CHU, Steve, Biophysics and bio-medicine in a new light, Applied Phys Colloq, May 21, 2013

Prof. Steven Chu, Nobel Laureate; former Sec of Energy for Obama 2009 to 2012.
Just returned to Stanford Med School in molec imaging program.

This is in Hewlett Teaching Center 201 at 4:15PM in Applied Physics Colloq.

intro: he did atomic physics  (and won a Nobel)

slide 1:  shows Roentgen:  first Nobel Prize winner.
first xray was his wife's hand with a wedding ring!
so,  medical imaging and physics go way back.

great list of imaging methods:

xray, EM, NMR, CR, Positron  PET, monoclonal Ab labeling, radionuclides,
EM staining,  GFP labeling,  single  fluor molec manipulation and imaging, etc.

OUTLINE for today:
1)  sub-wavelength resolution
2) bio-film imaging etc.

Ras-Raf imaging...

Kinesin and dynein....

he shows famous video clip of vesicle transport by a dynein molecule  walking along microtubule.

slide:  great  picture of  dynein:  from  Ahmet Yildiz...

how did they do imaging: put fluorophore on feet and watch as it walks....

300 nm is abt. the Abbe limit (of image separation)...
but consider a different issue:
can you find the center of a dot if u have enough light...
so, instead of 300 nm, u can do 30 nm (or smaller), if u have enuf light.

***********
next in the sub-wavelenth imaging of multiple probes...

Random excitation: PALM and STORM

now he shows 4 hills instead of just one (each is 300 nm at base)
find centroid and turn on each one in turn...
its as clear as your PALM or.... it took the world by STORM. (he quips)

2 groups: Eric Beitz  and also  Xiaowei Zhuang.
(we just heard XZ lecture last week, now at Harvard: stunning talk: also see 
my notes on that one)

can we do better than 10 nm localization?

to quote my former boss (Obama) (he quips)  YES WE CAN!

a single fluor molec is imaged onto a 4 by 4 CCD... but what is it?
difft pixels have sl difft photosensitivity...

move stage with PZT  (piezo) by just a few angstroms  and then centroid shfits
from one pixel to the next...

can get a mapping from your CCD array to what is actually there...

eg   take a 6Kbase dye-labeled DNA molec  550nm stretched by
optical tweezers holding a bead....

could image single fluor molec...

use Cy-3 fluor

their distance resolution was 0.7 nm = 7 angstroms!!!

but one of Xiaowei Z's post docs has a new dye that can give up millions of photons...

uses feedback correction...

submitted to Nature:  reviewers came back and said... show it to us in a living cell..
until then, this is just physics, not bio.

so, he shows applic of nanometer imaging to e-cadherin X dimer and trans dimer structures...

he shows nice picture of M;M dimer...

could get down to a few nm with real molecule...
  getting 10 to 20 nm resol  (and even sub nm) resol.

************
Veysel Berk etc al...
the assembly and structure of vibro cholerae biofilms...

bacteria protect themselves by developing biofilms...
sugar-starch polymers...  it's a protective cladding against attack of host...
he shows biofilm formation...

1908 Nobel... phagocyte... 
he shows photo of phagocyte devouring anthrax.

next, shows bio-fouling on marine vessles...
 need to dry dock them to clean off bio-fouling..

termites...
termites convert ligno-cellulose ito biofuels...

inside gut of termite are colonies of bacteria:
hundreds of difft types that do the digestion and
they also form a biofilm...

Yogi Berra:  (great American philosopher of the 20th century)...
if u don't go to your friend's funeral, he won't come to yours!

say u try to do in situ label of of bio-film matrix proteins using
antibodies to the protein exuded outside of cell...
but it's a blur...

next shows color stop motion stages of growth in bio-film labeling...
next shows confocal scope...  big incr in detail  but... at 200 to 300 you're at the limit...

but at super-res... u can see  indiv molecules... 
see polymer as it is being grown, molec by molec.
Xaoiwei Zhuang... did STORM  (see my notes on her lecture from last week)

now see "circulation system" of the bio-film... u see channels thru it where
nutrients go in and waste goes out...
new method of attack on biofilms... attack their circulation  (eg to kill vibrio)
*****************************
(new topic)

Cell-cell signaling/ imaging...

he shows video of cells dividing...

but they get signals from outside telling them when to divide and when to stop...
complex signaling network  (can't just divide willy nilly)

he shows tyrosine kinase receptors...
when they bind a ligand on the outside ...  but then the INSIDE
undergoes chain reaction...  RAS to RAF  to ERK

pathway is RTK to Ras to Raf to MEK to ERK...

but... if u've got a bad version of Ras or Raf  it will activate this signalling pathway autonomously
even without the ligand binding to RTK  => basis of some cancers...
uncontrolled proliferation..

worked for malig. melanoma  (rx based on RAF)

he shows diagrams of  BRAF-mutant melanoma
and RAS-mutant melanoma

these are mechs for  BRAF kinase inhibitor rx of malig melanoma  Plexxikon PLX4032...shs

used  CherryRed  infected cell with this red color...
can see the pairs...  shows slide with pairs at abt 40 nm apart...

this is with PAmCherry1...

but now look in a cell...

he shows  TIRF  total internal reflectance microscopy...

in cancer cell, you see dimers...  get RAF dimers...

previously used  EM = electron microscopy.. not v sensitive
cuz v low label efficiency...

now use Tetracycle to regulate expression...

link  TetOn with variable doses of Doxy  and lnked to  photofluor... so you can see it...

he shows PALM imaging  as u increase the dose of doxycycline (TCN )
to turn on system and suddenly see lots of RAF pais

so, now have a new path to drug discovery...

now we have a means  of  actually seeing whether small molecules ( possible drugs)
actually attaches and turns off signalling...

cancers mutate like crazy  (and they are survivalists)
 so they look for other means of proliferating;

he is age 65...have to think twice abt starting a " new family at age 65;"
he talks abt Axel.. collab at SLAC...

wants to get a dozen investigaotrs to form a hub to do similar imaging work...