ph880 topics in physics · 2010-10-12 · issues for real‐time (>30 hz) imaging in lscm...

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.


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


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.


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


Axial resolution issuecrosstalk between pinholes


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

ph880 topics in physics · 2010-10-12 · issues for real‐time (>30 hz) imaging in lscm...

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