Fluorescence

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Spinning Disk IIDemonstration© Science vol 300 David j. Stephens and Victoria J. Allen
Confocal Spinning DiscBeams 1Illumination 1 mWEmission rate 1.26 x 10 8 photons/secFluorophore saturation 63%Beams > 1000Illumination 0.4-0.6 μWEmission rate 1.72 x 10 5 photons/secFluorophore saturation 0.09%Point Scanner-ConfocalAcquisition Point by point -SLOWDetection PMT -Medium sensitivity(but signalamplification)Optical SectioningAdjustablePinholes -FLEXIBLE – lightloss; NOCROSSTALKSpinning DiscMany points -FASTEM-CCD -High SensitivityFixed Pinhole -INFLEXIBLE; lightloss; CrosstalkpossibleWidefieldOne frame at atime -VERY FASTCCD-EMCCD -Medium-to-highsensitivityNo sectioning -All light collectedhttp://biop.epfl.ch/pdf/PT-BIOPII_Live_cell_imaging-Feb2011-L_Gelman.pdf
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Praktikum zur Fluoreszenz- undKonfo
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Types of Light• Monochromatic•
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Lenses, Focus and AberrationsReason
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Types of microscopes andIlluminatio
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ObjectivesDipping objectives-physio
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Resolution• Definition: the small
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Resolution!!!!!Magnification identi
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Counts for transmitted and reflecte
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Depth of FieldThe axial range, thro
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Detection Systems Overviewhttp://bi
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DetectionDigital Cameras: Photons e
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Imaging - Triangle of Frustration©
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FluorescenceStokes (1852) - Jablons
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Principle of Fluorescence• Molecu
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Quantum Efficiency• Only emitted
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Fluorescent Dyes• Commercially av
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Organelle Lights ORGANELLESMitochon
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Quantum Dots“Whatever hue want”
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IMMUNOFLUORESCENCE-MICROSCOPYIndire
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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: Live „Confocal“ - Many Pinholes
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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