Localization Microscopy Theory, Sample Prep & Practical Considerations Patrina Pellett & Ann McEvoy Applications Scientist GE Healthcare, Cell Technologies May 27 th, 2015
Localization Microscopy Talk summary Theory Sample prep Minimize background Optimize everything Practical considerations What resolutions are even possible? Probe photophysical properties DeltaVision localization microscopy (DLM) Analysis tips Indiana Localization Workshop 27 May 2015 2
Localization Microscopy - Theory 3
Localization Microscopy How does this technique work? A lot of acronyms Image single molecules & determine localization by finding of center of it s image Requires special fluorophores that switch from an on to an off state Lateral resolution between 20 50 nm possible Indiana Localization Workshop 27 May 2015 4
Localization Microscopy Temporal separation of molecules Widefield Image Final Image Frame 21 Fit Find Centers Reconstruct 1) Switch on a few molecules 2) Find centers (Gaussian) 3) Localize centers of molecules 4) Build up final image Repeat 1000s of times Indiana Localization Workshop 27 May 2015 5
Localization Microscopy Acquire movie of blinking events Example DLM Acquisition Yin Lab, Harvard Indiana Localization Workshop 27 May 2015 6
Localization Microscopy How do we make organic fluorophores blink? Process of Fluorescence + O2 -> Photobleaching Excited state λ ex λ fl Triplet state Dark states Ground state 405 nm Buffer components: 1. Oxygen scavenger to help prevent photobleaching 2. Reducing agent to get dyes into stable dark state Indiana Localization Workshop 27 May 2015 7
Localization Microscopy How do we make fluorescent proteins blink? Photoactivators dark to bright State 1 λ ex State 2 Photoconverters convert to different fluorescent state State 1 λ ex State 2 Photoswitchers reversibly switch between states State 1 λ ex1 State 2 Standard Fluorescent Proteins add reducing agent State 1 λ ex2 Reducing agent Blinking Indiana Localization Workshop 27 May 2015 8
Localization Microscopy What to consider when choosing this method Advantages Disadvantages Down to 20 nm XY resolution Special probes/ imaging conditions required 2 color imaging More than 2 color imaging is tricky Live cell possible Bad temporal resolution Down to 70 nm Z resolution Limited z range TIRF region or very clean samples required Indiana Localization Workshop 27 May 2015 9
Localization Microscopy Sample Prep
Localization Microscopy Sample Prep Sample prep is key! Extremely clean labeling is required TIRF may be required Optimize all sample prep steps Excellent paper for starting point: Whelan & Bell, Nature Scientific Reports, (2015), 5, 7924 Indiana Localization Workshop 27 May 2015 11
Localization Microscopy Sample Prep Background looks identical to real signal DLM image of microtubules Are these small dots from the structure of interest or from background? Any molecule that blinks will be included in the final reconstructed image, so it is very important to minimize background (tips coming soon)! Indiana Localization Workshop 27 May 2015 12
Localization Microscopy Sample Prep Meticulous sample prep required DLM image of coverslip Incubated with secondary antibodies Indiana Localization Workshop 27 May 2015 13
Localization Microscopy Sample Prep Tips to minimize background Use extremely clean coverslips Optimize labeling conditions Filter sterilize all reagents Optimize fixative conditions Excellent blocking steps Long wash steps Optimize primary & secondary antibody concentrations Use very dilute concentrations, make your own labeled antibodies with low degree of labeling (good for small dense structures) Short secondary antibody incubations Try fluorescent proteins! Indiana Localization Workshop 27 May 2015 14
Localization Microscopy Sample Prep What to consider when choosing fluorophores Practical Concern Fluorescent Proteins Organic Dyes Targeting Genetically encodable Challenging Photon Emission Low (< 500 photons) High (> 1000 photons) Control of blinking Easy Limited External buffers required? No Yes # of switching cycles None to many Can switch many times Multicolor? Fewer choices Many choices May need to try many different labeling strategies/fluorophores for your application Indiana Localization Workshop 27 May 2015 15
Localization Microscopy Practical Considerations
Localization Microscopy Considerations Localization precision resolution Localization Precision Resolution Example: A single molecule with 20nm precision means it is located somewhere within these 20nm Example: The peak to peak distance of these 2 structures is 150 nm, i.e. the resolution of this image is 150 nm Line profile 0.8 Arbitrary Units 1.6 150nm 200 nm 100 200 300 Distance (nm) Localization precision is the certainty in the measurement of the fluorophore s position Resolution describes the minimum distance that 2 structures can be distinguished Indiana Localization Workshop 27 May 2015 17
Localization Microscopy Considerations High resolution requires high labeling density NNNNNNNNNNNNNN RRRRRRRRRRRRRRRRRRRR = 2, (llllllllllllllll dddddddddddddd) 1 DD Need data points every half the resolution you are claiming To achieve 10nm resolution, must have data points every 5 nm Or 2500 molecules/ diffraction limited area (2D) Or 250,000 molecules/ diffraction limited volume (3D) How abundant is your protein of interest? Indiana Localization Workshop 27 May 2015 18
Localization Microscopy Probe Considerations Sizes of common probes used for labeling Organic dye < 1 nm Fluorescent proteins 2 x 4 nm Self-labeling proteins ~ 2 x 4 nm Antibody ~ 15 nm Single domain antibody ~ 3 x 4 nm What is the size of the probe you are labeling your protein with? Indiana Localization Workshop 27 May 2015 19
Localization Microscopy Considerations What labeling densities are possible? Depends on size of probe & labeling efficiency If only considering a 1 nm wide probe, the highest labeling density possible is: 62,500 molecules/ diffraction limited area (2D) 3 x 10 6 molecules/ diffraction limited volume (3D) If only considering a 50 nm wide probe, the highest labeling density possible is: 25 molecules/ diffraction limited area (2D) 2500 molecules/ diffraction limited volume (3D) Indiana Localization Workshop 27 May 2015 20
Localization Microscopy Considerations High labeling efficiency required 100 nm circular structure with 10 possible fluorophore labeling sites Localization reconstruction of structure labeled with 30% efficiency Localization reconstruction of structure labeled with 70% efficiency Final reconstructed image Possible labeling site Labeling efficiency of structure of interest must be very high Indiana Localization Workshop 27 May 2015 21
Localization Microscopy Probe Considerations Photophysical properties vary ATTO488 Blinking AlexaFluor647 Blinking Dempsey et al., Nat Methods, (2011), 8(12), 1027-1036 Indiana Localization Workshop 27 May 2015 22
Localization Microscopy Probe Considerations Need dyes with low duty cycle Duty cycle = time in on state / time in off state 100 nm circular structure with 10 possible fluorophore labeling sites Duty cycle = 0.5 50% of the molecules will be on at any given time High duty cycle dyes have too many dyes on in a diffraction limited area Indiana Localization Workshop 27 May 2015 23
Localization Microscopy Probe Considerations Need dyes with low duty cycle Duty cycle = time in on state / time in off state 100 nm circular structure with 10 possible fluorophore labeling sites Duty cycle = 0.01 1% of the molecules will be on at any given time Low duty cycle dyes result in few dyes on at the same time Indiana Localization Workshop 27 May 2015 24
Multi-Color Localization Microscopy Advantages & disadvantages FPs Dye-pair Antibodies Single Dye Antibodies Minimal color choices Not commercially available Variable blinking kinetics Low number of photons Cloning required Good targeting Small size Dependent on primary antibody Same reporter dye Potential cross talk Large size Variable buffer conditions Dependent on primary antibody Commercially available Large size Some dyes blink in Vectashield Indiana Localization Workshop 27 May 2015 25
Localization on the OMX DeltaVision Localization Microscopy
DeltaVision Localization Microscopy Which probes can you use? Any fluorophore that matches the laser lines & emission filter settings! Available laser lines: 405, 488, 561 & 642nm Emission filter combinations Channel Emission Filters (nm) DAPI 420-450 FITC 500-555 mcherry 585-630 Cy5 660-705 Indiana Localization Workshop 27 May 2015 27
DeltaVision Localization Microscopy Key acquisition features Through TIRF light path Can image in deep TIRF or full epi Acquisition speeds of up to 100 Hz Powers of 1-5 kw/cm 2 (wavelength dependent) at the sample Progress monitor Power ramping Indiana Localization Workshop 27 May 2015 28
DeltaVision Localization Microscopy Key analysis features Down to 20nm resolution XY Proprietary mixed model algorithm Minimizes overlapping data exclusion Fewer acquisition scans required Good for very dense labeling Correlation algorithm & fiducial tracking for drift correction Indiana Localization Workshop 27 May 2015 29
DeltaVision Localization Microscopy Multi-emitter algorithm throws out less data Single Gaussian Fitting Multiple Gaussian Fitting Successfully localized Successfully localized Discarded or identified as single event May be localized with low precision Multi-emitter algorithm can successfully localize overlapping events DLM multiple Gaussian fitting algorithm can localize overlapping signals within a diffraction limited area, great for densely labeled samples Indiana Localization Workshop 27 May 2015 30
DeltaVision Localization Microscopy Correlative drift correction algorithm Not Corrected for Drift Corrected for Drift 1 µm Membrane labeling E. coli Used to correct for any lateral drift due to temperature fluctuations Indiana Localization Workshop 27 May 2015 31
Localization Microscopy Analysis tips
Localization Microscopy Analysis Tips How to process localization data 4 analysis phases 1. Localization 2. Correction 3. Tracking 4. Reconstruction Will cover each phase in detail during live demo Indiana Localization Workshop 27 May 2015 33
Localization Analysis Tips Do not have too many overlapping events Can get artifacts caused by more than one dye turning on in a diffraction limited volume Mislocalizations Armpit or webbing For small densely labeled samples 1 event per frame (centrosomes) For more spread out samples aim for 100-200 events per frame (microtubules) Indiana Localization Workshop 27 May 2015 34
Localization Analysis Tips Ensure that little drift occurs during experiment Can expect a few hundred nm of drift over 10 min acquisition Use drift correction tool to correct for drift during experiment If fails, increase time window Gives more events per bin Indiana Localization Workshop 27 May 2015 35
Localization Analysis Tips How to plot your localization events Different methods to filter & display data Filter by localization precision/ track length Ways to plot: 1. Gaussian weights each event based on LP 2. Histogram each event is plotted as a pixel Gaussian Histogram Indiana Localization Workshop 27 May 2015 36
Localization Microscopy Want more details? Come image with us! or Email me: Patrina.pellett@ge.com Indiana Localization Workshop 27 May 2015 37
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