Fluorescence

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DetectionDigital Cameras: Photons elicit electron hole pairs (photoelectric effect) –charge converted to voltage – this analogue signal is amplified andconverted into a binary image (AD-conversion)Digital Coding:Data Depth = levels of grey1 bit: 0,12 bit 00, 01, 10, 11BINNING: - speed increase by sampling 4, 9, 16 pixels as one- reduction of memory size- shorter illumination times
Nyquist-Sampling Theorem – or how many pixel do Ineed for a resolution representative image ?NYQUISTSAMPLINGCRITERION: the rate of samplingmust be at least 2-fold the samplefrequency to be able toreconstruct the analog signal froma digitalized one.sampling frequency is limited bythe pixel size of the chip!Calculation – the resolution at550nm with an objective 100x,NA=1,4 calculates to 230nm ->magnified by a factor of 100 =23µm ->on the chip the imagemust be large enough to cover 2pixels -> required pixel size is 11,5µm !R x M =2 x pixelsize½ inch chip is usually 6,4mm x 4,8mm: minimum # of pixels horizontally =6400 / 11,5 calculates to 557 pixelsLower mag objectives usually need more pixels for optimal resolution onthe CCD-chip (high resolution microscopes usually equipped with no morethan 1,3 Megapixel cameras)
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 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 40 and 41: IMMUNOFLUORESCENCE-MICROSCOPYIndire
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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