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Fluorescence Microscopy Phys 570V: Advanced Topics in Optics and Photonics Professor Tongcang Li Lecture 24: Optical imaging beyond diffraction limit Course website: http://www.physics.purdue.edu/academic- programs/courses/course_detail.php?SEM=fall2015&c=phys570V Syllabus, Lecture notes, etc. Purdue University Fall 2015 Physics 570V Room: Phys 331 Time: MW 2:30-3:45 PM 1 Generation of entangled photon pairs • Cascade in atomic transitions (eg. Ca) 2 Generation of entangled photon pairs • Down-conversion in nonlinear crystal 푛휔 푘 = 푐 Phase matching 3 This lecture: Optical imaging beyond diffraction limit The Nobel Prize in Chemistry 2014 Eric Betzig, Stefan W. Hell, William E. Moerner “for the development of super-resolved fluorescence microscopy" 4 Abbe’s Diffraction limit Abbe Resolutionx,y = λ/2NA 2 Abbe Resolutionz = 2λ/NA 5 Fluorescence microscopy https://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html 6 Content • 1. confocal microscope • 2. Near field scanning optical microscope (NSOM) • 3. Two-photon optical microscope • 4. Stimulated Emission Depletion Microscope (STED) • 5. Single-molecule localization microscopes: PALM, STORM, etc. 7 Confocal microscopy A pinhole in the back focal plane rejects the light coming from outside the focal plane. The pinhole size is a trade-off between good rejecting ability and sufficient light throughput (typically ~ 30 – 150 mm) wide field PMT confocal MPD CCD … image is scanned point by point dichroic whole image at once Resolution in confocal microscopy: Slightly higher resolution than in wide field microscopy (improvement ~ 1.4) when a very small pinhole is used. ~ 3D Gaussian profile The image is a convolution of the object and the PSF Confocal vs. Wide field microscopy: Wide-field: Confocal: Elimination of out-of-focus light improves contrast and, thus, resolution Confocal microscopy: Focusing only in one plane axial sectioning of the sample to ~ mm slices Near field scanning optical microscope (NSOM) Journal of Applied Physics 59, 3318 (1986); 12 Bell Jar Hallen lab, NC State 13 Signal Strength vs Resolution Resolution only depends on aperture, not wavelength Theoretical: 1/r6 scaling 50 nm practical limit Hallen lab, NC State 14 Scanning Probe Feedback Mechanism: AFM and NSOM same implementation 15 Near Field Scanning Optical Microscopy/Spectroscopy (NSOM) of Advanced Organic Thin Film Materials Joseph Kerimo, David M. Adams, David A. Vanden Bout, Daniel A. Higgins and Paul F. Barbara 16 Apertureless NSOM 17 Limitations • Shallow depth of view. • Weak signal • Difficult to work on cells, or other soft samples • Complex contrast mechanism • Slow 18 Two-photon optical microscope excited state excitatio emission emission n excitatio n excitatio n ground state One-photon excitation Two-photon excitation 19 The difference between single- and two-photon excitation Optical sectioning 20 Wide-field vs. confocal vs. 2-photon Drawing by P. D. Andrews, I. S. Harper and J. R. Swedlow 21 Resolution of 2-photon systems Using high NA pseudoparaxial approximations1 to estimate the illumination, the intensity profile in a 2-photon system, the lateral (r) and axial (z) full widths at half-maximum of the two-photon excitation spot can be approximated by2: 0.532 1 z 0.32 0 2 2 NA 0.7 2 n n NA 2 NA r0 0.325 NA 0.7 3 2 2 0.91 2 NA V2h r0 z0 1) C. J. R. Sheppard and H. J. Matthews, “Imaging in a high-aperture optical systems,” J. Opt. Soc. Am. A 4, 1354- (1987) 2) W.R. Zipfel, R.M. Williams, and W.W. Webb “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotech. 21(11), 1369-1377 (2003) 22 Practical resolution Effect of increased incident power on generation of signal. Samples of acid- fucsin-stained monkey kidney were imaged at a depth of 60 µm into the sample by confocal (550 µW of 532-nm light) and by multiphoton (12 mW of 1047-nm light) microscopy. Laser intensities were adjusted to produce the same mean number of photons per pixel. The confocal image exhibits a significantly narrower spread of pixel intensities compared to the multiphoton image indicating a lower signal to background ratio. Multiphoton imaging therefore provides a high-contrast image even at significant depths within a light-scattering sample. Images were collected at a pixel resolution of 0.27 µm with a Kalman 3 collection filter. Scale bar, 20 µm. Centonze VE, White JG. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys J. 1998 Oct;75(4):2015-24. 23 Bartek Rajwa Stimulated Emission Depletion (STED) Microscope Drive down to ground state with second “dump”pulse, Before molecule can fluoresce Quench fluorescence and Combine with spatial control to make “donut”, achieve super-resolution in 3D (unlike NSOM) 24 25 26 Setup 27 Excitation and deexcitation beams for 3D STED Hein B et al. PNAS 2008;105:14271-14276 Klar T A et al. PNAS 2000;97:8206-8210 28 Resolution improvement in STED Klar T A et al. PNAS 2000;97:8206-8210 29 Example: Subdiffraction resolution fluorescence imaging of microtubules Hein B et al. PNAS 2008;105:14271-14276 30 Single-molecule localization methods: PALM, STORM, etc. PALM: photoactivated localization microscopy STORM: stochastic optical reconstruction microscopy 31 Photo-active GFP G. H. Patterson et al., Science 297, 1873 -1877 (2002) This paper reported a photoactivatable variant of GFP that, after intense irradiation with 413- nanometer light, increases fluorescence 100 times when excited by 488-nanometer light and remains stable for days under aerobic conditions Native= filled circle Photoactivated= Open squares Wild-type GFP T203H GFP: PA-GFP 32 Photoactivation and imaging in vitro. G. H. Patterson et al., Science 297, 1873 -1877 (2002) 33 https://www.microscopyu.com/articles/superresolution/stormintro.html 34 Stochastic optical reconstruction microscopy (STORM) 35 36 3-D (z) resolution 37 Fig. 2. Three-dimensional STORM imaging of microtubules in a cell. Conventional indirect immunofluorescence image of microtubules 3D section (color coded) C-E zoom in of box in B B Huang et al. Science 2008;319:810-813 38 39 Final presentations: 11/30-12/9 Date Presenter 11/30 Eric Topel Mikhail Cong Wang Robert Shalaginov Sutherland 12/2 Dewan Woods Jaehoon Bang Yu Gong Zhuoxian Wang 12/7 Nirajan Mandal Di Wang Jonghoon Ahn Jin Cui 12/9 Ting-wei Hsu Jie Hui 40 Final presentations (12-minute talk + 3 minute Q&A). Grading guide: 1. Content (technical comprehension and explanation of the topic) 2. Organization and visual aids (Logical sequence, text, graphics) 3. Verbal presentation (speaking volume, rate, eye contact, body language, enthusiasm for the topic) 4. Overall impression (interesting talk, understanding and answering questions correctly, etc.) 41.
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