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Dr. Kees Straatman
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Image analysis Imaris
Volocity
ImageJ/Fiji
Huygens deconvolution
NIS-Elements (incl. deconvolution in RKCSB)
ScanR analysis
CellR analysis
Cell Profiler
FV1000/LAS
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Image analysis Intensity measurements
Size measurements
Organelle localization
Colocalization
Cell mobility
Distance measurements
FRAP, FLIP, FRET
ImageJ workshop Thursday 21 and Friday 22 July
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Intensity measurements
Absolute measurements of fluorophore concentration is almost impossible but relative measurements are possible.
Comparable and reproducible imaging conditions are needed.
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Be aware Sample problems:
Uneven dye loading/labellingLeakage of dyePhoto bleachingPhoto conversionUnequal cell thickness
Microscope set up: Lamp LifeLamp alignmentObjectives usedFilters used
Camera issues:Gain Offsets, Exposure timesEfficiency of converting Photons to electrons
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Noise 3 types of noise in imaging system:
•Dark Current: noise from heat and cosmic noise (exposure dependent)• Read Noise: noise of reading the signal (fixed)• Photon shot noise: detection of photons is a statistical process depending on the number of photons counted. This uncertainty has a Poisson distribution and is:
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Reduce noise
E-cad myo2a
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Mean filter Can be 3x3, 5x5, 7x7
Can be 3D
50 109 130 137 156
85 109 77 86 175
187 153 63 52 119
183 130 66 82 102
53 20 114 211 168
(50+109+130+ …… +114+211+168)/25 = 113
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Reduce noise
Mean filter (pixel 2 (5x5 kernel))
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Median filter Can be 3x3, 5x5, 7x7
Can be 3D
50 109 130 137 156
85 109 77 86 175
187 153 63 52 119
183 130 66 82 102
53 20 114 211 168
20-50-52-53-20-50-52-53-63-66-77-82-85-86-102-109-109-114-119-130-130-137-153-156-168-175-183-187-211
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Mean filter (pixel 2 (5x5 kernel)) Median filter (pixel 2 (5x5 kernel))
Reduce noise
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Overview Ch 1
0
1000000
2000000
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4000000
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8000000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Raw
Decon
Decon Nikon
3D_median
2D_median
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Raw
Decon
Decon Nikon
3D_median
2D_median
2D_Mean
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Overview Ch 1
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
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Raw
Decon
Decon Nikon
3D_median
2D_median
0
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
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Decon
Decon Nikon
3D_median
2D_median
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Overview Ch 2
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
16000000
18000000
20000000
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17
Raw
Decon
Decon Nikon
3D_median
2D_median
0
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Raw
Decon
Decon Nikon
3D_median
2D_median
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Reduce noise
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Complete Z-series
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1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33
raw
decon
3D-median
2DMedian
2D-MeanBackground
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Rolling ball background subtraction (ImageJ/scanR).
The subtraction is depending on surrounding.
The radius of the ball should be at least the size of the largest object that is not part of the background.
ROI in the same image. Don’t make the ROI too small.
The subtraction is a constant.
You can choose what is background.
Set an offset.
Sliders in software.
Background correction (fluorescence)
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Background correction (fluorescence)
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rollingbal
back -59
decon
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Background correction (fluorescence) Take an image with the light source blocked (Dark).
Take an image without sample but with the light source on (Light).
Take your images (Image)
Signal = (Image – Dark)/(Light – Dark) * bit factor
bit factor = 255 (8 bit)
4095 (12 bit)
65535 (16 bit)
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Background correction (Bright field) Take your images (Image)
Take an image without sample but with the light source on (Background)).
Subtract Background from Image
ImageBackground Background corrected
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FRAP controlsLeica: Widefield Super-resolution with Ground State
Depletion Leica SR GSD
Maximum resolution down to 20 nm
Standard fluorochromes can be used – no need to change your protocols
Ground state depletion (GSD) can be achieved by using high power lasers to transfer fluorophores into long-lived dark states – a non-fluorescent molecule state. Stochastically, as soon as single fluorophores return from the dark state, new bursts of emitted photons are recorded …...
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0
0.2
0.4
0.6
0.8
1
1.2
0
1.71
6
3.9
32
5.6
48
7.36
4
9.0
8
10.7
96
12.5
12
14.2
28
15.9
44
17.6
6
19.3
76
21.0
92
Ch1 Region2 (Intensity Average)
Ch2 Region2 (Intensity Average)
FRAP controls
Fixed material
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Materials and Methods Make, model and type of microscope used Type, magnification, and numerical aperture of the objective lens used Light source used Fluorochromes used Peak transmission and bandwidth of filter sets used (or the manufacturer part
number) Camera make and model Enviromental conditions during live cell imaging: temperature, CO2, buffer,
chamber, media, etc. Other motorized components used Acquisition software Name and version of image processing and analysis software used Details about image enhancement during image processing and analysis: type
of filters, background subtraction, gamma adjustments, type of deconvolution, 3D reconstructions, surface or volume rendering, etc.