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Introduction to Advanced Instruments
Multiphoton and Confocal Microscope System
1
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: [email protected]
黃兆祺
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Fluorescence Microscopy
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Why Fluorescence?• High contrast
• High specificity
• Quantitative
• Live cell
imaging
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Fluorescence
Quinine
Excitation
Emission
http://en.wikipedia.org/wiki/Quinine
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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.
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Fluorescence vs. Phosphorescence
10-9 sec 1~2 sec 10-3 sec
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Bioluminescence
Bioluminescent waves
Loghan Call and Jonathan Keena, 2001
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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.
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Fluorescence Spectra
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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
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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.
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Fluorescence Microscope Configuration
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Fluorescent Molecules
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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
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Traditional Fluorescent Dyes
Notice all of them are cyclic molecules!
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Cy Dyes
Enhanced water solubility, higher quantum yield, pH insensitivity, and photostability
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Alexa Fluor Dyes
Enhanced water solubility, higher quantum yield, pH insensitivity, and extremely
high photostability.
Mahmoudian
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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.
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Direct Targeting: DAPI Stained DNA
C. elegans
http://www.wormbook.org
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Fluorophore Conjugation:
Tubulin antibody conjugated with Alexa Fluor
Mouse neurons
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Confocal Microscopy
21
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Why Confocal?
Remove out of focus light!
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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
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Confocal vs Widefield Microscopy
confocalwidefield
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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
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Total Internal Reflection
Total internal reflection
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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
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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
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TIRF vs Widefield Fluorescence
Jaiswal and Simon, 2007
lysosomes
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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
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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
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Principles of Confocal Microscopy
http://www.bio.brandeis.edu/marderlab/microscopyB.html
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Confocal Microscope ConfigurationLaser Scanning Confocal Microscope (LSCM)
http://www.leica-microsystems.com/
Laser and combinerConfocal module
MicroscopeComputer
Vibration isolation table
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Confocal Microscope Components
http://www.olympusconfocal.com/theory/confocalintro.html
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Laser and Laser Combiner
http://micro.magnet.fsu.edu/primer/lightandcolor/lasersintro.html
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Photomultiplier Tube
What does the PMT see?
http://micro.magnet.fsu.edu/primer/java/digitalimaging/photomultiplier/endonpmt/index.html
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Galvanometer Mirror Scanner
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Galvanometer Mirror Scanner
https://www.youtube.com/watch?v=qCUiCs-aYNk
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Advantage of Confocal Microscopy
(1) Optical sectioningProduce thin (~0.6 μm) optical sections through thick (50~200 μm) specimen.
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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
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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
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Multiphoton Excitation Microscopy
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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
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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
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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!!
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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
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Multiphoton Excitation
Image by Steve Ruzin and Holly Aaron, UC Berkeley
Dilute FITC solution in a quartz cuvette (side view)
2-photonwidefield
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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
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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
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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
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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
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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
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Imaging Depth Limiting Factors
1. Objective lens working distance
2. Light penetration depth
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Tissue Clearing ReagentsSCALE reagent for tissue clearing
Hama et al., Nat Neurosci, 2011
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Absorption of Biological Tissues
http://en.wikipedia.org/wiki/Near-infrared_window_in_biological_tissue
WaterHemoglobin
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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
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Multiphoton vs. Confocal
https://www.microscopyu.com/articles/fluorescence/multiphoton/multiphotonintro.html
Signal Strength
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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
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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
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Application of Multiphoton Microscopy
Objective
neuron
blood vessel
neuron
blood vessel
Aβ
Objective working distance limited
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• 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