Fluorescence

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IMMUNOFLUORESCENCE-MICROSCOPYIndirect IFAlt.: Direct IFAdvantage direct IF: Detection more specific, since omission of unspecific Signals of secondary antibody.Advantage indirect IF: Signal amplification, due to binding of multiple secondary AB‘s to primary AB‘s.
Sample preparationTheoretically it‘s a Western blot – in situ on cells.What‘s different:Cells grow or have to be fixed on any kind of „support“ = which should beoptically transparentFixation neededPermeabilisation for antibody accessAntibodies coupled to fluorophorsDetection -> Microscope„Support“ – Material:Suspension cells:CytospinAdhesion Slides (BioRad): special coatingPoly-Lysine coated Coverslips: rarely suitable for suspension cellsAdherent Cells:Glas-CoverslipsPoly-Lysin-coated CoverslipsFilter-Inserts (porous Polycarbonat-Filter)Chamber-Slides / Chambered Coverglass
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 33: Fluorescent Dyes• Commercially av
Page 36 and 37: Organelle Lights ORGANELLESMitochon
Page 38 and 39: Quantum Dots“Whatever hue want”
Page 42 and 43: FIXIERUNG:variable-should be suited
Page 44 and 45: PERMEABILISIATION:For antibody diff
Page 46 and 47: • ProLong is good for Alexa dyes
Page 48 and 49: FLUORESCENCE MICROSCOPY
Page 50 and 51: Light Sources / 2Laser• Light amp
Page 52 and 53: Longpassfiltere.g. Longpass 420:Num
Page 54 and 55: Bandpassfiltere.g.: Bandpass 465/70
Page 56 and 57: Full Cube assembly Bandpass31001 (C
Page 58 and 59: Problem 2: cross emission (emission
Page 60 and 61: Pseudo-Confocal Microscopy - Struct
Page 62 and 63: Different grids - Differentsectioni
Page 64 and 65: Drawbacks - Troubleshooting• Phot
Page 66 and 67: DeconvolutionLimitations to the res
Page 68 and 69: Literature• J.Pawley, Handbook of
Page 70: Confocal Microscopywhat is differen
Page 73 and 74: Confocal: Functional ElementsDetect
Page 75 and 76: The Pinhole I• Light from below-f
Page 77 and 78: The Pinhole IIIA pinhole of 1 airy
Page 79 and 80: Confocal Overview II• A point lig
Page 81 and 82: Laser II - How are they used?• La
Page 83 and 84: Area Scanning - Point by Point
Page 85 and 86: Optics I: Overview
Page 87 and 88: Optics II: Selecting wavelengthsFil
Page 89 and 90: Confocal Overview IIIno interferenc
Page 91 and 92:
Configurations - Zeiss LSM510-71028
Page 93 and 94:
Live „Confocal“ - Many Pinholes
Page 95 and 96:
Confocal Spinning DiscBeams 1Illumi
Page 97 and 98:
Multiphoton MicroscopyPulsed lasers
Page 99 and 100:
What can you do?Z- stack including
Page 101 and 102:
Z-stack: Reducing Dimensions
Page 103 and 104:
Z-stack: Reconstruction• X/Y/Z im
Page 105 and 106:
Multicolor Labeling
Page 107 and 108:
Problem II: cross- emissionWhen emi
Page 109 and 110:
The Problems: How to they look like
Page 111 and 112:
Spectral Imaging
Page 113 and 114:
F-techniques - methodology
Page 115 and 116:
iFRAP
Page 117 and 118:
“Optical Highlighters”PHOTOACTI
Page 119 and 120:
Photoactivation (and yet another F-
Page 121 and 122:
Superresolution Ii.e. breaking the
Page 123 and 124:
Superresolution using Fitting Algor
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Fluorescence

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