CYTO 2014 Notes: Fluorescence Lifetime Workshop

Monday, May 26, 2014
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This is the first blog in a series covering CYTO 2014, held this May 17-22 in Ft. Lauderdale, Florida. Mainly in the form of raw notes, the intent of this series is to highlight talks, events, and trends from the annual cytometry congress and trade show that I found noteworthy. Clarifications, corrections, comments, and other suggestions welcome.

The Fluorescence Lifetime Workshop took place Monday, May 19. Co-chaired by Profs. Jessica Houston (New Mexico State University, Las Cruces, NM) and Silas Leavesley (University of South Alabama, Mobile, AL), the workshop featured the following panel of presenters: Patrick Jenkins (New Mexico State University, Las Cruces, NM); Prof. Miho Suzuki (Saitama University, Japan); Prof. János Szöllősi (Debrecen University, Debrecen, Hungary); and Dr. Ralph Jimenez (JILA/NIST and University of Colorado, Boulder, CO).

About 50 people attended the workshop. My notes of the presenters' talks follow--see also other pictures of the event.

Patrick Jenkins, NMSU  
Fluorescence Lifetime Flow CyTAUmetry
   - frequency domain FLT FC
   - FLT CVs in beads: ~ 5-20%
   - FLT CVs in cells: 15-30%
   - FLT CVs in yeast: 7%
   - yeast: tealFP strain and darkCitrine-tealFP FRET strain
   - FLT only difference (2 vs. 3 ns)
      heavy spectral overlap and similar FL intensity
   - sorted @ 60-99% purity on either strain

Miho Suzuki, Saitama U. 
Signal Transduction with FLT FC
   - chimeric FRET / FLIM based bioprobes
   - based on combining different biomaterials
   - organic dyes, FPs, QDs in different pairwise FRET combinations
   - cysteine – rare aminoacid
   - GFP has two instances of cysteine
      one inside the barrel and one on the outside
      latter suited for chemical mods
   - ID’d two aminoacids in GFP suitable for FRET
   - used Flicyme 300 FLT FC

János Szöllősi, Debrecen U. 
FLIM & TIRF
   - collaborator: György Vereb (fmr student)
   - what kind of bio questions need FRET-FLIM?
   - ErbB receptor tyrosine kinases and their associations
   - problems
   - uneven labeling of tissue
   - low intensity due to poor labeling locally
   - limited usefulness
   - ErbB1-4, HER1-4
   - ErbB1 also = epidermal growth factor receptor
   - ErbB2 – no ligand; if overexpressed, high chance for metastasis
   - ErbB3 – no kinase activity
   - involved in MAPK and PI3K signaling pathways
   - looking at heterogeneity of FLT signal within the cell (pixel by pixel)
   - FRET tells you if the proteins have associated
   - use Lambert LI-FLIM added to microscope
   - Olympus Microscope IX81 and IX2
   - Olympus 4-ch. CellTIRF
   - motorized stages
   - donor only: 3.44 ns; donor acceptor: 3.19 ns;
      donor acceptor + EGF: 2.89 ns
   - FLT shift under archazolid treatment: ~300 ps (FLT distrib ~ 1 ns wide)
   - phasor plot to look at stoichiometry of associated proteins

Ralph Jimenez, JILA/NIST/U. Colorado
Lifetime and Photobleaching for new FPs
   - organic dyes ~100x more stable than FPs
   - brightness of different FPs peaks at 532 and goes to 20% at 425 and 650
   - want to develop brighter, more stable FPs
   - photostability increased by O2 reduction
   - pulsed photobleaching assays on single HeLa cells (2 ms on, 8 ms off)
   - photobleaching needs to be studied at relevant intensities
      (10^2-10^5 W/cm2)
   - microfluidic channel with multiple bleaching
      532 nm beams in line source config
   - use optical trapping with 1064 nm beam to steer cells to “keep” path
   - studied mCherry (most photostable in mFruit family)
   - found mCherry mutant with 3x higher stability but 2x lower quantum yield
   - use additional laser beam to measure lifetime and stability simultaneously
   - Q (JS): saturation?
   - A (RJ): no, 1 photon every 20 ns



Image credits: CYTO/ISAC

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