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Fluorescence Microscopy – Avoiding the Pitfalls
Perkin-Elmer and McGill Imaging Facility Spinning Disk Confocal
WorkshopTuesday May 29th, 2007
http://jcs.biologists.org/cgi/content/full/120/10/1703 JCS 120:1703-1705, 2007
http://jcs.biologists.org/cgi/content/full/120/10/1703 JCS 120:1703-1705, 2007
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.
Fluorophore Cross Talk
Sequential
Simultaneous
Sequential
Simultaneous
SynaptotayminAlexa 488
DLG (Psd-95)Cy3
HRPCy5
Fluorophore Saturation5% 10% 30% 50%
5% 10% 30% 50%
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%
Reduced z-axis Resolution
Z-stack orthogonal views
Low Laser Powers
High Laser Powers
Offset0.1 0.05 0 -0.05 -0.1
Corrected0.1Offset
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
Detector Saturation
Increasing Laser Power
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
Camera Binning - Resolution
Resolution
Courtesy of David Hitrys QImaging
Under-Sampling
1x10.108 m/pixel
2x20.216 m/pixel
3x30.324 m/pixel
4x40.432 m/pixel
CameraBinning
SameDisplaySettings
DifferentContrast
Brightness
Under-Sampling1x1
0.108 m/pixel2x2
0.216 m/pixel3x3
0.324 m/pixel4x4
0.432 m/pixel
Software Resolution Issues
Go To Photoshop
Software Resolution Issues
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
Image Display - Colour
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.
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
1. Go To Photoshop
2. Go To ImageJ
Image Display – Gamma Factorbrightness contrast All Threelinear gamma 0.5gamma 2
Image Intensity
Dis
play
Int
ensi
ty
1. Go To MetaMorph
Imaging Facility
• http://www.lifesciencescomplex.mcgill.ca/imaging
• http://www.lifesciencescomplex.mcgill.ca/imaging/contact_us
Pattern FRAP