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PH880 Topics in Physics
Modern Optical Imaging (Fall 2010)Modern Optical Imaging (Fall 2010)
KAIST PH880 10/13/2010
Overview of week 7
• Monday
‐ Confocal microscopy: principle
‐ Resolution
‐ Setup
• Wednesday‐ Confocal microscopy: applications‐ Confocal microscopy: applications
‐ Spinning disk confocal
Applications‐ Applications
KAIST PH880 10/13/2010
Principle of Confocal Microscopybeam path (reflection geometry)
Wide‐fieldFluorescence
Confocalsectioning
KAIST PH880 10/13/2010Carl Zeiss webpage
Principle of Confocal MicroscopyPoint illumination & point detectionPoint illumination & point detection
Pinhole
KAIST PH880 10/13/2010
Overview of week 7
• Monday
‐ Confocal microscopy: principle
‐ Resolution
‐ Setup
• Wednesday‐ Confocal microscopy: applications‐ Confocal microscopy: applications
‐ Spinning disk confocal
Applications‐ Applications
KAIST PH880 10/13/2010
Florescence intensity in confocal microscopy
KAIST PH880 10/13/2010JA Conchelloe and JW Litchman, Nature Methods, 2005
3D PSF revisited
When NA < 0.5,
KAIST PH880 10/13/2010Carl Zeiss, Confocal Laser Scanning Microscopy
Pinhole size: tradeoff btw optical sectioning & SNR
Appropriate size of pinhole: 50‐80% of the diameter of the diffraction limited spot
Smaller pinhole: better sectioning, weaker signalLarger pinhole: worse sectioning, stronger signal
KAIST PH880 10/13/2010
g p g, g g
Resolution in confocal microscopy
Wide‐field Confocal
( )( , , ) ( , , ) ( , , )confocal excitation emission pinholePSF x y z PSF x y z PSF x y z= ⋅
Resolving power of the confocal scanning microscopy in approximately 1.4x betterResolving power of the confocal scanning microscopy in approximately 1.4x betterthen in a wide‐field fluorescence microscopy (When pinhole size is properly chosen)
KAIST PH880 10/13/2010
3D PSF in confocal microscopy
to consider both λexc and λem a mean wavelength was introducedto consider both λexc and λem, a mean wavelength was introduced.
KAIST PH880 10/13/2010Carl Zeiss, Confocal Laser Scanning Microscopy
3D PSF in confocal microscopy
Put together,g ,
,tot lateralFWHM XNAλ
=, 2 2( )
tot axialZFWHM
n n NA
λ=
− −( )
22ZnNA
λ(When NA < 0.5)
Y
X
KAIST PH880 10/13/2010Carl Zeiss, Confocal Laser Scanning Microscopy
Optical sectioning property in confocal microscopy
KAIST PH880 10/13/2010
Summary
Conventional microscopy
Confocal microscopy1 AU < PH <∞
Confocal microscopyPH < 1 AU
Optical slice thickness
not definable(optical sectioning is not
possible)
Axial resolution(Depth of Field in(Depth of Field in wave optics)
Lateral resolution
PH is the variable object‐side pinhole diameter in μm.
KAIST PH880 10/13/2010
3D fluorescent image from confocal microscopy
1 2D Laser scanning in the specimen1, 2D Laser scanning in the specimen. 2, a) repeat at the same focus a time series of imageb) step up/down the focus a 3D image stack
a 3D image stack of pollen
Reconstructed image
KAIST PH880 10/13/2010http://www.microscopyu.com
3D fluorescent image from confocal microscopy
1 2D Laser scanning in the specimen1, 2D Laser scanning in the specimen. 2, a) repeat at the same focus a time series of imageb) step up/down the focus a 3D image stack
a time series experiment with Kaede‐ptransfected cells.
* With the irradiation of UV light or violet light (350–400 nm), Kaede undergoes irreversible photoconversion from green fluorescence to red fluorescence.
KAIST PH880 10/13/2010http://www.microscopyu.com
Contrast in Confocal microscopy
1. Fluorophore: fluorescent dye, protein, quantum dotsfor targeting specific molecule
2. Autofluorescence: show fluorescence without labelingchlorophyll (in plant cells), collagen, elastin, fibrillin, flavin, indolamined l d d l l findolamine dimer, indolamine trimer, lipofuscin,
NADH (reduced form only), polyphenols (in plant cells), tryptophan
3. Elastic Light Scattering
4. Raman Scattering: confocal Raman scattering
KAIST PH880 10/13/2010
Contrast in Confocal microscopy
Backscattered light and autofluorescence signals combined:ll l & H G2 llcollagen gel & HepG2 cells
Image courtesy: J. Paul Robinson (Purdue university)
KAIST PH880 10/13/2010
g y ( y)
Summary: Widefield v.s. Scanning confocal
KAIST PH880 10/13/2010DJ Stephens and VJ Allen, Science, 2003
Other optical sectioning technique:Optical Projection TomographyOptical Projection Tomography
KAIST PH880 10/13/2010
Single point laser scanning
No chromatic aberration: design for all the confocal point‐scanning systems manufactured by Bio‐Rad since 1991
KAIST PH880 10/13/2010WB Amos et al, Biology of the Cell 95 (2003) 335–342
design for all the confocal point scanning systems manufactured by Bio Rad since 1991
Issues for real‐time (>30 Hz) imaging in LSCM
(servo‐controlled) linear galvanometerli i d i d 100 H f i id l‐ limited scanning speed: ~ 100 Hz for sinusoidal scan
‐ to raster‐scan 512x512 pix, it takes ~ 5 sec
Resonant galvanometer (aka counter rotation scanner)‐ Stored torsional E is used to oscillate the mirror in a sinusoid manner‐ Very fast: 1‐10 kHz‐ Video rate scanning is possible‐ Resonant Scanning Confocal Microscopy can gather images at 30 Hz
* Typical Excitation E need for Fluorophore is ~ 300 pJ[1]
If 512 512 i ll d i 1 h d ll f 1 / (512 512 ) 4‐ If 512 x 512 pixs are collected in 1 sec, the spot dwells for 1 s/ (512 x 512 ) ~ 4 μs‐ Need to delivery ~ 80 μW of light to each pix!
KAIST PH880 10/13/2010 1. Tsien R, Ernst L, & Waggoner A (2006) Fluorophores for confocal microscopy: photophysics and photochemistry. Handbook of biological confocal microscopy:338‐352
Scanning mechanisms for High Speed Confocal
KAIST PH880 10/13/2010http://www.olympusmicro.com
Spinning disk confocal microscopyNipkow disk, developed by German inventor Paul Nipkow in 1884
KAIST PH880 10/13/2010http://www.olympusmicro.com
Scanning Pattern in Spinning Disk
KAIST PH880 10/13/2010http://www.microscopyu.com
Axial resolution issuecrosstalk between pinholes
KAIST PH880 10/13/2010http://www.microscopyu.com
Yokogawa spinning disk
KAIST PH880 10/13/2010 http://www.microscopyu.comhttp://www.yokogawa.com
Detectors for spinning disk microscopy
KAIST PH880 10/13/2010
Fluorophore crosstalk (aka bleed‐through)
Solutions: ‐ choose a better combination of flourophores
l i ki
KAIST PH880 10/13/2010
‐multitracking‐ QDs
Spectroscopy + confocal microscopy
Atoms and molecules have an intrinsic energy‐band structurecan be examined with spectroscopy and provide useful info
e.g. a relationship btw fluorophore concentration and the fluorescence intensity (linear at low concentrations)
KAIST PH880 10/13/2010
Spectroscopy in confocal microscopy
KAIST PH880 10/13/2010
Overview of week 8
• Monday
‐ Digital Holography
‐ Quantitative Phase Microscopypy
• Wednesday (no class: mid‐term)Wednesday (no class: mid term)
KAIST PH880 10/13/2010
Reading List (wk 7 day 2)
h ll & i h ( ) i l i i i h d1. Conchello J & Lichtman J (2005) Optical sectioning microscopy. Nature methods 2(12):920‐931.
2. Kino G & Corle T (1989) Confocal scanning optical microscopy. Physics Today 42:55.3 T i T t l (2002) Hi h d 1 f / i f l i ith3. Tanaami T, et al. (2002) High‐speed 1‐frame/ms scanning confocal microscope with a
microlens and Nipkow disks. Applied optics 41(22):4704‐4708.4. Xi P, Rajwa B, Jones J, & Robinson J (2007) The design and construction of a cost‐
efficient confocal laser scanning microscope American Journal of Physics 75:203efficient confocal laser scanning microscope. American Journal of Physics 75:203.
KAIST PH880 10/13/2010