■Hä NIKON CORPORATION p^^fl Instruments Company Nikon NIKON CORPORATION Instruments Company Plan Apo Lambda series NIKON CORPORATION Instruments Company 60x Plan Apochromat Objective Manufacturer Flat-Field Correction Lateral Magnification Specialized Optical Properties Tube Length Cover Glass Thickness Range Cover Glass Adjustment Guage Figure 1 Nosepiece Mounting Thread Aberration Correction Numerical Aperture Working Distance Magnification Color Code Correction Collar Front Lens Element Assembly Common Objective Optical Correction Factors 10xAchromat 10x Fluorite 10x Apochromat i Lens ( j-Doublet-j Lens J Triplet-j Lens r Doublet- Front Lens- Lens Tripl i !^B3i Lens—\ Doublet I, Groups-4 mm* U ^■Meniscus-V! Lens Figure 2 If Numerical Aperture (a) NA = n • simoo (a) « = 7° NA = 0.12 (b) fx = 20° NA = 0.34 (c) a = 60° NA = 0.87 a Light Cone Figure 1 NA = n * sina November 13, 2020 5 NIKON CORPORATION Instruments Company f/ffm/f VC objective Conventional objective November 13, 2020 6 40x Air PA X - 0.95 NA, 210 u.m WD 40x Wl A A,S - 1.25 NA, 180 u.m WD B = Brightness D = Depth R = Resolution L= Live Cell Drosophila sp. embryo with DAPI stained nuclei, supplied by Dr. Jennifer Sallee, North Central College Microscope Image Formation NIKON CORPORATION Instruments Company An image is a huge array of sub resolution points Each point in the image is convolved by the objective to form a "Point Spread Function" (PSF). A PSF is unique to a particular objective and microscope configuration Focal Planes with Spherical Aberration Towards the Lens Paraxial Focal Point November 13, 2020 8 Microscope Image Formation NIKON CORPORATION Instruments Company ER Scanning 8-25-16-PUB 3 Feb 2017 4p Point Sources ® O © Convolved Point Sources Point sources of light spread in all direction Sources exist above and below focal plane At FOCAL plane Convolution November 13, 2020 9 NIKON CORPORATION Instruments Company Excitation light Airy Disk Intensity Focal Plane N.A. = 0.50 Numerical Aperture Airy Disk Intensity Focal Plane N.A. = 1.30 Numerical Aperture November 13, 2020 10 Resolution / Diffraction Limit Well resolved Just resolved Not resolved NIKON CORPORATION Instruments Company Nikon Figure 3 Ernst Abbe Diffraction Limits of Optical Instruments d = 2NA Lord Rayleigh Resolution Limits of Diffraction Limited Optical Instruments (Epifluorescence) Lord "Rayleigh (John Sfa.tt) (1842 191:9) R = 1.221 2NA November 13, 2020 11 Resolution Limits NIKON CORPORATION Instruments Company f/ffm/f November 13, 2020 12 x (nm) (NA=1.4, A=670nm) November 13, 2020 13 Resolution Limits Human eye Widefield microscopy Confocal microscopy Total internal reflection (TIRF) SUPER-RESOLUTION Atomic Force Microscopy Electron microscopy Approximate resolution 0.05 mm 250 nm (X,Y) 600-850 nm (Z) 250 nm (X,Y) 500 nm (Z) 50-100 nm (Z) < 100nm 1-50 nm < 1 nm November 13, 2020 14 Confocal vs. widefield microscopy NIKON CORPORATION Instruments Company Widefield Confocal *t 6 S Principle of Fluorescence NIKON CORPORATION Instruments Company Jablonski Energy Diagram Jablonski diagram Excitation (Absorption) 10"15 Seconds Excited Singlet States 5 Internal Conversion and Vibrational Relaxation (10~14-10"11 Sec) °- Fluorescence (109-10"7 Sec) }Vibrational Energy States Intersystem Crossing Quenching Non-Radiative § ■ Relaxation o i Delayed Fluorescence MA 5 Excited 2 Triplet 1 State o (TJ Intersystem * * Crossing Non-Radiative Relaxation (Triplet) Phosphorescence (10 3- 102 Sec) Ground State Figure 1 energy to excite is higher than the emitted energy this is named the "stokes shift" (a shift to longer wavelengths) November 13, 2020 17 Fluorescence filter block for confocal NIKON CORPORATION Instruments Company Principle of Excitation and Emission — Ultraviolet (UV) and Visible Light Emitted Blue — ! Light Fluorescent Specimen igure 3 emisní (bariérový) filtr těleso (Mok) filtru excitační (budicí) filtr příruba k upevněni filtru v tělesu dichrotcké zrcadlo (dělič chodu paprsků) 750-1050 Fluorescence filter block for confocal NIKON CORPORATION Instruments Company Principle of Excitation and Emission — Ultraviolet (UV) and Visible Light Emitted Blue — ! Light Fluorescent Specimen igure 3 emisní (bariérový) filtr těleso (Mok) filtru excitační (budicí) filtr příruba k upevněni filtru v tělesu dichrotcké zrcadlo (dělič chodu paprsků) 750-1050 Confocal laser scanning NIKON CORPORATION Instruments Company 066666666666'^ o -► ooooooooocoo -► r^^^^W ö66666666666r 066666666666^ oWSW OOOOOOOOOOOÖ OOOOOOOOOOOÖ OOOOOOOOOOOÖ 666666666666^ Confocal laser scanning NIKON CORPORATION Instruments Company Creating f.i. 512x512 image 2 - 30 fps / - 420 fps @ 512 x 64 pix Confocal v/s widefield microscopy NIKON CORPORATION Instruments Company .... Nikon Widefield Versus Point Scanning of Specimens Cover Glass - Specimen Microscope Slide Illumination Po,nt Scanning Beam Figure 2 Widefield Scanning Excitation occurs not only at focus point! Point scanning principle NIKON CORPORATION Instruments Company Pinhole Focal plane Sample out of / focus Point scanning principle NIKON CORPORATION Instruments Company ER Scanning 8-25-16-PUB 3 Feb 2017 Point Sources ® O © Convolved Point Sources Point sources of light spread in all direction Sources exist above and below focal plane Az X 1-> At FOCAL plane Convolution Point scanning principle NIKON CORPORATION Instruments Company ER Scanning 8-25-16-PUB 3 Feb 2017 Jisr Point Sources o © © Convolved Point Sources Point sources of light spread in all direction Sources exist above and below focal plane At FOCAL plane Convolution Point scanning principle NIKON CORPORATION Instruments Company ER Scanning 8-2S-16-PUB 3 Feb 2017 Pinhole Effect PH Pinhole? (7) A pinhole in the conjugate focal plane blocks signal At FOCAL plane Convolution Confocal v/s widefield microscopy Confocal Widefield ZO/12): -22-OlMm Z(lO/m-19.51ym ZCll/lÄ-17.01)jm Z(12/12>:-14.51)jm 3D data set Confocal vs. widefield microscopy NIKON CORPORATION Instruments Company Confocal vs. widefield microscopy Confocal vs. widefield microscopy NIKON CORPORATION Instruments Company Si • * 3D data set Confocal vs. widefield microscopy NIKON CORPORATION Instruments Company Kalibrace x:0.32 ^m, y:0.32 pm, z:2.50 \im Sifka:449.00 Vyska:335.46 \i\r\ Hloubka:75.00 prn 3D data set Pinhole shape NIKON CORPORATION Instruments Company Point spread function Pinhole size NIKON CORPORATION Instruments Company 30 urn 100 urn Hexagonal pinhole NIKON CORPORATION Instruments Company Nikon 30% brighter images Same optical sectioning performance November 13, 2020 39 NIKON CORPORATION Instruments Company f/ffm/f .'äff':?'- ' L-.1>.\. ••. Spectral imaging and Linear Unmixing NIKON CORPORATION Instruments Company Lambda Stack with Green, Yellow, and Orange Fluorescent Proteins 1 2 3 4 5 6 7 8 9 1011 12131415 16 1.0 { 0.8 - I 1 0.6 CO fl 0.4 E 0.2 -o (a) 450 1 1 Kb Iii» •10-Nanometer Waveband EGFP EYFP mKO 500 550 600 Wavelength (Nanometers) 650 700 - Diffraction Grating based Spectral imaging and Linear Unmixing Confocal Microscope Spectral Imaging Detector Configurations Diffraction Grating Dispersion November 13, 2020 44 Spectral imaging and Linear Unmixing NIKON CORPORATION Instruments Company Spectral imaging and Linear Unmixing Lambda Stack with Green, Yellow, and Orange Fluorescent Proteins 12345678 9 1011 12131415 16 450 500 550 600 650 700 Wavelength (Nanometers) A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 A13 A14 A15 A16 EGFP ■ ■ §9 ■ ■ ■ ■■■ ■ ■ EYFP (b) mKO ■ ■ ■ Combined □ ■ ■ Figure 6 Spectral detection NIKON CORPORATION Instruments Company NIKON CORPORATION Instruments Company f/ffm/f SAMPLE IMAGES Drosophila sp. Embryonic heart Mouse neurons Mosquito larvae nervous system XY Time plot of contracting isolated cardiomyocyte Whole cleared zebrafish head November 13, 2020 53 Simultaneous bleaching and imaging NIKON CORPORATION Instruments Company FRAP (Fluorescence Recovery After Photobleaching) Before photobleaching Right after photobleaching 0.3 seconds after photobleaching 1 second after photobleaching 1.3 seconds after photobleaching 2 seconds after photobleaching i>30 U«0 050 i:00 Time (minis) boar. 02 JO 2 GO 2 Point of photobleaching Simult. photostimulation and imaging NIKON CORPORATION Instruments Company Kaede Activation: 405nm Ex: 488nm /561nm Em: 525/50 & 595/50 Hi Speed PA-GFP NIKON CORPORATION Instruments Company Multiphoton confocal system NIKON CORPORATION Instruments Company MULTIPHOTON CONFOCAL MICROSCOPE Multiphoton confocal microscopy NIKON CORPORATION Instruments Company Two-Photon Jablonski Energy Diagram Excited Singlet State Single Photon Excitation Two-Photon Excitation 1 (a) Ultraviolet Excitation (350 nm) (b) Infrared Excitation (700 nm) }Vibrational Energy States Blue Fluorescence Emission J- b i Ground State Figure 1 Pulsed lasers pico (E12) to femto (E15) second pulses MP excitation Fluorophore Excitation in Multiphoton Microscopy Excitation area in confocal microscopy and multi photon microscopy MP excitation NIKON CORPORATION Instruments Company 1-photon vs. 2-photon Fluorescence from Fluorescence from out of focus planes focal spot only MP advantages NIKON CORPORATION Instruments Company Only excitation in focal point Much less bleaching Much less phototoxicity Use light in IR range (800-1300nm) 2 to 3 times deeper penetration; red light scattered less then blue light 488 light >7 times more scattered then 800nm No out of focus absorption Single and Two-Photon Scanning Profiles One-Photon Excitation Two-Photon Excitation x z Scan X-Z Scan November 13, 2020 63 Deep brain imaging in in vivo mouse NIKON CORPORATION Instruments Company B Frame screw -A Plastic ring Cranial window Bone screw 0 mm 0.5 mm 1.0 mm Plastic ring Frame screw Cranial frame VI • . 1 \ k Base plate 1.5 mm YFP Alexa594 A1R MP+ GaAsP Epi NDD detector unit NIKON CORPORATION Instruments Company deep brain imaging in in vivo mouse The brain of H-line 4-week-old mouse under anesthetize was studied with the open skull method. The entire shape of dendrites of pyramidal cells in layer V and hippocampal pyramidal cells can be visualized. Surprisingly dendrite of hippocampal pyramidal cells can also be imaged. Photographed with the cooperation of Dr. Terumasa Hibi,Dr. Ryosuke Kawakami,Dr. Tomomi Nemoto Research Institute for Electronic Science,Hokkaido University Objective Lens: APO LWD 25x 1.10 WD 2.0 mm Detector: EPI NDD GaAsP Detector Pyramidal cell in layer V 0.00 mm 0.10 mm 0.20 mm 0.30 mm 0.40 mm 0.50 mm 0.60 mm 0.70 mm 0.80 mm 0.90 mm 1.00 mm 1.10 mm 1.20 mm Hippocampus 3D zoom image Laser Spinning Disk Systems NIKON CORPORATION Instruments Company f/ffm/f Yokogawa Spinning Disk Unit Optical Configuration Lens Disc Shaped and Collimated — Laser Illumination Microlens Array Pinhole (Nipkow) Monochrome CCD Camera 1 kamera = 1 FL kanál LSCM Scan Pattern Spinning Disk Scan Pattern Confocal Microscope Scanning Patterns Figure 4 Sample Oil Objective Covers lip Wide Field Fig. 1 a Laser Scanning Fig, 1b Spinning Disk Fig. 1 c TIRF - Total Internal Reflection Fluorescence NIKON CORPORATION Instruments Company Nikon Snell's Law: n2 (water) n 1 (glass) Glass to air Water to air Glass to water ^crit - ^crit - 9Crit ~ 41. 1° 48.6° 61.0° n1 sin61=n2sin62 sin9c = n2 n1>>n2 Evanescent Wave NIKON CORPORATION Instruments Company Total Internal Reflection Fluorescence Evanescent Wavefront Aqueous Medium (n = 1.33- 1.37) Fluorophores Interface^ Laser Excitation- Incident Angle Excited Fluorophore Glass — Microscope Slide (n = 1.518) Reflected Fiaure 1 Lightwaves figure i E,= E0exp(-z/dp) cL=- znrt./We-CiVn,)* Penetration depth: - wavelength - Diff. n1 and n2 - Incident angle laser beam (> critical angle) - Typically between 50 200nm Why is this useful for FL microscopy? Excitement of Fluorophores within only 50-200nm of coverslip No background Fluorescence from rest of specimen Very high increase in S/N Measurement single molecule fluorescence possible Coverslip Sample Immersion Oil NIKON CORPORATION Instruments Company TIRFM Specimen Illumination Configurations Laser Specimen prjsm ^Keflecti Light Figure 2 Specimen High Numerical Aperture Objective TIRFM Cellsf"*2/^c Aqueous TIR Surface |,V Medium Immersion Oil Cover Glass Peripheral Objective Figure 3 Rays Off-Axis Distance Laser Beam Front Lens Objective Rear Focal Plane Evanescent wave thickness; axial resolution t 200nm 1 t lOOnm t 60nm NA1.40 NA1.46 NA1.49 Widefield vs Confocal vs TIRF Widefield Confocal S.E. Ledevedec Leiden University (GFP-dSH2) TIRF NIKON CORPORATION Instruments Company Neuronal Growth Cone DIC/TIRF Alexa488 Actin Imaged by Andy Schaefer, Paul Forscher Lab., Yale University ■Hä NIKON CORPORATION pp^<9 Instruments Company Nikon uper-Resolution Microscopy - 2014 Nl=sz: November 13, 2020 75 Super-Resolution Microscopy NIKON CORPORATION Instruments Company Nikon Small Molecule Super-resolution Optical microscopy resolution i-11- Protein Virus Bacteria 1 Cell Hair Ant 1 1 I 1 J 1 nm 10 nm 100 nm 1 |jm 10 100 |jm 1mm November 13, 2020 76 Super-Resolution Microscope System Stochastic Optical Reconstruction Microscopy Developed by Dr. Xiaowei Zhuang and colleagues - Howard Hughes Medical Institute, Harvard University Enhanced resolution that is more than 10 times greater than conventional optical microscopes N-STORM reconstructs high resolution fluorescence 2D (20 nm) or 3D (50nm) images from precise localization information of individual fluorophores (SMLM) N-STORM enables molecular understanding of the specimen 78 STORM - Principle NIKON CORPORATION Instruments Company f/ffm/f Structures within cells are mostly in the magnitude of 5-100 nm and they are lying close to each other. 2 nm urn November 13, 2020 80 Optical Localisation NIKON CORPORATION Instruments Company Why not switching on the fluorophores individually - one after the other? To reconstruct the sample structures one has to repeat this process a few thousand times... November 13, 2020 81 Optical Localisation NIKON CORPORATION Instruments Company Why not switching on the fluorophores individually - one after the other? To reconstruct the sample structures one has to repeat this process a few thousand times... November 13, 2020 82 N-STORM with pairs of dyes The N-STORM works with photoswitchable dyes. These are dye pairs that consist of one shorter wavelenght activator dye and a second dye with longer wavelenght as reporter Activator Reporter Binding Site Flurophores November 13, 2020 83 STORM Principle 1) „Switch off" all reporters with a strong pulse of light at 647 nm. DARK STATE 2) Low excitation of the activator with 561 nm. The activator stochastically enables only some reporters to emit fluorescence. ACTIVATION 3) Normal Imaging at 647 nm. The positions of the emitting molecules are recorded. IMAGING & LOCALISATION 4) Repeat this sequence several 1.000 times. Localizing the PSF Centroid NIKON CORPORATION Instruments Company A El "I 1 500 nm Diffraction Limited Spot of the emission of a single Reporter on DNA during a single cycle (/> c o o Q. CD Ü C CD Ü CO Q) i_ O DZ + Centroid of the PSF after Gaussian Fit ,000 (h/y.. -500' ^ -1,000^1,000 Point Spread Function (PSF) of the diffraction limited spot November 13, 2020 86 STORM; super-resolution by localization NIKON CORPORATION Instruments Company Conventional fluorescence ■ _ Raw images DAotitisrtitian STORM Image Localization + + 2x real time Stochastic Optical Reconstruction Microscopy = STORM Rust, Bates &Zhuang, Nat. Methods, 2006 Bates, Huang, Dempsey & Zhuang, Science, 2007 Images of microtubules in a mammalian cell Source: http://zhuang.harvard.edu/storm.html Images adapted from Science 317, 1749-1753 (2007) November 13, 2020 89 November 13, 2020 90 Multicolor N-STORM NIKON CORPORATION Instruments Company Alexa405 Alexa647 CY2 Alexa647 CY3 Alexa647 Difference in Excitation Same Imaging Wavelenght 3 kinds of "Activator" and 1 kind of "Reporter" are available. Alexa405 - Alexa647 Compound CY2 - Alexa647 Compound CY3 - Alexa647 Compound 400 450 500 Wavelength (nm) 550 600 Sequential activation of Alexa647 NIKON CORPORATION Instruments Company Nikon Fluorescent 03 > H—' Ü 03 O H—' o Q_ o 03 > H—' Ü 03 CD Q 647 nm Alexa647 405 457 532 Cy3 6ZZ All channels are imaged with Alexa647 No chromatic abberations! Multi-color without filter change! 9 dye pair combinations currently available Max of 3 dye pairs sequentially during acquisition November 13, 2020 92 ">s '/■[■ M\:*.X>xi\^/ ■'>•■'•'>''• I Cy3 / Alexa 647: Clathri / ry ^ ?! / -;- "' ' Cy2 / Alexa 647: Microtubule L' /r -i> ti M Wt<- ámfmáw N-STORM Applications NIKON CORPORATION Instruments Company Physics A Pure Physics Dye behavior Calculation (SOFI) 3D (SLM) £*i Neuroscience Synapse Receptors Spine Cell Biology Cytoskeleton Colocalization DNA mapping Diagnosis SR FISH Abnormal Chromosome November 13, 2020 96 N-STORM 10x resolution increase NIKON CORPORATION Instruments Company 0.5 |jm , ... .. :,f 4. 0.5 um I-1 - 0.1 |jm - • • • * t *6 November 13, 2020 97 3D STORM NIKON CORPORATION Instruments Company ffflfQn Above Focus In Focus Below Focus z (nm) -600-400-200 0 200 400 600 z (nm) Above Focus In Focus Below Focus EMCCD Molecules localized in Z • Molecules above focus maintain symmetry in Y • Molecules below focus maintain symmetry in X • Fitted to Gaussians similar to XY Huang, Zhuang et al, Science (2008) 10 November 13, 2020 3 N-STORM Summary NIKON CORPORATION Instruments Company Stochastic Optical Reconstruction Microscopy (2D, 3D) Photo switchable dyes Localization of each fluorescent molecule with nanometer precision Construction of a Super-Resolution image from these points Activator excitation with 3 lines possible (405, 457, 561) Imaging with high power 647nm laser (300 mW) 3D STORM using astigmatic (cylindrical) lens 10-fold increase in resolution (XY: 20 nm, Z: 50 nm) Acquisition speed - Minutes N-STORM Instrumentation NIKON CORPORATION Instruments Company f/ffm/f N-STORM Instrumentation NIKON CORPORATION Instruments Company N-SIM NIKON CORPORATION Instruments Company N-SIM SUPER-RESOLUTION MICROSCOPE SYSTEM s N (Nikon) - SIM ("Structured Illumination Microscopy") Two times better resolution than diffraction limit: SIM ~ 100 nm / Wide Field 200 nm minate with diffraction-limited grid pattern - Image reconstruction Licensed from UCSF Developed by Dr. Mats G. L. Gustafsson, Dr. John W. Sedat and Dr. David A. Agard of UCSF 10 November 13, 2020 9 Structured light patterns u November 13, 2020 0 ^63110663170062^^^611 An unknown object (a) is illuminated by a known pattern of light (b) resulting in a moire pattern (c). From Moire pattern and known pattern it can be concluded to the unkown pattern! J.B.J. Fourier provides the „key" to the significant improvement of resolution! 11 November 13, 2020 1 From Real Image to Fourier Image NIKON CORPORATION Instruments Company Frequency in Y Frequency in X Frequency in Y Frequency in X 11 November 13, 2020 2 From Real Image to Fourier Image. NIKON CORPORATION Instruments Company Frequency in Y Frequency in X Frequency in Y Frequency in X 11 November 13, 2020 3 From Real Image to Fourier Image... NIKON CORPORATION Instruments Company z B 1/ E f * 4 r H 0 K •4- 11 vember 13, 2020 4 November 13, 2020 6 N-SIM Illuminator / SIM Image Set NIKON CORPORATION Instruments Company Diffraction limited grid Shifted over 3 positions (2D/TIRF-SIM) 5 positions (3D-SIM) Rotated to 3 orientations I Resulting in 9/15 images 9 (3 shifts x 3 rotations) 15 (5 shifts x 3 rotations) Reconstruction in reciprocal space Acquire images with 3 pattern angles x 3 phases. Separate the 7 information components Reassemble into an extended resolution image Resolution is extended Objective aperture radius Theoretical extended radius after reconstruction processing is 2x original High frequency (higher resolution) information is further from origin New resolution is 2x original New Resolution Limit using SIM Resolution is extended NIKON CORPORATION Instruments Company • Final step is to Re-transform extended resolution Fourier image back to real space • Includes additional processing to compensate for the point spread function of the system. Image From - Mats Gustafsson - UCSF N-SIM image has twice resolution compared to conventional microscope. November 13, 2020 Principle of SIM - Diffraction NIKON CORPORATION Instruments Company Nikon Resolution depends on Numerical Aperture (NA). Diffracted light of the fine structure of sample cannot be captured by objective lens. N.A. Structured Illumination However, the fine structure of the sample can be captured as moire pattern by illumination of structured light. N.A.X2 As a result, we can get images with double NA. November 13, 2020 N-SIM applications Flat or sparse samples Samples features details between 100 and 200 nm Dynamics - seconds or slower (0.6 sec/frame - 2D SIM) (1.0 sec/frame-3D SIM) Actin/Cyto skeleton Microtubuli Mitochondria Bacteria Conventional vs. SIM November 13, 2020 Mitochondria - Acquisition time 1.8sec November 13, 2020 Conventional vs. SIM (Multicolor) Colocalization of VEGF signal receptor (Cy3) and ubiquitin E3 ligase (FITC) Conventional November 13, 2020 November 13, 2020 Conventional vs. SIM (Multicolor) NIKON CORPORATION Instruments Company ffflfQn November 13, 2020 Drosophila polytene chromosome squash Nikon N-SIM system, 3D-SIM NIKON CORPORATION Instruments Company Nile Red DivIVA-GFP merge r • ^ J * * * * V < ••' i ."• V Bacillus subtilis bacterium stained with membrane dye Nile Red (red), and expressing the cell division protein DivlVA fused to GFP (green). The superior resolution of N-SIM system allows for accurate localization of the protein during division. Henrik Strahl & Leendert Hamoen, Centre for Bacterial Cell Biology, Newcastle University 13 November 13, 2020 2 N-SIM Summary NIKON CORPORATION Instruments Company Structured Illumination Microscopy ( 2D, 3D, TIRF) Creation of a diffraction pattern on the sample Interaction of the overlying pattern with fine patterns in the sample creates Moire effect (= interference pattern) Based on this information, the original pattern can be determined computationally Resolution: 2-fold increase in Resolution XY: lOOnm, Z: 250 nm (~85 nm with TIRF) Common staining procedure (standard fluorophores) Suitable for live cell imaging Up to 5 Laser Lines (from: 405/457/488/515/532/561/647) November 13, 2020 13 3 STED (Stimulated emission depletion) NIKON CORPORATION Instruments Company STED-microscopy ŕ.ii• lutr 6ŕjm H-Quenching bwr J^^^Ě beam B Standard Donut Suppressed Effective PSF PSF depletion outer PSF Fluorescence M Phase Plate Depletion Laser Activation Laser Pinhole Detector • e • m m Increasing intensity of depletion laser 200 nm Decreasing PSF 50 nm 13 November 13, 2020 5 Děkuji za pozornost