Far-field Super-resolution Optical Microscopy

Photo-Activated Localization Microscopy(PALM)

Stochastic Optical Reconstruction Microscopy(STORM)

卢锦胜 2015.10.21

Question before Presentation

Isn't it right that the detail of arbitrarily small things can be see clearly as long as the magnification of the optical microscopy is large enough?

Diffraction limit

23/5/3 2

What is the diffraction limit?

(1) From the perspective of imaging

Lateral resolution:

Axial resolution:

What is the diffraction limit?

(2) From the perspective of PSF (Point Spread Function)

PSF ： In optical microscopy, any point source of light smaller than the diffraction limit appears with a fixed size and shape represented by an airy disk pattern (Gaussian distribution) known as the point-spread function (PSF).

Optical system is actually a low-pass filter!

How to improve resolution within the diffraction limit?

Far-field: Confocal and multi-photon microscopy

4Pi and I5M microscopy

Near-field: Total Internal Reflection Fluorescence (TIRF)

How to transcend the diffraction limit?

Near-Field Scanning Optical Microscopy (NSOM, SNOM)

The evanescent wave has been limited both laterally and axially to within 20 nm and thus transcended Abbe’s limit by an order of magnitude in all dimensions (Betzig and Trautmann, 1992).

What about Far-field?

Far-field Diffraction limit

（ STED）

Space

Time

（ PALM & STORM）

Single-Molecule Localization （ SML ）

N: number of photons gathereda: pixel sizeb: standard deviation of the backgroundsi: standard distributionΔ: diffraction limit

Single-Molecule Localization （ SML ）

Single-Molecule Localization （ SML ）

Three important criteria must be satisfied to achieve SML:

a)The number of photons detected for each fluorophore needs to be high enough;

b)Fluorophore mobility needs to be slow enough, as compared to the image acquisition time;

c)Particle PSFs cannot overlap extensively.

Green Fluorescent Protein(GFP)

405

488

It can, however, be activated by irradiation at 413 nm and then displays fluorescence when excited at 488 nm. Eventually, after intense irradiation at 488 nm the mutant is irreversibly inactivated by photo bleaching. （ Patterson and Lippincott-Schwartz, 2002）

（ PALM）

Acquisition Steps

Three independent steps:

(a)pre-activation bleach—where cells are illuminated by a strong excitation laser;(b)activation—the light-induced activation of an extremely reduced number of fluorophores;(c)readout—excitation and imaging of the activated fluorophores followed by bleaching.

（ PALM）

Set-up（ PALM）

Acquisition Steps

single particle tracking PALM (sptPALM)

Fluorescence PALM (FPALM)

PALM with independent running acquisition (PALMIRA)

STORM

dSTORM

Photo-switching fluorescent dyes

Genetically encoded fluorophores

Photo-switching Process（ STORM）

Acquisition Steps（ STORM）

Set-up（ STORM）

Results(PALM & STORM)

The microtubules and clathrin-coated pits in a BS‑C‑1 cell are immunostained with Cy2-Alexa 647-labelled and Cy3-Alexa 647-labelled antibodies, respectively.

3D- 多色成像：

BS-C-1细胞中的微管及线粒体的多通道成像 (Scale bars, 3 um (a,b) 500 nm (c–e))

CONCLUSIONS

• Resolution ：• Slow imaging time, high stability of microscopy• Positioning accuracy affected greatly by SNR

PALM/STORM contrast• Fluorophores• Imaging time(100)

Episode ：

北京大学副教授的席鹏在这个科研领域颇有发言权，他表示：“庄小威没能获奖，我大感意外。”

资料背景为瑞典皇家理工学院博士徐磊发表博文直称：“诺奖获得者埃里克·贝齐格在超分辨率显微镜方面的贡献不如庄小威”。他认为“贝齐格在技术的持续推动上后继无力，只有庄小威真正推动了这种方法的巨大发展”。

Episode ：

Episode：

REFERENCES

• Henriques R, Griffiths C, Hesper Rego E, et al. PALM and STORM: Unlocking live‐cell super resolution[J]. Biopolymers, 2011, 95(5): 322-331.‐

• http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html• Scientific Background on the Nobel Prize in Chemistry 2014 • https://en.wikipedia.org/wiki/Photoactivated_localization_microscopy• http://zeiss-campus.magnet.fsu.edu/articles/superresolution/palm/

introduction.html• http://baike.baidu.com/link?url=5hV76eiexcR8xYuSu-fDIT86QDGzGr0jgkc-zp-

JVMM1XnbBipg0IJCbRFITYF_0qf5oT-bHpUnJJ0zq8TsEBFIyE9NlfeYrdQ9WP5HDvo3fxERg9auEh8gjFaYWO5KgChA9IMUErMhGE3DZuUphH_-U2mw8KvII3hupuvygTAz3dbmlrnUGr2XiEUwAEn_J

• http://wenku.baidu.com/link?url=qX28KE6L2bvUJNG77gfQVf-dLnLdOuKDNutmh-IfID7vwLwXIxuuP4dtTNLhRo55HbxhywiYD7DV7l0udCYgqFsn0k5JfepqY8Rt-xc-aba

• Teacher’s ppt