Seeing at the Nanoscale New Microscopies for the Life Sciences Copyright Springfield Republican Dr.
Download ReportTranscript Seeing at the Nanoscale New Microscopies for the Life Sciences Copyright Springfield Republican Dr.
Seeing at the Nanoscale New Microscopies for the Life Sciences Copyright Springfield Republican Dr. Jennifer Ross, Department of Physics University of Massachusetts Amherst July 1, 2010 Visualizing Living Cells Biological systems are transparent and difficult to see Visualizing Living Cells We can use a variety of optical tricks to enhance the contrast Phase Contrast Visualizing Living Cells We use fluorescence to see components inside cells Red = mitochondria Green = actin Principles of Fluorescence Use a light microscope to illuminate and observe fluorescence Fluorescent molecules excites more Violet (higher energy) emits more Red (lower energy) High energy Low energy E = h*f Energy is Planck Constant times the frequency of light Common Fluorophores Rhodamine, Fluoroscein, Cy-dyes, Alexa-dyes Green Fluorescent Protein and numerous derivatives (won Nobel Prize in Chemistry 2008) Fluorescence - How it works Excitation 10-15 s Vibrational Relaxation 10-14 - 10-11 s Fluorescence 10-9 - 10-7 s QuickTime™ and a decompressor are needed to see this picture. Fluorescence microscopy Anatomy of modern inverted microscope QuickTime™ and a decompressor are needed to see this picture. Olympus Microscopy Resource Center website QuickTime™ and a decompressor are needed to see this picture. Nikon MicroscopyU website Fluorescence microscopy Epi-illumination path Fluorescence Cube with filters and dichroic QuickTime™ and a decompressor are needed to see this picture. Fluorescence microscopy - Modern Detection QuickTime™ and a decompressor are needed to see this picture. CCD Charged-coupled device (CCD) camera Each pixel detects photons, which are translated to a number that is displayed as a grey value on a computer Fluorescence microscopy - Multiple Colors Transmitted light microscopy (phase contrast) CCD is black and white, so we switch filter sets to see different colors Fluorescence microscopy - Multiple Colors Fluorescence microscopy with green emission filter set CCD is black and white, so we switch filter sets to see different colors Fluorescence microscopy - Multiple Colors Fluorescence microscopy with red emission filter set CCD is black and white, so we switch filter sets to see different colors Fluorescence microscopy - Multiple Colors False color red and green overlay CCD is black and white, so we switch filter sets to see different colors Most two-color imaging is not simultaneous, but rather sequential Nanoscale in Biology 25 nm Proteins (2-5 nm) Protein, DNA, RNA filaments (2-25 nm) Molecular complexes (5-25 nm) Membranes (4 nm thick) 13 nm 4 nm 4 nm Vale, et. al. Cell 2003 Visualizing the Nanoscale Attaching fluorescent molecules to these objects allows us to see them and watch their dynamics Qui ckTi me™ and a decompressor are needed to see this pictur e. QuickTime™ and a H.264 decompressor are needed to see this picture. Microtubules outside of cell, Ross Lab Microtubules inside of cell, Wadsworth Lab Visualizing Single Proteins Attaching fluorescent molecules to these objects allows us to see them and watch their dynamics QuickTime™ a Singleand motor proteins decompressor are needed t o see t his pict ure. walking along microtubules Quic kT ime™ and a dec ompres sor are needed to s ee this pic ture. QuickTime™ and a decompress or are needed t o see this pict ure. How do we Visualize Single Molecules? When you illuminate a sample in epi-fluorescence, a rather large volume is illuminated Causes background fluorescence Many molecules in the field How can we see single molecules? Slide Cover glass Inverted objective Visualizing Single Molecules 1) Dilute the sample Visualizing Single Molecules 1) Dilute the sample 2) We could use a confocal spot with apertures to block out-ofplane fluorescence Visualizing Single Molecules 1) Dilute the sample 2) We could use a confocal spot with apertures to block out-ofplane fluorescence 3) Total Internal Reflection Fluorescence My method of choice Total Internal Reflection Fluorescence Microscopy Focus laser on back-focal plane of objective It comes out collimated Total Internal Reflection Fluorescence Microscopy Move focused spot to edge of back focal plane Collimated beam tilts Total Internal Reflection Fluorescence Microscopy More details on how to do TIRF can be found at: J.L. Ross and R. Dixit, “Two color single molecule TIRF imaging and tracking of MAPs and motors,” Microtubules in Vitro, Methods in Cell Biology, Eds. J. Correia and L. Wilson (2010). Move to far edge so that angle > critical angle for total internal reflection Total Internal Reflection Fluorescence Microscopy Zoom in on Evanescent Wave Decays exponentially in z Only about 100 nm into sample Brighter is closer to cover glass Only molecules within 100 nm are visible Total Internal Reflection Fluorescence Microscopy More details on how to do TIRF can be found at: J.L. Ross and R. Dixit, “Two color single molecule TIRF imaging and tracking of MAPs and motors,” Microtubules in Vitro, Methods in Cell Biology, Eds. J. Correia and L. Wilson (2010). Total Internal Reflection Fluorescence Microscopy More details on how to do TIRF can be found at: J.L. Ross and R. Dixit, “Two color single molecule TIRF imaging and tracking of MAPs and motors,” Microtubules in Vitro, Methods in Cell Biology, Eds. J. Correia and L. Wilson (2010). Resolution Limits to Imaging Single Molecules A motor protein takes an 8 nm step, can we measure that in our single molecule assay? Yes and No. Ideally, the motor is only about 4 nm, so an 8 nm step should be visible Resolution Limits to Imaging Single Molecules A motor protein takes an 8 nm step, can we measure that in our single molecule assay? Yes and No. Ideally, the motor is only about 4 nm, so an 8 nm step should be visible But it’s not resolvable… Resolution Limits to Imaging Single Molecules A motor protein takes an 8 nm step, can we measure that in our single molecule assay? Yes and No. The objective diffracts the light, because it is a wave. d 1.22 2NA This is the diffraction limit NA = n*sinmax n = index of refraction Resolution Limits to Imaging Single Molecules Diffraction limited spot for a high-NA objective 508nm d 1.22 1.22 208nm 2NA 2*1.49 8 nm steps are not observable How can we improve our resolution? Smaller Larger NA? Super-resolution Use math tricks! Intensity of diffraction-limited spot highest at center Intensity Actually a Bessel function, but is well-fit by a 2-D Gaussian m e™ and a m pr e s so r s ee t hi s p ic tu re . Fit the shape of the intensity to find the center with high accuracy FIONA: Fluorescence Imaging with One Nanometer Accuracy QuickTime™ and a decompressor are needed to see this picture. We can fit every image of the single molecule to find the center with 10-20 nm “resolution.” FIONA: Fluorescence Imaging with One Nanometer Accuracy Replace the fuzzy spot with a 2-20 nm dot More photons (brighter spot) leads to better “resolution” and a smaller dot. Effect of pixel size on resolution Here, resolution is limited by pixel size - not fundamental properties of light Not resolvable Large enough to be resolved Gaussian fitting gives better than 1 pixel accuracy FIONA: Fluorescence Imaging with One Nanometer Accuracy Follow the motor for multiple frames More photons, better fitting The diffraction limit is broken! Total Internal Reflection Fluorescence Microscopy on Cells cells Epi-fluorescence image at surface TIRF image at surface http://www.microscopyu.com/articles/fluorescence/tirf/tirfintro.html Total Internal Reflection Fluorescence Microscopy on Cells Drawbacks: Can only image at surface If there are many molecules in cell, still can’t see single molecules because cell is crowded mCherry-tubulin (green) GFP-Eg5 (red) epi-fluorescence TIRF TIRF on mitotic cell expressing GFP-Eg5 Wadsworth Lab Total Internal Reflection Fluorescence Microscopy on Cells Drawbacks: Can only image at surface If there are many molecules in cell, still can’t see single molecules because cell is crowded QuickTime™ and a decompressor are needed to see this picture. TIRF on mitotic cell expressing GFP-Eg5 Wadsworth Lab Single Molecule Imaging in Cells FPALM and STORM - super-resolution imaging FPALM: Fluorescence Photoactivation Localization Microscopy STORM: Stochastic Optical Reconstruction Microscopy 1 2 0 0 1 0 0 0 Intesiy 8 0 0 6 0 0 4 0 0 2 0 0 0 0 5 0 1 0 0 1 5 0 D is t an c 2 0 0 2 5 0 3 0 0 e Techniques based on switchable fluorophores and FIONA analysis. are ne Patricia Wadsworth, UMass Amherst Biology Sam Hess, UMaine Orono Physics Single Molecule Imaging in Cells FPALM and STORM Inside a cell, there are many many proteins of various types. Fluorescence microscopy allows us to localize proteins in cells. Genetic fluorescent labels allow us to watch dynamics in live cells. Pat Wadsworth, GFP-tubulin LLPCK cell line QuickTime™ and a Animation decompressor are needed to see this picture. Single Molecule Imaging in Cells FPALM and STORM To watch dynamics, you can “turn-on” fluorescence Photo-activatable fluorophores Dynamics of tubulin in the mitotic spindle QuickTime™ and a H.264 decompressor are needed to see this picture. Pat Wadsworth, PAGFP-tubulin LLPCK cell line Single Molecule Imaging in Cells FPALM and STORM Photo-activatable fluorophores GFP derivatives and other organix compounds High energy photon “activates” quantum state to allow fluorescence Normal fluorescence after activation QuickTime™ and a decompressor are needed to see this picture. Single Molecule Imaging in Cells FPALM and STORM Now, the trick is to only “turn on” a few molecules at a time They have to be spaced far apart, so we can see single molecules Very low power laser illumination => single photons Single Molecule Imaging in Cells FPALM and STORM Build the image from the images of single molecules Single Molecule Imaging in Cells FPALM and STORM QuickTime™ and a decompressor are needed to see this picture. http://www.umaine.edu/sensorigert/faculty/profile.php?id=307 Single Molecule Imaging in Cells QuickTime™ and a decompressor are needed to see this picture. FPALM and STORM We can build a super-resolution image by combining all the locations into one single image. Single Molecule Imaging in Cells FPALM and STORM Single Molecule Imaging in Cells FPALM and STORM Single Molecule Imaging in Cells FPALM and STORM Single Molecule Imaging in Cells FPALM and STORM Single Molecule Imaging in Cells FPALM and STORM and Single Molecule Dynamics Or, we can watch the dynamics of single molecules are they move in the complex cellular environment QuickTime™ and a decompressor are needed to see this picture. Our new FPALM/STORM microscope at UMass 2 EM-CCD cameras for fullscreen two-color GFP-mCherry (red-green) EOS-mCherry (red-red) Cy3-Cy5 (red-dark red) Our new FPALM/STORM microscope at UMass Funded by the National Science Foundation, Major Research Instrumentation Program (Ross, Wadsworth) Our new FPALM/STORM microscope at UMass Nikon TiE inverted EM-CCD slave Perfect Focus Illumination level Imaging level EM-CCD master Funded by the National Science Foundation, Major Research Instrumentation Program (Ross, Wadsworth) Our new FPALM/STORM microscope at UMass Illumination level Imaging level Argon-ion laser Funded by the National Science Foundation, Major Research Instrumentation Program (Ross, Wadsworth) Our new FPALM/STORM microscope at UMass Thank You! Funded by the National Science Foundation, Major Research Instrumentation Program (Ross, Wadsworth)