Fluorescence Microscopy – Avoiding the Pitfalls Perkin-Elmer and McGill Imaging Facility Spinning...
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![Page 1: Fluorescence Microscopy – Avoiding the Pitfalls Perkin-Elmer and McGill Imaging Facility Spinning Disk Confocal Workshop Tuesday May 29 th, 2007.](https://reader036.fdocuments.in/reader036/viewer/2022062805/5697bfef1a28abf838cba025/html5/thumbnails/1.jpg)
Fluorescence Microscopy – Avoiding the Pitfalls
Perkin-Elmer and McGill Imaging Facility Spinning Disk Confocal
WorkshopTuesday May 29th, 2007
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http://jcs.biologists.org/cgi/content/full/120/10/1703 JCS 120:1703-1705, 2007
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http://jcs.biologists.org/cgi/content/full/120/10/1703 JCS 120:1703-1705, 2007
![Page 4: Fluorescence Microscopy – Avoiding the Pitfalls Perkin-Elmer and McGill Imaging Facility Spinning Disk Confocal Workshop Tuesday May 29 th, 2007.](https://reader036.fdocuments.in/reader036/viewer/2022062805/5697bfef1a28abf838cba025/html5/thumbnails/4.jpg)
Fluorophone Cross Talk
Tubulin BIODIPY-FL
Sequential Simultaneous
actin
tubu
lin
actin
tubu
lin
Actin Texas Red
Sequential Simultaneous
Confocal Simulator
Overlay colour and colour blindness.
![Page 5: Fluorescence Microscopy – Avoiding the Pitfalls Perkin-Elmer and McGill Imaging Facility Spinning Disk Confocal Workshop Tuesday May 29 th, 2007.](https://reader036.fdocuments.in/reader036/viewer/2022062805/5697bfef1a28abf838cba025/html5/thumbnails/5.jpg)
Fluorophore Cross Talk
Sequential
Simultaneous
Sequential
Simultaneous
SynaptotayminAlexa 488
DLG (Psd-95)Cy3
HRPCy5
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Fluorophore Saturation5% 10% 30% 50%
5% 10% 30% 50%
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Distance (m)
0 2 4 6 8 10
Inte
nsity
0
50
100
150
20010%10%30%50%
Fluorphore Saturation
Distance (m)
0 2 4 6 8 10
Inte
nsity
0.0
0.2
0.4
0.6
0.8
1.0 10%10%30%50%
Intensity Data Normalized Intensity
5% 10% 30% 50%
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Reduced z-axis Resolution
Z-stack orthogonal views
Low Laser Powers
High Laser Powers
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Offset0.1 0.05 0 -0.05 -0.1
Corrected0.1Offset
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Offset
Distance (m)
0 5 10 15 20
Inte
nsity
0
200
400
600
800
1000
1200
1400
1600Offset 0.1 Offset 0.05 Offset 0 Offset -0.05 Offset - 0.1
Raw Intensity Data
Distance (m)
0 5 10 15 20
Inte
nsity
0
200
400
600
800
1000
1200
1400Offset 0.1 Offset 0.05 Offset 0 Offset -0.05 Offset - 0.1
Corrected Intensity Data
Distance (m)
0 5 10 15 20
Inte
nsity
0
200
400
600
800
1000
1200
1400Offset -0.1 Offset 0.1 85% Brighter
320% Brighter
Error Due to Offset
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Detector Saturation
Increasing Laser Power
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Distance (m)
0 2 4 6 8 10 12 14 16 18
Inte
nsity
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Normalized Intensity
* *
Detector Saturation
Distance (m)
0 2 4 6 8 10 12 14 16 18
Inte
nsity
0
1000
2000
3000
4000
Intensity Data
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Camera Binning - Resolution
Resolution
Courtesy of David Hitrys QImaging
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Under-Sampling
1x10.108 m/pixel
2x20.216 m/pixel
3x30.324 m/pixel
4x40.432 m/pixel
CameraBinning
SameDisplaySettings
DifferentContrast
Brightness
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Under-Sampling1x1
0.108 m/pixel2x2
0.216 m/pixel3x3
0.324 m/pixel4x4
0.432 m/pixel
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Software Resolution Issues
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Go To Photoshop
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Software Resolution Issues
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Over Sampling
Distance (m)
0.0 0.5 1.0 1.5 2.0
Inte
nsi
ty
0
200
400
600
800
1000
1200
1400
1600
1800Zoom 1Zoom 3Zoom 6Zoom 8Zoom 10
Zoom 10.140 m/pixel
Zoom 30.047 m/pixel
Zoom 60.023 m/pixel
Zoom 80.017 m/pixel
Zoom 100.014 m/pixel
http://www.lifesciencescomplex.mcgill.ca/imaging/links
~0.050 m/pixel63X NA 1.4 Oil
Nyquist Sampling Calculator
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Image Display - Colour
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Copyright ©2005 Society for Neuroscience
Zhang, H. et al. J. Neurosci. 2005;25:3379-3388
Figure 3. Active PAK localizes to synapses
If possible only usecolour in overlays.
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Image Display – Brightness & Contrast
Distance (m)
0 5 10 15 20
Pix
el I
nten
sity
0.0
0.2
0.4
0.6
0.8
1.0
1.2100 ms 400 ms 700 ms 1000 ms
100 ms 400 ms 700 ms 1000 ms
Distance (m)
0 5 10 15 20
Pix
el I
nten
sity
0
100
200
300
400
500
600100 ms 400 ms 700 ms 1000 ms
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1. Go To Photoshop
2. Go To ImageJ
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Image Display – Gamma Factorbrightness contrast All Threelinear gamma 0.5gamma 2
Image Intensity
Dis
play
Int
ensi
ty
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1. Go To MetaMorph
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Imaging Facility
• http://www.lifesciencescomplex.mcgill.ca/imaging
• http://www.lifesciencescomplex.mcgill.ca/imaging/contact_us
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Pattern FRAP