1. STED: Nanoscale 3D Optical Imaging Digvijay Raorane & Arun Majumdar Department of Mechanical Engineering Department of Materials Science University of California, Berkeley Materials Sciences Division Lawrence Berkeley National Laboratory

2. Outline Motivation Introduction: Conventional Optics Near Field Imaging STED -Theory -Previous experimental work On-going experiments

3. Virus Microtubules Cell Organelle Biological Imaging TMV 16.5 nm ER canaliculi dia. 40-60 nm Microtubule 25 nm

4. Need for High Resolution Optical Technique Biomolecules that require imaging are typically 1- 50 nm in size Far-field optics (e.g. confocal) limited to resolution > 200 nm, which cannot directly resolve molecular-scale phenomena Atomic Force Microscopy cannot be used inside a cell Optical/fluorescence imaging is most-widely used approach for real-time intracellular visualization NSOM (Near-field Scanning Optical Microscope)

5. Optical Imaging at a Glance Far-Field Optics Near-Field Scanning Optical Microscope (NSOM) d Aperture-limited spatial resolution d ~ 50 nm Diffraction-limited spatial resolution ~ λ /2*NA

6. NSOM Limitations Single fiber is limited to the field of view to ~ 50 nm. It is difficult to maintain the tip at the constant distance from the sample within few nms. Tip can get damaged by the thermal stress due to the light. Scanning a whole cell area (10  m x 10  m) takes time. Multi-location imaging and dynamics cannot be observed. Fiber-drawing and aperture fabrication is not repeatable, producing different imaging conditions each time. Tip may get clogged when biological sample is in its buffer medium. Tip Profile http://micro.magnet.fsu.edu Tip Damage Rosa et al., Appl. Phys. Lett. 67, (18), 2597-2599 (1995)

7. What is Stimulated Emission Depletion (STED) Microscopy?

8. Spontaneous Emission - Fluorescence Wavelength, Absorption Vibrational Relaxation Absorption Emission ex Spontaneous Emission sp-em  ~10 ns

9. Fluorescence Imaging Diffraction-limited spatial resolution ~ λ /2*NA

10. Stimulated Emission Wavelength, Absorption Vibrational Relaxation Absorption Emission ex st

11. Physical Realization Conceptual Set-up Avalanche Photodiod e Fluorescence 645-715 nm λ/2 Phase Plate Hell S. et al.,Nature Biotech., 21(11), 2003 Excitation Spot STED Spot Fluorescence Imaging Spot, d<< /2*(NA)

12. Point Spread Function Engineering Weiss S. et al., PNAS, 97 (16),pg. 8747–8749,2000 Non Linear Optical Effect - STED laser quenches tail of PSF due to excitation laser => reduction in FWHM of resultant focal spot incident on fluorescence sample

13. Resolution Resolution (FWHM) dependence on Intensity For typical experiment, FWHM (Δr)

14. STED Imaging Wavelength, Absorption Vibrational Relaxation Absorption Emission ex st img

15. Proof of Concept Westphal et al, APL, 82(18), 3125 - 3127 (2003) Westphal et al,PRL 94, 143903 (2005) Al 2 O 3 matrix wetted by Polymethyl Methacrylate

16. Experimental Set up LPC Ti:Sa Laser OPO PS CH DC 1DC 2 SAMPLE Detector Delay Fluorescence ex st img

17. Collaboration: Prof. Costas Grigoropoulos (ME Dept, UCB)

18. Quantum dot as substitute to fluorescent tags - To test compatibility of Q Dots with STED microscopy to overcome photobleaching of fluorescent labels Hines et al,Advanced Materials,15, 1845, 2003 Alivisatos et al, Nature Biotech., 22, 47 – 52, 2004 Nucleus with actin fibres On-Going Work

19. Advantages of STED Microscopy High resolution can be achieved routinely ( < 50 nm) No need of probe/tip Signal can be collected at far-field Better than confocal microscope in terms of resolution Multiple areas can be probed by forming multiple spots on the sample Resolution depends on the laser intensity ( FWHM = f( Intensity)) Incorporates most widely used fluorescence technique by biologists Can image live biological sample Optical system is simple to understand Can scan the sample in z direction for 3 D image Hell et al, J. Opt. Soc. Am. A, 9(12), 2159 – 2166, 1992 Yeast Mitochondria Mammalian Golgi 3 D Image