Fluorescence

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Fluorescent Dyes• Commercially available from many companiesEmissionspectra of the Alexa SeriesAlexa Fluor488• Usually planar polycyclic organics with conjugated double bondsFluorescein
Page 1 and 2: Praktikum zur Fluoreszenz- undKonfo
Page 4 and 5: Types of Light• Monochromatic•
Page 6 and 7: Lenses, Focus and AberrationsReason
Page 8 and 9: Types of microscopes andIlluminatio
Page 10 and 11: ObjectivesDipping objectives-physio
Page 12 and 13: Resolution• Definition: the small
Page 14 and 15: Resolution!!!!!Magnification identi
Page 16 and 17: Counts for transmitted and reflecte
Page 18 and 19: Depth of FieldThe axial range, thro
Page 20 and 21: Detection Systems Overviewhttp://bi
Page 22 and 23: DetectionDigital Cameras: Photons e
Page 24 and 25: Imaging - Triangle of Frustration©
Page 26 and 27: FluorescenceStokes (1852) - Jablons
Page 28 and 29: Principle of Fluorescence• Molecu
Page 30 and 31: Quantum Efficiency• Only emitted
Page 35 and 36: Single stainingsCounter-StainsDoubl
Page 37 and 38: FUNCTIONAL ANALYSESApoptosis TdT(te
Page 39 and 40: SAMPLE PREPARATIONconventional Work
Page 41 and 42: Sample preparationTheoretically it
Page 43 and 44: Fixation ChemistryFormaldehyde:Note
Page 45 and 46: EMBEDDING:Your last washing step sh
Page 47 and 48: Check-List• Only use #1.5 (0.17mm
Page 49 and 50: Light Sources / 1Halogen LampsMercu
Page 51 and 52: Types of Filters• Longpass-filter
Page 53 and 54: Shortpassfiltere.g. Shortpass 500:N
Page 55 and 56: Strahlenteiler = Beamsplitter• Sp
Page 57 and 58: Problem 1: cross-excitationa fluoro
Page 59 and 60: Filter set for simultaneous detecti
Page 61 and 62: Pseudo-confocal Microscopy-„Apoto
Page 63 and 64: WIDEFIELDAPOTOME-MODE63x oil, nA=1,
Page 65 and 66: Apotome - Advantages• Simple syte
Page 67 and 68: The Point-Spread FunctionFor this r
Page 69 and 70: Thank you for your attention
Page 72 and 73: Depth of Focus
Page 74 and 75: Light source:Extended vs Point illu
Page 76 and 77: The Pinhole IIoptical sectioning po
Page 78 and 79: Tight junctionsAdherens junctions©
Page 80 and 81: Laser I• Monochromatic• Linearl
Page 82 and 83:
Laser III - How are they used?
Page 84 and 85:
Photomultiplier Tube (PMT)+ no „f
Page 86 and 87:
Optics I: Splitting wavelengthsMBS:
Page 88 and 89:
Optics III: Spectral DetectionGrati
Page 90 and 91:
Practical ExamplesCLSM + spectral d
Page 92 and 93:
Live Confocal - „Pin“slit vs Pi
Page 94 and 95:
Spinning Disk IIDemonstration© Sci
Page 96 and 97:
Spinning Disc• Advantages:‣ Fas
Page 98 and 99:
1P vs MP Microscopy1 Ph MP Multipho
Page 100 and 101:
Z-stack
Page 102 and 103:
Z-stack: Animation
Page 104 and 105:
Objective behaviour - Imaging Thick
Page 106 and 107:
Problem I: cross- excitationThe exc
Page 108 and 109:
Problem III: Problem I + Problem II
Page 110 and 111:
Spectral Imagingl-scanDefine fluoro
Page 112 and 113:
The F-techniques• FRAP: Fluoresce
Page 114 and 115:
FRAPUsing FRAP, one can determineth
Page 116 and 117:
FLIPA part of a cell at a distance
Page 118 and 119:
OverviewPA PS PC
Page 120 and 121:
http://www.nature.com/focus/cellbio
Page 122 and 123:
Superresolution II - RESOLFTREversi
Page 124:
(Fluorescenct)-PhotoActivationLocal
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Fluorescence

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