Eric Betzig: Imaging Life at High Spatiotemporal Resolution
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- čas přidán 14. 03. 2015
- In this lecture, held on 3/9/15 at UC Berkeley, Nobel Laureate Eric Betzig, describes three areas focused on addressing the challenges of high resolution imaging: super-resolution microscopy; plane illumination microscopy using non-diffracting beams; and adaptive optics to recover optimal images from within optically heterogeneous specimens.
I really love it when brilliant scientists cuss. Not sure why but it just hits differently.
I started to watch for a few minutes. Here I am, the video is over, and still needing more of his talks.
0:00 Intro by Berkeley physics chair
4:42 Talk begins - what is super resolution? Why fluorescence?
7:34 Eric’s interesting history - near-field, Bell labs
10:09 Two inspired experiments - Moerner 1989, cryo temp spectral signature of single molecule; Harald Hess, probe switch, resolve by color
13:10 Frustration with near-field, hiatus, an idea struck, back to the family business
20:40 Searching for meaning, the GFP light bulb, building the microscope; Optical lattice, photoactivated fluorescent protein, PALM - Nobel Prize, the 4th dimension is time; La Jolla Lab, Jennifer Lippincott-Schwartz, 200nm to 20nm,
Howard Hughes
31:13 PALM at HHMI, 2-color, spatial segregation of core promoter (Tjian), actin dendritic spine
33:21 Sick of PALM, labeling density, dead fixed cells, overexpression, 10,000x brighter than natural light phototoxic, Stefan Hell STED
36:44 Structured illumination (SIM), beat frequency, Moire fringes doubles diffraction-limited resolution, faster, much less intensity, spatial resolution (pixel, exposure) temporal resolution light intensity, Gustaffson, live imaging
40:02 Improve SIM scope objective high 1.7 numerical aperture + TIRF -> 80nm, endocytosis, no photoswitching -> multicolor
42:52 Nonlinear SIM, higher harmonics by saturating fluorescence/photoswitch -> 60nm, early endosome, EM validation
45:05 Sacrificing spatial resolution for 4th dimension time resolution, challenges of 3D imaging: time resolution must improve at the same rate as space (or image will smear), most microscopies are focused on 2D resolution (scans a flat surface; z is poor), unlike nature, also harder to compromise intensity for signal in 3D (1 extra dimension); in short, live much harder than fixed, 3D much harder than 2D
48:43 3D currently just blasting cells with wide field or death star with confocal, most important innovation in microscopy: plane illumination (cylindrical lens from the side, thin sheet of light coincident with plane in focus and illuminate whole plane at once, like a camera), no bleaching, quick imaging; problem: 4 um sized light sheet too big to study cell; solution: diffraction free/Bessel beam illuminates objective with a ring creates tiny little pens of light, like the supermarket checkout scanner, doesn’t diffract/diverge, plane by plane buildup (1000 planes or several volumes/sec)
Another problem: in cross-section looks like a bullseye with concentric rings, as you sweep the light along side lobes create out of focus excitation; solution: 2-photon microscopy to excite just the focus
52:56 Another solution: step light discretely instead of continuously, creates grating pattern, with structured illumination use side lobe to create patterned excitation, poke beyond diffraction limit in 3D
More problem: took too long to step the beam to create pattern; solution: fan the beam into 7 to fix speed but also makes it less damaging to cell, instantaneous intensity much more important than total dose of light: plane > line > point; confocal is a deathray
More “problem:” More split side lobes get too close and interfere,
but separation magic: at certain splits all side lobes destructively interfere
with each other and solves the problem itself; old theory sees light ->
lattice light sheet microscopy, faster, very noninvasive (protein turnover
faster than photobleaching)
57:09 Lattice sheet so thin that it’s thinner than detection objective -> only focus plane excited -> fantastic signal to noise in thick sectioning; gets rid of dye labeling problem with PALM: -> PAINT (Hochstrasser): molecules moving so fast but when they bind to sample then they fix and localizes to spots (good reserve of fluorophore for density but hard to work with high SNR) -> works well with lattice sheet to provide the labeling density
59:45 Lattice sheet with 4D imaging: light sensitive cell division, etc.
1:03:59 Even more problem: scrambled signal due to optical aberration from sample/equipment; solution: adaptive optics -> flat distorted wave front AO system bounce off mirror pick a bit from sensor that determines the deformation and the computer changes the shape of mirror to correct distortion to create a flat wave front; doesn’t gain resolution, recover resolution and signal lost from scattering; historical connection between microscopy and astronomy in that astronomy innovation comes first; in astronomy: light from distant galaxy not very bright (not enough signal to measure error) laser excites sodium atoms to create artificial star by fluorescence -> bright enough and close enough for correction; transparent organism like zebrafish it’s applicable: zebrafish big enough and enough variation in the refractive index across the organism so you have to create many corrections with AO (200um); lattice sheet with both excitation and detection AO and PALM/SIM; Mouse brain scatters like tofu (like observing star through the clouds), but can do AO correction (Na Ji)
1:13:05 Problem with academia: people get too wound up in titles and prizes and papers; it’s subjective; the things that matter is the accomplishment/application/insight; most people won’t be professors, but there are other ways to contribute; tech transfer to ubiquitous use difficult -> solution: set up a center for collaborations with biologists, document the build, work with microscope company (can buy lattice sheet); Betzig lab only 3-5 people
1:19:30 Q&A
Thanks for organizing
Thank you for uploading the video here. It's been a pleasure to watch it and learn from this great person, developer, leader and physicist. Don't know if he ever going to read this, but Sir, I want to thank you for your message to us, the young people, I'm a Genomic Biotechnologist and entrepreneur from Mexico, your words really help me to continue fighting impact my society.
Cheers from Mexico!
My PhD project was about this tec. Thanks Dr Betzig!
this is a major breakthrough for regular scientists.... can you imagine having 10000 biologists, cooridinating on understanding quantum cellular communication using this tool..
I am truly mesmerized. you are amazing Sir Betzig. Hope to meet you one day.
this guy inspires me.
Saved the URL to this. Have to show this to my oldest niece ("the molecular mechanisms of diseases")
I love this speech! Very inspirational and exciting!! - recent Duke BME grad and Harvard Med Student
Standing ovations !
This should have more views.
I FUCKING love the FUCKING swearing. lol. this is amazing work.
loving the language lmao
the talk is fucking amaizing