Nanoscale measurement examples in biology: Sub-diffractive imaging & the fly brain challenge

Transcription

1 Nanoscale measurement examples in biology: Sub-diffractive imaging & the fly brain challenge Harald Hess, HHMI Janelia Farm Introduction to HHMI Janelia Farms Sample: Nerves, Worm Brains, Fly Brains, Rodent Brains The data challenge! Probes and Microscopies: Existing Electrophysiology Fluorescent labels Microscopes Innovating Examples Electrophysiology Labels Enhancing contrast Improving Resolution PALM & ipalm

2 Goals at Janelia Farms Two synergistic objectives: Identify general principles that govern how information is processed by groups of neurons Develop enabling imaging technologies

3 Core Janelia Computation, Imaging Genetic & Chemical Probe Engineering Genetic Computation Analysis Imaging, Microscopy Application Electrophysiology Application Behavior Innovation Imaging, Microscopy, Electrophysiology

4 The Sample One Neuron Worm C. Elegans A Fly A Rodent

5 The Sample Nerve Cell Size Synapse 100 nm Dendrites mm Soma(body) mm Axons 0.1 >10 mm dendrites synapse Worm C. Elegans 302 Neurons 60 mm dia ~8000 Synapses Connectivity Maps.

6 Imaging Volume of Fly Brain Electron Microscope Resolution 300 mm 5 nm 300,000 Neurons 1 Billion Synapse 200 Tera Voxels 1 Tera Voxel

7 Fly Brain Inspection : Semiconductor Chip Inspection If Fly Brain were Pentium 4 Dual Core sectioned to 0.07 micron and spread out Die Fly 5 cm 2 5 cm 2 65 nm Wire Axon Tiled Fly Brain Sections 200 Million Transistors Synapse 200,000 CLB(FPGA) Nerve Cell

8 Genetic Variants Multiple Dimension Challenge History Variants, Training, Toxins, Samples=S D 30 samplings on each of 6 axis 1 billion samples x trillions of voxels Scale Up! Need Help!

9 Existing tools to probe Electrophysiology Special Fluorescent Probes Fluorescent Proteins, (FP) Functionality in FP s Ca++ imaging Channel rhodopsin Uncaging Scanning confocal Small section EM

10 Patch Clamp - Silicon Tetrode Probes

11 Fluorescent Protein Labels For Cell Red Fluorescent Protein Label + excited + fluorescent Cell Protein A Yellow Fluorescent Protein Label Cell Protein B + excited + Red Labels Protein Yellow Labels Protein fluorescent

12 Why Flourescence Microscopy for Cell? ~10,000 component Proteins Fluorescent labels! E.g. Green Fluorescent Protein & others Effective light bulb to label a protein of choice =

13 Functionalized Fluorescent Proteins Ca++ Sensitive FP s Uncage Neurotransmitters Voltage Sensitive Membrane FP s Activable Fluorescent Proteins

14 Block face scanning electron microscopy (SEM) imaging of rat cortex work of Winfried Denk

15 Innovating tools to probe Electrophysiology Customized-designer probes Harness/enhance Fluorescent Probes Designer fluorescent proteins sense Action Pot Splitter Designer caged dyes Activatable FP s more wavelengths PALM a sub diffractive microscopy ipalm a 3D sub diffractive microscopy Higher throughput serial section EM Combine EM & Optics sample prep protocols

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18 Triangulate on a Nerve Sci Fi Horror Movie

19 Innovating tools to probe Electrophysiology Customized-designer probes Harness/enhance Fluorescent Probes Designer fluorescent proteins sense Action Pot Splitter Designer caged dyes Activatable FP s more wavelengths PALM a sub diffractive microscopy ipalm a 3D sub diffractive microscopy Higher throughput serial section EM Combine EM & Optics sample prep protocols

20 Improving FP performance Na Ji, Eric Betzig Pulse Splitter: Motivation two-photon signal a I 2 photodamage a I b b~ Pulse Splitter: Design Concept 1-to-4 split one pulse into N sub-pulses: damage reduced by N 1-b/2 (e.g., damage N-fold less for b= 4) 1-to-10 t n 1 d 1 n 0 d 0 q 0 50% 50% 0% 33% 40% 50%

21 Innovating tools to probe Electrophysiology Customized-designer probes Harness/enhance Fluorescent Probes Designer fluorescent proteins sense Action Pot Splitter Designer caged dyes Activatable FP s more wavelengths PALM a sub diffractive microscopy ipalm a 3D sub diffractive microscopy Higher throughput serial section EM Combine EM & Optics sample prep protocols

22 Practical Resolution Limit High NA Immersion microscopy The Abbe Limit (1873) a l/n n sin(a) = NA l resolution= d = ~ 200 nm 2nsin( a) biomolecular scale ~ 1 nm

23 Bio-Molecular vs. Optical Scale Diffraction Limited Spot GFP Green Fluorescent Protein 200 nm 1 nm

24 Molecular Localization Gaussian Center Fit Accuracy: = 200 nm ~ Photons

25 Density Challenged: spacing < diffraction limit??

26 Photoactivatible Fluorescent Proteins unactivated activated + + absorb 405 nm to activate non-absorbing fluorescent

27 1) Establish Sparse Subset with Fractional Activation Excite & Bleach Excite & Bleach Excite & Bleach Switchable Flourescent Protiens: Kaede, Eos 2)Localize: Fit Point Spread Function s ~ 200 nm Center Location w Error ~ sen ~ 10 nm

28 New Microscope Total Internal Reflection Fluorescence (TIRF) w 405 nm & 561 nm laser & single photon sensitive CCD courtesy: Molecular Expressions

29 TIRF image 70 nm Section Lysosomes FP: Kaede Marker: CD63 cell: COS mm

30 PALM image 70 nm Section Lysosomes FP: Kaede Marker: CD63 cell: COS mm

31 Mitochondria Section Eos in lumin, TIRF image 1 mm

32 Mitochondria Section Eos in lumin, PALM image

33 Mitochondria Section Eos in lumin, TEM image

34 PALM TEM Composite Image

35 Dual Color PALM: Application to Adhesion Complexes DIC Dual Color PALM of Cytoskeletal and Adhesion Complex Proteins in HFF Cells TIRF PALM red = Dronpa / a-actinin green = tdeos / vinculin partial co-localization observed between a-actinin & vinculin H. Shroff, et al., PNAS 104, (2007)

36 Innovating tools to probe Electrophysiology Customized-designer probes Harness/enhance Fluorescent Probes Designer fluorescent proteins sense Action Pot Splitter Designer caged dyes Activatable FP s more wavelengths PALM a sub diffractive microscopy ipalm a 3D sub diffractive microscopy Higher throughput serial section EM Combine EM & Optics sample prep protocols

37 Accessing vertical locations Defocus & combine with PALM Hess Betzig Patent, Xiaowei Zhuang, Sam Hess, Joerg Bewersdorf Interferometry nm Phase Shift Interferometric Microscopy What is Interferometry???? How it works Performance & Demo Interferometry (z) + PALM (x,y) ~10-20 nm imaging 3D Demonstrations Limitations and planned extensions Call for samples, challenges

38 Z position by defocus symmetric or asymmetric Estimate from Spot Size & Shape B. Huang et al., Science 319, 810 (2008). M. F. Juette et al., Nature Methods 5, 471 (2008). other 3D R. Schmidt, et al. Nat. Methods 5, 539 (2008). M. G. L. Gustafsson et al., Biophysical J. 94, 4957 (2008).

39 Single Photon Interferometric Fluorescence Imagining Mirror CCD j 1 I 1 = ½ * (E 1 +E 2 ) 2 = A 2 * (1+sin(2kd)) L 1 L o S d L 1 L 2 50:50 beamsplitter nm Mirror CCD j 2 I 2 = ½ 0.5 * (E 1 -E 2 ) 2 = A 2 * (1+sin(2kd+p))

40 Single Photon Interferometric Fluorescence Imagining Mirror CCD j 1 I 1 = ½ * (E 1 +E 2 ) 2 = A 2 * (1+sin(2kd)) L 1 L o S d L 1 L 2 50:50 beamsplitter nm Mirror CCD j 2 I 2 = ½ 0.5 * (E 1 -E 2 ) 2 = A 2 * (1+sin(2kd+p))

41 Next Single Photon Fluorescence Interferomety Highest resolution Z position + PALM = 3D SuperRes Best Sensitivity for Endogenous Fluorescent Proteins Instrument Extend Z range - Modulo signal with 250 nm 2 nd color for IPALM Convenient Sample Loading/Throughput Sample mounting Improve EM Compatibility Labels & Sample Preparation Brightness # molecules On/off contrast ratio Bio Compatibility Other contrast Fixation: Ultrastructure and Fluorescence preservation Applications - Samples Open bio-relevant problems

42 Innovating tools to probe Electrophysiology Customized-designer probes Harness/enhance Fluorescent Probes Designer fluorescent proteins sense Action Pot Splitter Designer caged dyes Activatable FP s more wavelengths PALM a sub diffractive microscopy ipalm a 3D sub diffractive microscopy Higher throughput serial section EM Combine EM & Optics sample prep protocols

43 Collaborators PALM Eric Betzig HHMI Michael Davidson NHMFL FSU Scott Olenych NHMFL FSU Jennifer Lippencott Schwartz NIH George Patterson NIH Rashid Sougrat NIH Juan Bonifacino NIH Wolf Lindwasser NIH Gleb Shtengel Hari Shroff Suliana Manley Rick Fetter Derik Greenfield Anne McEvoy Jen Gillette Clare Waterman Tony Pakorn ipalm New NanoFab Probes Herschel Marchman EM Rick Fetter Mehdi Bolorazedhi HHMI HHMI NIH HHMI Berkeley Berkeley NIH NIH NIH HHMI HHMI HHMI