TIRFm. New at the OCS Microscopy Core. (Total Internal Reflectance Fluorescence Microscopy) Michael Cammer. Lunch Talk March 2015
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1 TIRFm (Total Internal Reflectance Fluorescence Microscopy) New at the OCS Microscopy Core Lunch Talk March 2015 Michael Cammer
2 Nikon Eclipse Ti microscope
3 History 2010: Purchased with lasers for TIRF by Dr. Michael Dustin 2014: Transferred to Microscopy Core and upgraded by OCS for epifluorescence (LED light sources, new computer, additional lenses & scmos camera)
4 Technical Details Nikon Eclipse Ti inverted microscope Environment chamber with heat unit Motorized stage for tiling and multiple fields imaging Autofocus stability NIS-Elements software
5 Technical Details Lens Type N.A. Plan Fluor 40x DIC M N Apo TIRF 100x Oil DIC N Plan Fluor 10x Ph1 DLL 0.30 Plan Apo 20x DIC M N S Plan Fluor ELWD 40x Ph2 ADM 0.60 Plan Apo λ 60x Oil Ph3 DM 1.40
6 Excitation wavelengths nm Technical Details Standard Epifluorescence with Andor Zyla scmos Camera Dichroics CFP/YFP/Dsred Dapi/FITC/ TxRed/Cy5 Emission wavelengths nm 435/26 475/20 515/30 540/21 595/40 632/60 700/75
7 Technical Details TIRF with Andor DU897 Camera Excitation Lasers 405 nm 488 nm 561 nm 641 nm Chroma C-TIRF zet405/488/561/635x quad-band cleanup/excitation filter zt405/488/561/640rpc Filters in external emission wheel: ET450/40M ET525/50M ET600/50M ET700/75M
8 Before we discuss TIRF, highlights of the Nikon microscope in standard modes.
9 Field of view with 40X air lens MAP2 Larisa Gofman
10
11 Large Tissue Scanning
12 Multiple Field Timelapse Multiple Colors Multiple fields Timelapse
13 Multiple Field Timelapse Multiple Colors Multiple fields Timelapse
14 Multiple Field Timelapse Multiple Colors Multiple fields Timelapse 9 fields, 2 colors + transmitted, 2 min intervals for 8 hours (12 GB data) Evgenia Korol in Mamta Tahiliani lab
15 Live Cell Imaging Techniques Workshop Friday, April 24 th, :00 p.m. 2:00 p.m. 2 nd floor, Conference Room 2:00 p.m. 5:00 p.m. Hands-On Demonstrations (Microscopy Core) The microfluidic platform is designed to enable perfusion based microenvironment control for long term, high quality live cell microscopy. Continuous perfusion of culture medium to the cells recreates the physiologic mass transport condition for optimized cell health, giving a suitable growth environment for long-term experiments from 4-72 hours on the microscope stage. The system enables single or multi-cell tracking while automated perfusion controls washout, drug changes, and dynamic solution profiles. Temperature and CO2 control is maintained by an on-chip microincubator.
16 TIRF (Total Internal Reflectance Fluorescence) Standard Epifluorescence TIRF Higher contrast of molecules at substrate
17 50 to 200 nm Z Axis Resolution Technically, the spatial resolution in the Z axis isn t improved. The energy activating the fluorescent molecules is limited to a depth of 200 nm maximum. Effectively, the result is imaging molecules only within 50 to 200 nm of the substrate, or effective resolution of nm in the Z axis. How do we do this?
18 Total Internal Reflectance Fluorescence Microscopy is based on an evanescent field that is produced at the critical angle between two interfaces of different refractive indexes. Three Worlds 1955 by M.C. Escher
19 Approaching Critical Angle
20 Schematic from Duke microscopy website coverslip How It Works Internal Reflection
21 Chromatic Aberration Different colors of light focus on different focal planes. This is a problem in microscopy where you want to take pictures of violet through near infra-red fluorescent emitters. When you focus on the green fluorescence, with all microscopes to some extent the violet and far-red images are out of focus. Problem Solved
22 Practicalities # 1.5 coverslip Not #0, not #1
23 Example #1.5 Coverslip Bottom Chambers
24
25 Sequential Colors To go faster, need to use fewer imaging modes and fewer colors. Easy to sequence different conditions; run fast single color then slower multiple channels. Time Intervals Seconds
26 Example Quantification: Radial Intensity Plots Show Locations of Molecules Per Cell Compartments En face view of the synapse with csmac, psmac and dsmac and en face view of a kinapse. From Dustin, 2011 intensity Radial intensity plot from cell center (left) to periphery (right) um
27 Primary Cilia Primary cilium in fibroblasts (marker acetylated α tubulin) unpublished Linda Schneider (P. Satir & S.T. Christensen labs) & Michael Cammer
28 Primary Cilia Primary cilium in fibroblasts (marker acetylated α tubulin)
29 Allows for high contrast imaging of MT or associated proteins (motors, receptors, etc.) in intact primary cilia unpublished Linda Schneider & Michael Cammer
30 From standard epifluorescence to TIRF in an f-actin in vitro assay Rob Eddy unpublished
31 Anchor points closer to substrate are brighter
32 TIRF FRET Active WASp is localized in podosomes and its activity is required for podosome maintenance. WASp is active in podosomes. RAW/LR5 cells transfected with a WASp biosensor, fixed and stained with Alexa Fluor 568-phalloidin and imaged by TIRF microscopy. Dovas A, Gevrey JC, Grossi A, Park H, Abou-Kheir W, Cox D. Regulation of podosome dynamics by WASp phosphorylation: implication in matrix degradation and chemotaxis in macrophages. J Cell Sci Nov 1;122(Pt 21): doi: /jcs
33 Hi Lo may be useful for thick samples Approaching Critical Angle
34 Early C. elegans embryo imaged with near-tirf illumination to overcome the problem of the 200nm-thick eggshell that makes it difficult to use the true TIRF optics. The molecule here is Par-6-GFP a polarity protein. This method allows measuring the exchange rate and mobility of the single molecules at the membrane. Yuliya Zilberman in Nance lab 20 FPS
35 Correlative TIRF and TEM g. T cell with centrally accumulated GAG-GFP resuming motility and releasing GAG-GFPcontaining microvesicles. h. Higher magnification image of boxed region in g. showing internal juxta-membrane density in GAG-containing microvesicles. Arrowhead, plasma membrane. Modified from Choudhuri K, Llodrá J, Roth EW, Tsai J, Gordo S, Wucherpfennig KW, Kam LC, Stokes DL, Dustin ML. Polarized release of T-cell-receptor-enriched microvesicles at the immunological synapse. Nature Mar 6;507(7490): doi: /nature12951.
36 Official Website: MC s Personal notes site: This talk without movies at: (draft as of _1344) Michael Cammer
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