Dyversity and Typhoon 9400 Imaging of CyTM dye 1D and 2D gels
Purpose
To compare the dynamic range, linearity, sensitivity, and image quality of the Dyversity imaging system with the TyphoonTM 9400 laser scanner (GE Healthcare).
Executive Summary
Imaging with the Dyversity imaging system is faster compared to the Typhoon laser scanner for similar resolution and this has been demonstrated for 1D and 2D gels (for
example, for the Cy3 1D gel imaging is 4X faster and for the Cy3 2D gel imaging is 10x faster). For the BSA protein, the dynamic range, linearity and sensitivity are
identical for both imaging systems. Examination of the image quality by viewing the background for 1D gel images (background corrected and noise filtered) and the signal
for the raw/uncorrected 2D gel images in 3D mode reveals the presence of background artefacts for the Typhoon image only. For the raw 2D gel images there is no
apparent difference in the image quality indicating comparable performance.
The main advantage of the Dyversity imaging system compared to the Typhoon laser scanner is fast acquisition times.
Materials
Typhoon 9400 laser scanner: lasers 488, 532 and 633; emission filters 520BP40, 580BP30, 670BP30
Dyversity imaging system: 6.3M pixel camera KAF6303E with 50mm f1.4 lens; Cy dye lighting module and band pass emission filters, sample end mirror.
GeneSnap – image acquisition
GeneTools software – 1D gel software analysis
Dymension 3 software – 2D gel software analysis
ID Gel
12% acrylamide gel with 5% acrylamide for the stacking gel, 18cm x 16cm. Two loaded proteins: Sigma carbonic anhydrase (29kDa) and Sigma bovine serum albumin
(66kDa); loading buffer containing 300mM of DTT. 3x50 microgram of stock solution of each protein labeled with Cy2TM, Cy3TM or Cy5TM (minimal dyes), hence dilution of
stock solution for titration series. Titration: 0.05, 0.1, 0.5, 1, 2, 5, 10, 50 and 100ng of each protein and for each Cy dye.
2D Gel
3x 50 microgram total soluble proteins from Arabidopsis cell suspension were labeled with Cy2, Cy3 or Cy5 (minimal dyes) and loaded in the first dimension gel (non linear
pH 3-10 IPG strip). The IPG strip was then applied to the second dimension (12% acrylamide gel 18cm x 16cm).
CyTM is a registered Trademark of GE Healthcare.
Imaging Times
From the user’s perspective the most significant difference between using Dyversity and Typhoon is in the imaging times. The scan times on Typhoon are solely
determined by the scanning resolution, and the sensitivity is determined by the PMT (photo multiplier tube) gain. On Dyversity the scan times are only dependant on the
sensitivity.
Typhoon
For an 18cm x 16cm gel at 100 micron resolution the scan time is 8 minutes per channel. Between scans the system takes around 1 minute to stabilize, so a 100 micron
scan for all 3 dyes takes 27 minutes. To avoid saturating the images it is often necessary to take some “pre-scan” images at a lower resolution. At 1000 micron resolution
for an 18cm x 16cm gel this takes 2 minutes per channel plus the 1 minute stabilization times before each scan, - a total of 9 minutes. These scan times will scale linearly
with gel size, so a 24cm x 18cm gel would take approx 3.3 times as long, i.e. 26 minutes per channel at 100 microns, and 7 minutes for a “pre-scan” per channel at 1000
microns. During the scanning the computer is essentially ‘locked’ i.e. no other software can be used at the same time.
Dyversity
As Dyversity images the whole gel at once the scan times are not dependant on sample size but solely on the exposure time required to achieve the desired sensitivity.
1x1 binning gives a resolution of 90 microns per pixel. However, using 8x8 binning it is possible to pre-scan the gels checking for saturation with exposure times of 1 or 2
seconds. This gives a “live” saturation preview mode. Note that the exposure times used during the Dynamic Range Assessment, especially for the saturated images,
longer exposures were used than those used on typical 2D gels, as we were testing the limits of the system’s sensitivity. On “normal” 2D Cy dye gels the imaging times
typically seem to be under 60 seconds per channel.
Results
1) Dynamic Range Assessment
A 1D gel was imaged using non saturating imaging conditions and the following parameters were used:
Dyversity, 90micron resolution Typhoon 9400, 100micron resolution
Cy2 2mins exposure Cy2 PMT 525V, 8mins scanning
Cy3 2mins exposure Cy3 PMT 550V, 8mins scanning
Cy5 30secs exposure Cy5 PMT 530V, 8mins scanning
Images were compared in Dymension with background correction and noise filtering applied to both Typhoon and Dyversity images in order to assess the data
quantitatively.
The red box marks the BSA protein loaded at 0.5ng and for imager and dye this band is more visible for the Cy5 dye.
In order to examine the linearity of the signal a GeneTools analysis was performed for the Cy5 images using automatic track location, rolling disk correction (radius 30). All
the bands were assigned quantities (for those visible bands) ie 0.5, 1, 2, 5, 10, 50 and 100ng and a quantity calibration curve generated. The closer the R value is to 1 the
more linear the data.
In addition the dynamic range was calculated.
Dynamic range 100ng to 0.5ng: 2.3 orders of magnitude for BSA labelled with Cy5 using the Dyversity or Typhoon imaging system.
2) Sensitivity Assessment
A 1D gel was imaged using saturating imaging conditions and the following parameters were used:
Dyversity, 90micron resolution Typhoon 9400, 100micron resolution
Cy2 8mins exposure Cy2 PMT 735V, 8mins scanning
Cy3 4mins exposure Cy3 PMT 750V, 8mins scanning
Cy5 2mins exposure Cy5 PMT 775V, 8mins scanning
Comparing these images, the 7th lane from the right (0.5ng BSA) is clearly visible. However on the Typhoon image the peaks at the right of image are more saturated
(truncated). This suggests that Dyversity is capable of imaging a greater dynamic range in a single image.
3) Image Quality Assessment
For the 1D gel images (background correction and noise filtering turned on in Dymension) the background was examined and for the 2D images both correction methods
were turned off.
In both the Cy2 and Cy3 images generated on the Typhoon ‘swirls’ can be seen. Cy3 image only shown below.
The background looks ‘even and flat’ for the Dyversity image, but ‘swirls’ are present in the Typhoon image and these are inexplicable, though these have been seen on
other Typhoon systems.
Another 2D gel was imaged using non saturating imaging conditions and the following parameters were used:
Dyversity, 90micron resolution Typhoon 9400, 100micron resolution
Cy2 45secs exposure Cy2 PMT 500V, 8mins per scan
Cy3 45secs exposure Cy3 PMT 525V, 8mins per scan
Cy5 10secs exposure Cy5 PMT 500V, 8mins per scan
Images were compared in Dymension with background correction and noise filtering turned off for both Typhoon and Dyversity images in order to compare the quality of
the raw images.
There appears to be no difference in image quality for the 2D images between the two scanners.
All Trademarks and Trade names are acknowledged
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