PH880 Topics in Physics
Modern Optical Imaging (Fall 2010)Modern Optical Imaging (Fall 2010)
KAIST PH880 10/11/2010
Review of week 6
• Monday
‐ Fluorescent
‐ Fluorescent microscopypy
• WednesdayWednesday‐ Live cell imaging & fluorescent proteins (FPs)
‐ Advanced techniques in fluorescence‐ Advanced techniques in fluorescence
‐ FCS
‐ FRET
‐ etc
KAIST PH880 10/11/2010
Cell structures and functions
n∼1.34‐1.4
n∼1.4‐1.5
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FCS: fluorescent correlation spectroscopy
2
( ) ( )( )
I t I t dtG
I
ττ
+= ∫
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FRAP, FLIP, and so on
Nature Reviews Molecular Cell Biology 5, 855‐862 (October 2004)
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gy , ( )
Overview of week 7
• Monday
‐ Confocal microscopy: principle
‐ Resolution
‐ Setup
• Wednesday‐ Confocal microscopy: applications‐ Confocal microscopy: applications
‐ Spinning disk confocal
Applications‐ Applications
KAIST PH880 10/11/2010
Problems in (wide‐field) fluorescent microscopy
B i HiImage
Brain Hippocampus
Depth of focus (thickness of image plane)
http://www.olympusfluoview.com
Depth of focus (thickness of image plane)~ 300 nm (for high NA obj lens)c.f. sample thickness: mammalian cell 5‐15 um
Sample For a given 2D fluorescence image, more than 90% of fluorescent is out‐of‐focus light
KAIST PH880 10/11/2010
Optical sectioning is needed
Depth of focus & Depth of fieldDepth of Field : the range of distances (in object space) for which object points are
Depth of field vs. NAPSF (in x‐z plane)
imaged with acceptable sharpness with a fixed position of the image plane
Note:Depth of focus refers to image space, and depth of field refers to object space
KAIST PH880 10/11/2010
(often used interchangeably with each other)
Principle of Confocal Microscopybeam path (reflection geometry)
Wide‐fieldFluorescence
Confocalsectioning
KAIST PH880 10/11/2010Carl Zeiss webpage
Optical Sectioning microscopy
Confocal microscopy‐ Laser scanning confocal microscopy‐ Spinning‐disk confocal microscopyLi i‐ Line scanning
Deconvolution (computational optical microscopy)Deconvolution (computational optical microscopy)
Structured Illumination Microscopy
Optical Projection TomographyOptical Coherent MicroscopyOptical Coherent Microscopy
KAIST PH880 10/11/2010
Principle of Confocal MicroscopyPoint illumination & point detectionPoint illumination & point detection
Pinhole
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Principle of Confocal MicroscopyPoint illumination & point detectionPoint illumination & point detection
b i id lLaser beam is ideal: all energy in a
collimated coherent lplane wave
Point illuminationPoint detection
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First confocal microscopypatented in 1961patented in 1961
Prof Marvin Minsky (MIT)Prof. Marvin Minsky (MIT)
KAIST PH880 10/11/2010
History of confocal microscopySummarized from WB Amos et al Biology of the Cell (2003)Summarized from WB Amos et al, Biology of the Cell, (2003)
•The word ’confocal’ has been first used and confocal microscopy was demonstrated pyexperimentally by (Brakenhoff et al., 1979)
•The underlying physics was understood (Wilson and Sheppard, 1984)
•A spinning Nipknow disk was introduced to illuminate the specimen with multiple points, (Petran et al, 1968)
•The first biologically‐convincing results were obtained by Brakenhoff et al (1985) (images of nuclei in which the chromatin, stained with a fluorescent dye)
Refs:Brakenhoff, G.J., Blom, P., Barends, P., 1979. Confocal scanning microscopy with high‐aperture lenses. J. Microsc. 117, 219–232232.Wilson, T., Sheppard, C., 1980. Theory and Practice of Scanning Optical Microscopy. Academic Press, London.Petran, M., Hadravsky, M., Egger, D., Galambos, R., 1968. Tandem scanning reflected light microscope. J. Opt. Soc. Amer. 58, 661–664Brakenhoff G J van der Voort H TM van Spronsen E A Linnemans WA M Nanninga N 1985 Three dimensional
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Brakenhoff, G.J., van der Voort, H.T.M., van Spronsen, E.A., Linnemans, W.A.M., Nanninga, N., 1985. Three‐dimensional chromatin distribution in neuroblastoma cell nuclei shown by confocal scanning laser microscopy. Nature 317, 748–749
Scanning schemes
Ill i ti iS l i Illumination scanningSample scanning
Move specimen
Avoid off‐axis aberrationVibration issue
Off‐axis aberration(astigmatism, coma and field curvature)Little Vibration
KAIST PH880 10/11/2010JA Conchelloe and JW Litchman, Nature Methods, 2005
Scanning schemes
(slow)
Horizontal: fast scanVertical : slow scan
“4‐f system”(fast)
y
Problems: chromatic aberration in 4‐f system, rotating polygon does not produce
KAIST PH880 10/11/2010WB Amos et al, Biology of the Cell 95 (2003) 335–342
the ideal purely‐rotatory movement, noise issue in the polygon
Scanning schemes
No chromatic aberration: design for all the confocal point‐scanning systems manufactured by Bio‐Rad since 1991
KAIST PH880 10/11/2010WB 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
Setup for (reflection) fluorescent confocal microscopy
Note: scanning and descanning are done by the same oscillating mirror
KAIST PH880 10/11/2010JA Conchelloe and JW Litchman, Nature Methods, 2005
Photomultiplier tubes (PMT)multiply the current produced by incident light by as much as 100 million times (i.e., 160 dB), in multiple dynode stages.
‐photoelectric effect: electrons are emitted from matter as a consequence of their p qabsorption of energy from photon. ‐Secondary emission: when primary incident particles of sufficient energy (charged electrons or ion) hit some material, the electrons are emitted.
KAIST PH880 10/11/2010It enables individual photons to be detected (when the incident flux of light is very low.)
Florescence intensity in confocal microscopy
KAIST PH880 10/11/2010JA Conchelloe and JW Litchman, Nature Methods, 2005
3D PSF revisited
When NA < 0.5,
KAIST PH880 10/11/2010Carl Zeiss, Confocal Laser Scanning Microscopy
Pinhole size
Appropriate size of pinhole: 50‐80% of the diameter of the diffraction limited spot
Smaller pinhole: better sectioning weaker signalSmaller pinhole: better sectioning, weaker signalLarger pinhole: worse sectioning, stronger signal
KAIST PH880 10/11/2010
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 10/11/2010
3D PSF in confocal microscopy
to consider both λexc and λem, a mean wavelength was introduced.
KAIST PH880 10/11/2010Carl 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 10/11/2010Carl Zeiss, Confocal Laser Scanning Microscopy
Optical sectioning property in confocal microscopy
KAIST PH880 10/11/2010
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 10/11/2010
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 10/11/2010
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 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.
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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 10/11/2010
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)
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g y ( y)
Summary: Widefield v.s. Scanning confocal
KAIST PH880 10/11/2010DJ Stephens and VJ Allen, Science, 2003
Other optical sectioning technique:Optical Projection TomographyOptical Projection Tomography
KAIST PH880 10/11/2010
Overview of week 7
• Monday
‐ Confocal microscopy: principle
‐ Resolution
‐ Setup
• Wednesday‐ Confocal microscopy: applications‐ Confocal microscopy: applications
‐ Spinning disk confocal
Applications‐ Applications
KAIST PH880 10/11/2010
Reading List (wk 5 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.
KAIST PH880 10/11/2010
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