremely

high photostability.

Mahmoudian

Fluorescent Molecule Targeting

Direct targeting

Certain fluorescent molecules can target specific DNA, RNA,

proteins, or organelles (e.g. DAPI, propidium iodide,

MitoTracker, LysoTracker).

Fluorophore conjugation

Attaching fluorescent molecules to antibodies or targeting

molecules that can recognize specific cellular components.

Fluorescent protein fusion

Fusing a fluorescent protein to a protein of interest.

Direct Targeting: DAPI Stained DNA

C. elegans

http://www.wormbook.org

Fluorophore Conjugation:

Tubulin antibody conjugated with Alexa Fluor

Mouse neurons

Confocal Microscopy

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Why Confocal?

Remove out of focus light!

Confocal vs Widefield Microscopy

widefield

confocal

medulla muscle fiber pollen

http://zeiss-campus.magnet.fsu.edu/tutorials/opticalsectioning/confocalwidefield/index.html

http://www.olympusconfocal.com/theory/confocalintro.html

Confocal vs Widefield Microscopy

confocalwidefield

Confocal vs Widefield Microscopy

~0.6 µm >2 µmAxial

resolution<0.2 µm ~0.8 µm

http://zeiss-campus.magnet.fsu.edu/articles/livecellimaging/techniques.html

Total Internal Reflection

Total internal reflection

Total Internal Reflection Fluorescence

At the critical angle, the beam of excitation light is totally reflected from the glass/water interface. The

reflection generates a very thin electromagnetic field (< 200 nm) in the aqueous medium, which has an

identical frequency to that of the incident light. This field, called the evanescent wave, undergoes

exponential intensity decay with increasing distance from the surface.

n2 x sin(θc) = n1 x sin(90º) = n1

sin (θc) = n1/n2 = 1/n2

n1

n2

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html

TIRF Penetration Depth

Fluorescence Intensity versus Penetration Depth

I(z) = I(o) e-z/d

I(z): intensity at a distance z

I(o): intensity at interface

d: penetration depth

n1

n2

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/tirf/tirfintro.html

TIRF vs Widefield Fluorescence

Jaiswal and Simon, 2007

lysosomes

交大共儀-全反射螢光倒立顯微系統

Nikon Ti-TIRF-E System

雷射系統

操作及應用分析軟體活細胞觀察系統

Confocal Microscopy

The principle of confocal imaging was

developed in 1950s by Marvin Minsky and

aims to eliminate out-of-focus signal.

As only light produced by fluorescence very

close to the focal plane can be detected, the

image's optical resolution, particularly in the

sample depth direction, is much better than

that of widefield microscopes.

Marvin Minsky

Principles of Confocal Microscopy

http://www.bio.brandeis.edu/marderlab/microscopyB.html

Confocal Microscope ConfigurationLaser Scanning Confocal Microscope (LSCM)

http://www.leica-microsystems.com/

Laser and combinerConfocal module

MicroscopeComputer

Vibration isolation table

Confocal Microscope Components

http://www.olympusconfocal.com/theory/confocalintro.html

Laser and Laser Combiner

http://micro.magnet.fsu.edu/primer/lightandcolor/lasersintro.html

Photomultiplier Tube

What does the PMT see?

http://micro.magnet.fsu.edu/primer/java/digitalimaging/photomultiplier/endonpmt/index.html

Galvanometer Mirror Scanner

Galvanometer Mirror Scanner

https://www.youtube.com/watch?v=qCUiCs-aYNk

Advantage of Confocal Microscopy

(1) Optical sectioningProduce thin (~0.6 μm) optical sections through thick (50~200 μm) specimen.

Advantage of Confocal Microscopy

(2) 3D reconstruction of specimenOptical sections can be used to re-construct the specimen in 3D space.

https://vimeo.com/sciinstitute

When Do You Need to Use LSCM?

Analyzing the expression level of a cytosolic protein No

Analyzing the intracellular localization of a protein Yes

Analyzing the localization of a protein in the lamellipodium No

Analyzing protein level in phenol red-containing medium Yes

Fluorescent live cell imaging No

Analyzing protein co-localization Yes

Analyzing fluorescent protein dynamics in ms scale No

Multiphoton Excitation Microscopy

Principle of Multiphoton Excitation

• Predicted in 1930 by Maria Göppert-Mayer.

• Simultaneous absorption of two photons in a single quantized

event (about 10-18 second or one attosecond).

• Need high photon flux (0.1 – 10 MW/cm2).

http://micro.magnet.fsu.edu/primer/java/multiphoton/jablonski/index.html

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/multiphoton/multiphotonintro.html

Multiphoton Excitation Is Rare

In bright day light a good photon absorber absorbs

in a 1-photon process: once a second

in a 2-photon process: once every 10 million years

Leica Multiphoton Microscopy

Titanium-sapphire Laser

http://en.wikipedia.org/wiki/Ti-sapphire_laser

The average power is ~1 W, but the peak power is over 150,000 W!!

Multiphoton Excitation

• Two-photon absorption by a fluorophore is a function of the

square of the excitation intensity.

• Pulsed laser beam intensity drops as the square of the distance

from the focal plane.

http://micro.magnet.fsu.edu/primer/java/multiphoton/excitationregion/index.html

http://micro.magnet.fsu.edu/primer/techniques/fluorescence/multiphoton/multiphotonintro.html

Multiphoton Excitation

Image by Steve Ruzin and Holly Aaron, UC Berkeley

Dilute FITC solution in a quartz cuvette (side view)

2-photonwidefield

Multiphoton Photobleaching

• Due to the decreased excitation volume, the sample will not photobleach

outside of the focal volume.

• The focal volume is usually 0.3 μm in lateral direction and 1 μm in axial

direction.

• Other techniques (such as confocal microscopy) bleach the sample

throughout the light path.

http://micro.magnet.fsu.edu/primer/java/multiphoton/excitationbleaching/index.html

顯微鏡本體

共軛焦掃描器

操作及應用分析軟體

雷射系統

交大科技部貴儀-多光子共軛焦顯微系統

Leica TCS SP5 X + Two photon system

Multiphoton vs. ConfocalBulky Object

Egner et al., J. Microsc 2002

Comparison of the optical sectioning capability of the multiphoton and the spinning disk

confocal microscopes for an extended, bulky object. Surface rendered 3D-image of two

pollen grains of similar diameter (~25 µm). The multiphoton image possesses a much

lower background than its spinning disk confocal counterpart.

confocal2-photon

Multiphoton vs. ConfocalSmall Object

Comparison of the optical sectioning capability of the multiphoton and the spinning disk

confocal microscopes for small objects. Surface plot of GFP-labeled mitochondria

(~6 µm). In the case of a small object, multiphoton and spinning disk confocal show

almost identical optical sectioning capabilities.

confocal2-photon

Egner et al., J. Microsc 2002

Multiphoton vs. ConfocalImaging Depth

A shark choroid plexus stained with FITC collected at 80 μm below the specimen surface

(200 μm is the maximal imaging depth for confocal microscopy). The overall image

contrast in the confocal image is greatly reduced by the presence of background fog.

confocal 2-photon

https://www.microscopyu.com/articles/fluorescence/multiphoton/multiphotonintro.html

Imaging Depth Limiting Factors

1. Objective lens working distance

2. Light penetration depth

Tissue Clearing ReagentsSCALE reagent for tissue clearing

Hama et al., Nat Neurosci, 2011

Absorption of Biological Tissues

http://en.wikipedia.org/wiki/Near-infrared_window_in_biological_tissue

WaterHemoglobin

Near-infrared (NIR) Window

http://en.wikipedia.org/wiki/Near-infrared_window_in_biological_tissue

Effective penetration depth for breast tissue

NIR window = 626~1316 nmNIR window = 626~1316 nm

Multiphoton vs. Confocal

https://www.microscopyu.com/articles/fluorescence/multiphoton/multiphotonintro.html

Signal Strength

Multiphoton vs. ConfocalSignal Strength

A shark choroid plexus stained with FITC collected at 140 μm below the specimen

surface. It is clear that the 2-photon image in the middle is fully saturated in many areas,

demonstrating the improved signal collection.

confocal 2-photon

1000 V 750 V1000 V

https://www.microscopyu.com/articles/fluorescence/multiphoton/multiphotonintro.html

Multiphoton vs. ConfocalResolution

The image resolution obtained with multiphoton excitation is usually

WORSE than that achieved in a confocal microscope. If a biological

structure cannot be resolved in the confocal microscope, it will similarly

not be resolved in a multiphoton excitation microscope.

The utilization of longer excitation wavelengths (such as red or infrared),

although an advantageous aspect of two-photon excitation, actually results

in a larger resolution spot.WHY?

Resolution = 0.61 • λ/NA

Application of Multiphoton Microscopy

Objective

neuron

blood vessel

neuron

blood vessel

Aβ

Objective working distance limited

• Reduced photodamage and photobleaching

• Increased imaging depth in specimen

• Selective excitation of fluorophores by two and three photons

• Improved signal strength (elimination of confocal pinhole)

Advantages of Multiphoton