You can observe a lot by just watching! Aaron Taylor, PhD Managing Director BRCF Microscopy Core Sample provided by Puck Ohi, CDB Microscopes are a very old technology! Rene Descartes James Maxwell Ernst Abbe August Kohler Light Geometric Optics Wave Optics Resolution Theory Illumination Theory Microscopy circa 1900 Election Microscopy circa 1930 Ernst Ruska Electron Optics So what’s new? Every thing else! Microscopy circa 2010 Image Analysis Computer control Advanced Lasers Advanced New dyes and optoelectronics Microscope probes (old!) Cutting-edge ‘microscopes’ are complex physics experiments! Lattice Light Sheet Focused Ion Beam – Scanning Electron Microscope Interferometric PALM Why do we need microscopes? Our eyes are designed to focus on distant objects spread over a large field of view. Human Eye Retina 1500 FOV Lens What is our angular resolution? The curvature of our lens limits Human Eye angular resolution. 0.020 Angular Resolution (0.3 m at 1000 m for 20/20 vision) What is our spatial resolution? The lens can change curvature, such that we can see smaller details in closer objects, but eventually ‘near point’ is reached, when the rays are too divergent to be focused. θ > θ Light blurry 2 1 on the retina θ1 θ2 Near Point = 25 cm 25 cm x 0.0003 o/a ratio = 0.0075 cm spatial resolution A Microscope Magnifies and Resolves A microscope bends highly divergent light so that the rays become nearly parallel, as if coming from a large, distant object. “Microscope” Parallel rays; Image occupies large fraction of visual field Magnified image on Lens 1 retina θ Lens 2 Divergent rays; The angle between these rays determines resolution Optics have a resolution limit Light (and electron beams) have wave properties that limit resolution. Diffraction 1D Profile 2D Profile Interference Intensity θ 0.61 λ Lens NA NA = n sinθ Airy Disk 0.61 λ 0.61 (500 nm) = = ~300 nm NA 1 Biological materials that microscopes let us see lumenlearning.com Cryo-EM/ET Unaided Eye (Vitreous) Light Microscopy Electron Microscopy (Chemically stained) Super-Resolution Light Microscopy Correlative LM-EM Practice of Electron Microscopy Electron microscopy provides highest resolutions, but samples must be fixed and staining is typically non-specific. Electrons have a very Lenses are electric or Sample is embedded short wavelength magnetic fields in a plastic h λ = mv Electrons can only travel in a vacuum! Transmission Electron Microscopy (TEM) transmits electrons through a very thin section (70 nm) of the sample and measures where the electrons were absorbed. Scanning Electron Microscopy (SEM) raster scans a focused electron beam across the surface of a sample and measures how the electrons are reflected or ejected. FIB-SEM can image an entire volume Mauricio Torres, Physiology; Sample prepared in Microscopy Core and imaged at MC2 Practice of Light Microscopy Light microscopy is compatible with living samples and allows for staining of specific biomolecules at endogenous levels. Most all modern light microscopy Many tricks can be played with images fluorescence both the excitation and emitted light to gain new capabilities GFP FITC • Spectral and life-time imaging • PSF engineering • Optical sectioning • 2-photon (non-linear) imaging • Light sheets Fluorescence is quantifiable (unlike colorimetric stains) and dark ‘background’ provides up to single molecule sensitivity. Single molecule localization microscopy can provide EM-like resolution for specific proteins TIRF Mode See also: UMICH SMART Center (Szymborska, Science, 2013) SMLM and EM can be combined for truly correlative light and electron microscopy 1 um (Paez-Segala, Nat Meth, 2015) Tissue clearing and Expansion allow super resolution without special microscopes (Ku, Nat Biotech, 2016) Point-scanning microscopy allows in vivo animal imaging Confocal / Two-Photon (Weigert, JCB, 2013) Light can be used to control protein activity or localization (Wu, Nature, 2009; Yoo, Dev Cell, 2010) Lattice light sheet microscopy allows fast and gentle 3D cell imaging Lattice Light Sheet (Chen, Science, 2014) Gaussian light sheet microscopy allows gentle whole embryo imaging Multi-View Gaussian Light Sheet (Tomer, Nat Meth, 2012) Use image analysis to quantify the data Neha Shrestha, Physiology Thank you! Find out more about the Microscopy Core: Google “Umich Microscopy” Email: [email protected] • Microscopes • Expert Advice • Custom Image Analysis • Expansion Microscopy and Tissue Clearing • Educational Workshops.