fluorescence microscopy – avoiding the pitfalls perkin-elmer and mcgill imaging facility spinning...

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