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My Favorite Fluorophore Von Helmholtz

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My Favorite Fluorophore Von Helmholtz 5/17/21 My Favorite Fluorophore Borondipyrromethene dyes emit visible light with high fluorescence quantum yields upon excitation lEx BODIPY Photoactivatable BODIPYs Designed to Monitor the Dynamics of Supramolecular Nanocarriers Zhang, Y.; Swaminathan, S.; Tang, S.; Garcia-Amorós, J.; Boulina, M.; Captain, B.; Baker, J. D.; Raymo, F. M. J. Am. Chem. Soc. 2015, 137, 4709–4719 von Helmholtz von Helmholtz recognized the possibility of replacing transmitted light with emitted light to image objects Carl Zeiss AG started the production Hermann L. F. von Helmholtz of optical microscopes based on Humboldt University transmitted light and the design of Ernst Abbe in 1872 Die Theoretischen Grenzen für die Leistungsfähigkeit der Mikroskope von Helmholtz, H. L. F. Ann. Phys. 1874, 557–584 1 5/17/21 Heimstadt Heimstadt developed the first fluorescence microscope for the Austrian manufacturer Carl Reichert Oskar G. Heimstädt Carl Reichert Das Fluoreszenzmikroskop Heimstadt, O. Z. Wiss. Mikrosk. 1911, 28, 330–337 A Few Definitions Epi-illumination = Excitation and emission are the same (epi) side of the sample Trans-illumination = Excitation and emission are opposite (trans) sides of the sample Wide-Field Microscope = Excitation is focused on a relatively large area of the sample and the emission coming from all the excited area is detected Laser-Scanning Microscope = Excitation is focused on a relatively small area of the sample, the emission coming from the excited area is detected and the excited/emitting area is scanned across the sample 2 5/17/21 Confocal Laser-Scanning Microscope Total Internal Reflection Microscope 3 5/17/21 Multi-Photon Microscope Fluorescence Lifetime Imaging 4 5/17/21 Coons Antibodies can be tagged with fluorescent labels to probe their interactions with complementary antigens Tagged Antibody Antibody + Labeling Albert H. Coons Harvard University Fluorescein Isothiocyanate The Demonstration of Pneumococcal Antigen in Tissues by the Use of Fluorescent Antibody Coons, A. H.; Creech, H. J.; Norman Jones, R.; Berliner, E. J. Immunol.1942, 45, 159–170 Chalfie, Shimomura and Tsien The Nobel Prize in Chemistry 2008 was awarded for the discovery and development of the green fluorescent protein GFPMartin was isolatedChalfie from CellsOsamu expressing Shimomura GFP Rabbit expressingRoger GFP Y. Tsien theColumbia jellyfish University Aequorea Marine Biological Laboratory University of California, victoria and Boston University Medical School San Diego The Nobel Prize in Chemistry 2008 www.nobelprize.org 5 5/17/21 Watching Cells in Multicolor African Green Monkey Kidney Fibroblast Cells Bovine Pulmonary Artery Endothelial Cells Chinese Hamster Ovary Cells Human Bone Osteosarcoma Cells Human Brain Glioma Cells Human Lung Carcinoma Cells Nikon MicroscopyU www.microscopyu.com/galleries/confocal Focusing and Diffraction Diffraction prevents the focusing of light into a single point and instead distributes the radiation intensity … Focal Plane Airy Pattern Objective Lens r Glass l Slide r ~ 2 Fluorophore … on the focal plane of a lens in the form of a pattern with dimensions controlled by the radiation wavelength Zhang, Y.; Raymo, F. M. Methods Appl. Fluoresc. 2020, 8, 032002 6 5/17/21 Diffraction and Resolution Fluorophores can be resolved only if their center-to- center distance is greater than the radius of their Airy disk d > r d = r Fluorophore Fluorophore Fluorophore Fluorophore Resolved Fluorophores Unresolved Fluorophores Fluorophores must be hundreds of nanometers away from each other to be resolved Betzig, Hell and Moerner The Nobel Prize in Chemistry 2014 was awarded for the development of super-resolved fluorescence microscopy Eric Betzig Stefan W. Hell William E. Moerner Janelia Research Campus Max Planck Institute for Stanford University Howard Hughes Medical Institute Biophysical Chemistry The Nobel Prize in Chemistry 2014 www.nobelprize.org 7 5/17/21 Stimulated Emission Depletion Switching and Localization Switchable fluorophores can be differentiated in time and localized individually with nanometer precision I I 0 0 I I x0 x0 x x I0 I0 I I FluorescenceFluorescence OFF ON Fluorescence ONOFF 0 0 y y y y Imaging Intracellular Fluorescent Proteins at Nanometer Resolution Betzig, E.; Patterson, G. H.; Sougrat, R.; Lindwasser, O. W.; Olenych, S.; Bonifacino, J. S.; Davidson, M. W.; Lippincott-Schwartz, J.; Hess, H. F. Science 2006, 313, 1642–1645 8 5/17/21 Fluorescence Switching Ultraviolet ms Activated Area PMMA 10 µm Fluorescence Nanoscopy Incubation + Antibody Cell 2 µm Vero 500 nm 9 5/17/21 Bleaching and Diffusion Fluorescence recovery after photobleaching offers the opportunity to monitor dynamic events optically … Bleached Imaged Area Area lEx lEx lEx Bleaching Excitation Diffusion Fluorophores … but only under harsh illumination conditions and in the presence of significant amounts of molecular oxygen Ishikawa-Ankerhold, H. C.; Ankerhold, R.; Drummen, G. P. C. Molecules 2012, 17, 4047–4132 Activation and Diffusion Fluorescence activation in a region of interest within an imaging plane and sequential image acquisition permit … Activated Imaged Emissive Area Area State lAc lEx lEx Activation Excitation Diffusion Nonemissive State … the monitoring of the translocation of the activated species and the quantification of their diffusion rates Raymo, F. M. Phys. Chem. Chem. Phys. 2013, 15, 14840–14850 10 5/17/21 Color Switching The photoinduced switching of the fluorescence color permits the imaging of the sample before and after … Imaged Activated Imaged Area Area Emissive Area State B A B lEx lAc lEx Excitation Activation Excitation B lEx Emissive Diffusion State A … the activation event in addition to the subsequent monitoring of the translocation of the activated species Color Switching l Ac + PBMA Activated Area 100 µm 11 5/17/21 Blastoderm Photoactivation Activated Area Activated Area 100 µm Blastoderm Dynamics Activation 3 6 9 12 15 min 50 µm D = 5.3 × 10–8 cm2 s–1 Activation 30 60 min 92 µ m 167 µm 12.
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