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Imaging2D gelsImaging2D gelsESSENTIAL TIPS AND TRICKS
EXPLORING LIFE
Imaginggels
Imaginggels
ESSENTIAL TIPS AND TRICKS
2D gel electrophoresisalso demandingproteomic analysisand needs hardsample preparationprotocol, choosingtechnique, andon the way
Introduction
on the waygeneration ofadequate imaging
The imagingsuccessful analysisprevious steps
This guide hasinformationthe best possibleimage analysisincluded e.g.
1 - http://www
electrophoresis is an extremely valuable butdemanding technique for performing global
analysis. Making your own gels takes timehard work. After weeks of adapting the
preparation setup, optimizing the gel runningchoosing the corresponding gel stainingand separating the samples, the final step
way to computerized analysis is the
Introduction
way to computerized analysis is theof images from your 2D gels by using the
imaging technology.
imaging step is at least as important for theanalysis of your experiment as each of the
steps.
has been created to help you preserve thethat is contained in the gel and produce
possible input for computerized 2D gelanalysis with modern software technology, as
. in Delta2D1.
//www.delta2d.com
Here is our checklist for creating 2D gelachieve the best possible input for computerizeddescribed in more detail on the following
• Create grayscale instead of colorimages.
• Try to span the complete available
2D Gel Imaging: Checklist
• Try to span the complete availablegrayscale range for each dye youuse.
• Choose the image resolution suchthat the smallest spots you want toanalyze have a diameter of at least5 pixels.
• Process all gel images using thesame orientation.
• Place each gel at the same positionon the imaging device. If possible,only create an image of a region ofinterest.
• Use the same parameters for allgels in your experiment to make theimaging process easy andimaging process easy andreproducible. Save and reuseprofiles that include theseparameters and your region ofinterest.
Do you know any tips or tricks to be included
Please send suggestions, questions or comments
gel images. We believe that it helps tocomputerized image analysis. Each item is
pages.
• For multiplex imaging adjust theparameters for each dye separately.
• Limit post-processing of images to• Limit post-processing of images tocrop, mirror, and rotation by 90,180, or 270 degrees.
• Use the software for image post-processing that came with yourimaging device. Avoid third partysoftware like Photoshop for imageprocessing if you do not exactlyknow the effects on quantitativeanalysis.
• Never use JPEG files forquantitative analysis.
• Avoid TIFF files if you can createand process calibrated image fileformats such as GEL or IMG/INF.
included in the next version of this guide?
comments to support@decodon.com.
Scientific scannersinstruments.backgroundimaging your
The modesimple: lighttransformed
Which Type of Imaging Device?
transformedmeasured bymeasured forinto a numberThe numbersimage file.
The imagingfrom the lightnext page):
• For visiblepasses throughthe detectorreflection modesource andsource andis reflectedeventually
• Fluorescenceparticularwith specificExcitationillumination
scanners and cameras are complex. The following section provides someinformation to make the right choices for
your gels.
of operation of an imaging device islight is emitted from a light source,
transformed by the gel, and then the resulting light is
Which Type of Imaging Device?
transformed by the gel, and then the resulting light isby a detector. The signal intensity is
for each position on the gel and convertednumber by an A/D (analog to digital) converter.
numbers are subsequently processed into a final
imaging mode indicates how the light travelslight source to the detector (see figure on
stains, in transmission mode the lightthrough the gel and is directly measured by
detector on the opposite side of the gel. Inmode the gel is located between the light
and a reflector. Light travels through the gel,and a reflector. Light travels through the gel,reflected and again traverses the gel before it
is detected.
Fluorescence dyes are excited by light with awavelength/energy level and emit light
specific longer wavelength/lower energy level.is achieved either by direct or edge
illumination.
A/D Converter
Data processing
File
Light SourceSource Optics
Detector
Detector Optics
Sample (Gel)Reflector
Glas Panel
Spot
Transmission Reflexion Direct
Visible Stains
Imaging principles for visible light (transmission, reflection
For high quality data we recommend transmissioneffects are less likely than with reflectiondifferences in higher spot intensities arecenters become completely black. In reflectionbecause the light travels through the gelof complete absorption and saturation effectsfor details).Consideration of the scan mode is importanttypes of Coomassie or silver nitrate. For fluorescenceless relevant because the detected lightgel.
Subsequent manipulation of the imagesoftware not designed for quantitative imageVisually enhanced images often show truncationsregions. Gel image files are not only picturesdata. Only flipping, mirroring, rotating inthe original data intact. Free rotating, enlargingkind of gray level, contrast or gamma adjustmentand may damage your image files (also seeCalibration").
Spot Spot
Direct Illumination Edge Illumination
Fluorescence
Optical
Filter
light (transmission, reflection) and fluorescence.
transmission mode because saturationreflection mode. Saturation means that
are not properly resolved - dark spotreflection mode this happens more easilygel twice, which increases the likelihoodeffects (see section "Saturation Effects"
important with light-absorbing stains, e.g. allfluorescence scanning the scan mode is
is emitted by the proteins within the
image data by using image processingimage analysis may ruin your images.
truncations in the high and low intensitypictures but also collections of quantitative
in 90 degrees steps and cropping leaveenlarging or reducing, down scaling, anyadjustment distort the quantitative datasee section "Data Reduction and Image
During thedecomposed(pixels). Smalleri.e. more details
Resolution isare micron (1 micrometer),
Image Resolution
1 micrometer),inch) or in the
Higher resolutionsthe increasethreshold resolution
On the otherresults in aScanning takestake up moremore memory
Image resolutionbe adapted in
the imaging process the 2D gel isdecomposed into a mosaic of equally sized squares
Smaller pixels mean higher image resolution,details are visible on the image.
is measured in various units. Typical units(1 micron corresponds to a pixel width of
micrometer), ppi / dpi (pixels per inch / dots per
Image Resolution
micrometer), ppi / dpi (pixels per inch / dots perthe metric system pixels per millimeter.
resolutions yield increased precision. However,increase in precision is marginal once a certain
resolution is exceeded.
other hand, every doubling of the resolutionfourfold increase of the image file size.
takes more time at higher resolutions, filesmore disc space, and image analysis requires
memory and time.
resolution is part of the parameters that canin the imaging software.
The following table shows the relation betweensizes for some typical resolutions.
ima
ge
re
solu
tio
n[d
ots
pe
r in
ch]
pix
el
wid
th
ima
ge
re
solu
tio
n
dia
me
ter
[pix
el]
of
a
dia
me
ter-
spo
t
are
a [
pix
el]
of
a
dia
me
ter-
spo
t
ima
ge
re
solu
tio
n[d
ots
pe
r in
ch]
pix
el
wid
th[i
nch
]
ima
ge
re
solu
tio
n[m
icro
n]
dia
me
ter
[pix
el]
of
a
1m
m-d
iam
ete
r
are
a [
pix
el]
of
a
circ
ula
r1m
m-d
iam
ete
r
100 0.0100 254 4 12
150 0.0067 169 6 27
200 0.0050 127 8 49
300 0.0033 85 12 110
400 0.0025 64 16 195
Typical image resolutions and corresponding spot and file sizes.
For image analysis of protein spots, the smallestdiameter of about 5 to 10 pixels. As an example,diameter of about 5 to 10 pixels. As an example,very small spot with 1 mm in diameter willthe resulting image.
Increasing the resolution after imaging doeswhile reducing resolution entails loss ofprovided that the diameter of the smallest5 pixels.
between resolution, spot sizes and file
dia
me
ter
[pix
el]
of
a
dia
me
ter-
spo
t
are
a [
pix
el]
of
a
dia
me
ter-
spo
t
wid
th o
f a
20cm
ge
l im
ag
e [
pix
el]
file
siz
e [
MB
]8 b
it g
rey
le
ve
ls
file
siz
e [
MB
]16 b
it g
rey
le
ve
ls
dia
me
ter
[pix
el]
of
a
5m
m-d
iam
ete
r
are
a [
pix
el]
of
a
circ
ula
r5m
m-d
iam
ete
r
wid
th o
f a
20cm
ge
l im
ag
e [
pix
el]
file
siz
e [
MB
]8 b
it g
rey
le
ve
ls
file
siz
e [
MB
]16 b
it g
rey
le
ve
ls
20 304 787 0.6 1.2
30 685 1181 1.3 2.7
39 1217 1575 2.4 4.7
59 2739 2362 5.3 10.6
79 4869 3150 9.5 18.9
Typical image resolutions and corresponding spot and file sizes.
smallest spots should have a minimumexample, with a resolution of 200 dpi aexample, with a resolution of 200 dpi a
will have a diameter of about 8 pixels in
does not add information to the image,data. However, this loss is acceptable
smallest spot of interest does not drop below
Enlargingbackgroundgeneration.backgroundfully exploitThe more signalimage the more
Dynamic Range
There are severalrange:
Cutoff highinitial imaginglowest andmeasured signalrange of interestrespectively,of interest arethe maximumthe A/D converterlevels thanalternative approachthe A/D converterthe A/D convertercurves (seeCalibration")
the distance between signal andis one of the major tasks in imageMaximizing the distance between
and top intensity in an image means toexploit the dynamic range signal recognition.
signal intensity levels are recorded in themore accurate is the quantitative analysis.
Dynamic Range
several ways to increase the dynamic
high background by a preset. In theimaging step the user can usually specify the
highest intensity signals of interest. Thesignal intensities below or above this
interest are set to zero or to the maximum,respectively, while signal intensities within this range
are evenly distributed between zero andmaximum for the bit depth. This makes sense if
converter can discriminate more intensitycan be stored in the image file. An
approach is transforming the data fromconverter by using gradation / calibrationconverter by using gradation / calibration
also section "Data Reduction and ImageCalibration").
Controlling the conversion from colordevices create grayscale images by convertingimage file based on average intensities ofimprove the resulting image in some situationsgrayscale conversion yourself, e.g. by extractingabsorption or (even better) with the highest
• An image of a Coomassie blue stained• An image of a Coomassie blue stainedblue color channel from the color imageimage.
• A similar approach can be used for theefficiently absorb blue light, so extractingexcluding red and green) gives the best
Increasing the voltage of the lightthe light detector may be increased if thecovered by signal intensities in a fluorescencesignals overall, but also the absolute distancestrongest intensity signal on the gel imagePlease be aware of saturation effects! (See
Increasing the exposure time. Similarly,Increasing the exposure time. Similarly,prolonged if the intensity range is not completelyin a camera system. As for the approachsignal intensities and the absolute distancestrongest intensity signal on the gel imagePlease be aware of saturation effects! (See
color to grayscale. Most imagingconverting a color image to a grayscale
of the different color channels. You cansituations by controlling the color-to-
extracting the color channels with highhighest dynamic range:
gel can be improved by excluding thegel can be improved by excluding theimage before converting it to a grayscale
the brownish silver stains. They mostextracting the blue color channel (and
results.
detector. The voltage (sensitivity) ofthe intensity range is not completely
fluorescence scan. This not only increases thedistance between background signal and
image.(See also section "Saturation Effects".)
Similarly, the exposure time may beSimilarly, the exposure time may becompletely spanned by signal intensities
above, this increases both the overalldistance between background signal and
image.(See also section "Saturation Effects".)
The intensitydifferentiationfluorescenceare absolutelynormally lasershighly concentratedfluorescent dyesdifferent lasers
Light Intensity andDetector Sensitivity
different lasersexcitation andlamps andtheir highest
The light harvestingcollect the lightemitted frominto computerelement orelectrical signalA/D (analogstored in the
intensity of the light source directly influencesdifferentiation of high density spots. Especially forfluorescence imaging high performance light sources
absolutely necessary. In this field of imaginglasers or UV lamps are used. Lasers deliver
concentrated light and can effectively excitedyes. Depending on the fluorescent dyes
lasers with specific colors as well as
Light Intensity andDetector Sensitivity
lasers with specific colors as well asand emission filters are needed. Usuallylasers need a warm-up phase to reach
highest performance.
harvesting detector and the A/D converterlight that has passed through or that is
from the gel and translate this informationcomputer readable signals: The detector (CCD
PMT) converts the light intensity to ansignal that is subsequently digitized by the
(analog to digital) converter. These data arethe image file.
Scanners. It is common to use office documentdyes (Coomassie, silver, X-ray film).differentiation of gray levels up to an opticalsufficient for gel documentation purposesdifferentiation of high density spots reaching
Document scanners resolving ODs up to 3or professional product lines of well knownor professional product lines of well knownlamp before starting the scan process isbecome more powerful when they reachusage of the scanner causes aging of theintensity.
In many high performance scanners for fluorescencesignals can be detected. In these scanners(PMT - photo multiplier tube) can be adaptedsensitivity for very weak signals but mayUnfortunately increasing PMT voltage alsoPMT voltage should be modified withenhancement of strong signals "boosting"saturation threshold.
Cameras. Camera systems usually cannotCameras. Camera systems usually cannotintensity. The system sensitivity can be modifiedsimilarly to changing PMT voltage for acameras is different from scanners, homogenousgel is an important quality aspect. Some camerainhomogenous illumination, which has to
document scanners to detect absorbingThe manufacturers usually claim a
optical density (OD) of 2.0. This is mostlypurposes. However, it is insufficient forreaching optical densities near 3.7 to 4.0.
3.7 or 4.0 can be found in the premiumknown manufacturers. Warming up theknown manufacturers. Warming up theis highly important because the lamps
reach their working temperature. Extensivethe light source resulting in weaker light
fluorescence detection also very weakscanners the voltage of the light detector
adapted. Higher voltages increase themay also increase image background.
also results in amplifying noise. Thereforecare. Another pitfall is simultaneous
"boosting" very intense spots beyond the
cannot be adjusted to changing lightcannot be adjusted to changing lightmodified by varying the exposure time,
scanner. Because the construction ofhomogenous illumination of the complete
camera systems can compensate for anto be adjusted frequently.
Saturation effectsbecause theyintensity spotsquantities ofseveral reasons
Intense staininglight passing
Saturation Effects
light passing
Dyes can appearstaining techniqueshigh intensityintensity correlationtechniques basedstaining (silver
Avoid saturation
• reducing staining
• loading less
Heavily fluorescingHeavily fluorescingdetector, oroverload thedifferentiateis overexposedphosphorimaging,accumulatemay be exhausted
A perfect spot without any saturation effect.
effects heavily affect quantitative datathey virtually truncate the peaks of high
spots. These truncations distort theof spots. Saturation effects may occur for
reasons:
staining can cause complete absorption ofpassing through the spot.
Saturation Effects
passing through the spot.
appear in very high concentration. Sometechniques accumulate pigment on top of
intensity spots losing the linear amount-to-correlation. This can be observed in all
based on the photographic silver nitrate(silver staining, X-ray film).
saturation effects in the lab by
staining or increasing destaining time, or
less protein on the gel.
fluorescing gels may overload the lightfluorescing gels may overload the lightintensively excited light detectors may
the A/D converter, which can no longerdifferentiate between strong signals. Similarly, if a gel
overexposed to a phosphorscreen duringphosphorimaging, the capacity of the screen to
energy from radioactive decay eventsexhausted.
Saturation effects usually have a planarsoftware packages highlight saturated imageimage they can also be observed.
A saturated protein spot.
To reduce or eliminate saturation effects,
• increase light intensity (not for fluorescence
• reduce the PMT voltage (scanner), and/or
• reduce the exposure time (camera, phosphorscreen)
For classical silver staining techniquestypical "doughnut spots". Silver stainingproteome analysis because the relation
Silver stain: A "doughnut protein spot" with strongly
proteome analysis because the relationprotein amount is usually not linear.
planar appearance. Many imaging deviceimage regions. In the 3D view of the gel
repeat the imaging and
fluorescence staining),
and/or
phosphorscreen).
saturated spots generally appear asstaining should not be used in quantitativerelation between silver stain intensity and
strongly diminished central staining intensity.
relation between silver stain intensity and
Avoiding background,They may interfereGel breakingthe spot detection,spots etc.process. Thethe effect oflow signals
Artifacts:Improve Device Handling
low signalslow sampleradioisotopesfrustratingperforming analysis
Backgroundfluorescing glassLow fluorescenceproblems. Alsofilters forbackground.filters are suitable
Noise canenhancing alsoenhancing alsovoltage solvesscreens thataccumulatebefore exposurethe imagingmanipulation
background, artifacts and noise is essential.interfere with the spot detection process:
breaking separates spots, sprinkles may misleaddetection, noise can cover low intensity
Artifacts also affect the quantitationThe weaker the signals are the stronger is
of artifacts on quantitative analysis. Verymay be observed due to faint spots, very
Artifacts:Improve Device Handling
may be observed due to faint spots, verysample amounts or poor incorporation of
radioisotopes. Frequently, it is better and lessto repeat the experiment instead ofanalysis of suboptimal images.
may be caused by insufficient destaining,glass plates, gel coverings and backings.
fluorescence equipment minimizes suchAlso inappropriate selection of optical
fluorescence imaging may cause. Reevaluate which light sources and
suitable for your analysis.
be produced by high PMT voltagesalso random signals. Adapting the PMTalso random signals. Adapting the PMT
solves this problem. Similarly, phosphorthat have not been used for a longer time
noise. Erasing the screen immediatlyexposure solves this problem. Try to optimize
imaging procedure and avoid downstreammanipulation of your images.
• Exclude regions containing artifacts during
• Image all gel images using the same orientation
A common trick is to cut off one cornercorner. Make sure that this corner becomesfor every gel you scan. This has the addedhave the orientation that is usually requiredincreasing from left to right, Mw decreasingincreasing from left to right, Mw decreasingto rotate or flip the image we recommendyour imaging device. General purpose imagePhotoshop) may not preserve the vendorinformation in the image file.
• Ghost images or spot shadows
Do not move the gels during exposureaware of device vibrations during theprocess. Fix the gel on the device surfacesuction cups).
• Horizontal or vertical lines
Sometimes single CCD elements in CCDdesktop scanner fail to work. The correspondingline of the image remains empty. The onlysolve this problem is replacing the CCDsolve this problem is replacing the CCDthe whole scanner.
• Artifacts or noise from phosphorscreens
Decontaminate or replace phosphorscreensscreens before use to locate and removebefore exposure to eliminate accumulated
• Artifacts and scratches on glass panels
Handle your imaging devices with carewood free tissue (do not use recyclingpanels clean and free of dust and fat.
during imaging
orientation
of the gel. Cut off the low Mw, low pIbecomes the lower left corner in the image
added advantage that your images willrequired by the proteomics journals (pI
decreasing from top to bottom). If you needdecreasing from top to bottom). If you needrecommend using the software that came with
image processing software (e.g. Adobevendor specific grayscale calibration
Ghost images are problematic because
or scan. Bethe imagingsurface (use
CCD bars of acorresponding
only way toCCD element or
problematic because they are hard to recognize.
CCD element or
phosphorscreens; scan auto-exposed phosphor-remove contaminations. Erase the screen
accumulated noise.
care and only use a mild detergent andrecycling tissue!) for cleaning. Keep all glass
Proteinfärbung / Detektion680
660
640
Legendemission
Stoke Shift
excitation
Coomassie
R250Coomassie
Excitation / Emission of Fluorescent Dyes
400
600
500
480
460
440
420
580
560
540
520
640
620
G100
G200
G300
Coomassie
G250
Wav
elen
gth
in n
m
G-Dyes
300
380
360
340
320
280
TryptophanPhenyl
alanine
Tyrosin
amino acidsautofluorescence
Proteinfärbung / Detektion
ProQMultiplex
Dyes
Excitation / Emission of Fluorescent Dyes
G100
G200
G300
Coomassie
Fluor Orange
ProQ
Diamond
Phosphate
ProQ
Amber
TMHs
ProQ
Sapphire
Poly His
Dyes
Dyes
Cy2
Cy3
Cy5
Cy-Dyes
ProQ
Emerald
Glycosyl
Tryptophan
Proteinfärbung / Detektion
Sypro
Red
Sypro
TangerineGlycoprotein
Single Channelfluorescent Dyes
Sypro
Orange
Coomassie
Fluor Orange
Sapphire
Poly His
Sypro
Ruby
Sypro
Rose
Epicocconone
LavaPurple
Proteinfärbung / Detektion680
660
640
Krypton
Infrared
Krypton
Glycoprotein
Single Channelfluorescent Dyes
Legendemission
Stoke Shift
excitation
400
600
500
480
460
440
420
580
560
540
520
640
620
Krypton
Flamingo
Wav
elen
gth
in n
m
300
380
360
340
320
Epicocconone
LavaPurple
280
Currently variousgeneral twobinding dyesbefore electrophoreticDyes, etc.) andbinds electrostaticallyelectrophoresis)
Appropriate Protein Dyes
Besides lowthe followingchoosing a protein
• Is the dyerange ofdynamic range?
• Does thesaturation
• Has anybeen reported
• Does theavailable imagingand emissionand emission
• Is the stainingminor handlingvariances of
various protein dyes are available. Intwo types can be distinguished: covalentlydyes (the dye chemically binds to the protein
electrophoretic separation – e.g. G-Dyes, Cy-and non covalently binding dyes (the dye
electrostatically to proteins afterelectrophoresis).
Appropriate Protein Dyes
protocol complexity and attractive pricefollowing aspects should be considered when
protein dye:
dye suitable for the protein concentrationinterest, the expected sensitivity and
range?
dye tend to show artifacts (sprinkles,effects, noise, background)?
significant protein-to-protein variabilityreported for the dye?
dye match the characteristics of theimaging system, e.g. in terms of excitation
emission wavelengths?emission wavelengths?
staining protocol for the dye tolerant againsthandling deviations to avoid unacceptable
of resulting signal intensities?
The following figure depicts the dynamicprotein variation of different post-electrophoretically
100
1000
10000
Dye specific signal intensities for different proteins
1
10
100
1000
10000
1
10
100
10 100 1000 10000 10 100
concentration [ng protein / mm
sig
na
l [1
03
cou
nts
]
1 – Varying amounts of proteins (bovine serumalbumin) were stained with the indicated dyesof the Institute of Microbiology – Greifswald University
dynamic ranges, sensitivity and protein-to-electrophoretically applied dyes.
β-LGCSA
BSACoomassie BB
BSALava Purple
proteins and concentrations1.
β-LGCSA
BSA
Flamingo
β-LGCSA
BSA
Krypton
β-LGCSA
BSA
Coomassie SB
β-LGCSA
BSASypro Ruby
β-LGCSA
BSA
1000 10000 10 100 1000 10000
concentration [ng protein / mm2 gel]
serum albumin, beta-lactoglobulin, chicken serumin a scientific study of the proteomic group
University (Kuzinski, Greifswald, 2007).
Light Sourceits specific excitationlower energy,intensity oflight from thethe excitation
Because common
Detection of Fluorescent Dyes and Multifluorescence
Because commonfixed wavelengthsalso violet,of laser andcompromise.continuouslimited rangeexcitation. Theinterest unfortunatelylaser scanners
Emissionfilters) are usedlight as possiblewavelengthmore light ofmore light ofthe higher aresignal detectiononly one dyewavelength
Source. Each fluorescent dye absorbs light ofexcitation wavelength and emits light of
energy, i.e. of a longer wavelength. Thethe emitted signal is maximized if the
the imaging device exactly corresponds toexcitation maximum of the fluorescent dye.
common laser scanners contain lasers of
Detection of Fluorescent Dyes and Multifluorescence Setups
common laser scanners contain lasers ofwavelengths (red, green, blue – in some devices
blue-green or infrared), the combinationand dye (excitation maximum) is always a
. Devices using light sources with aspectrum provide more flexibility if a
range of the spectrum can be selected forThe light intensity of the spectral band of
unfortunately is much weaker than that ofscanners.
Filters. Optical long pass filters (LPused to detect as much of the emitted
possible. LP filters are specified by theabove which light can pass the filter. The
of the emission spectrum passes the filter,of the emission spectrum passes the filter,are the sensitivity and dynamic range of
detection. Thus in all experimental setups withdye an LP filter slightly above the excitation
should be employed.
In multiplex designs (like the DIGE approachabsolutely mandatory to avoid detectingdifferent dyes. This can already happen eventhe light emitted by this dye might excitesecondary fluorescence. When using andetected, regardless of whether it originatesother one(s). Such unwanted secondary fluorescencepass (BP) filters.pass (BP) filters.
BP filters are specified by the wavelengthor wavelength range for which the filter is500/20 BP filter is most transmissive at 500490 to 510 nm can pass. High quality BPacross the whole specific band width. Narrowthe detection (emission) wavelength; however,bands appropriate broad band pass filters
To summarize, the selections of dyesinterdependent. The light source needswavelengths of the dyes. Furthermore aseparate detection of the specific light signals
Imaging time. Multiple scanning of multiplexImaging time. Multiple scanning of multiplex(up to 20 minutes per scan in establishedshrinkage owing to evaporation of gelpositions may be shifted, making it difficultanalysis. This can be avoided by keeping theusing Delta2D's innovative approaches includingcomplete expression profiles1. Furthermore,shorter exposure time.
1 - http://www.delta2d.com
approach using G-dyes or Cy-dyes) it isdetecting a mixture of light being emitted by
even if only one dye is excited, becauseexcite one of the other dyes causing
an LP filter all emitted light would beoriginates from the dye of interest or the
fluorescence is blocked by using band
wavelength at which light transmission is highestis transmissive. For instance, an optical
500 nm, but also light in the range fromBP filters are almost equally translucentNarrow band filters may exactly confinehowever, for dyes with broad emission
filters enhance the detection sensitivity.
dyes and the imaging device areto be compatible with the excitationfilter set should be used that permits
signals emitted by each dye.
multiplex gels is rather time-consumingmultiplex gels is rather time-consumingestablished systems) and may result in gel
gel buffer or water. Consequently spotdifficult to match spots during the image
the scan surface wet enough and/or byincluding image warping and creating
Furthermore, camera systems require a much
Several artifactshandling:
• Air bubbles
Use enoughbe awaredevice. Silicone
Artifacts:Improve Gel Handling
device. Siliconeproblematic
• Newtonian
Try to avoidplates. If thisinside the gel
• Artifacts on
Try to focusimaging device
• Fingerprints
Use (powderfree)panels of your
• Labels on the
Bubbles may interfere with quantitation.
Using permanentsometimessubsequentdoes notpattern.
artifacts can be avoided by appropriate gel
bubbles and water droplets
enough water between glass panel and gel butof water seeping into your imaging
Silicone sealants may help to close
Artifacts:Improve Gel Handling
Silicone sealants may help to closeproblematic grooves.
Newtonian rings
avoid them using Kapton® tape or glassthis is not possible focus the laser beamgel.
on the gel surface
focus the laser beam inside the gel if yourdevice supports this feature.
Fingerprints on the gel
(powderfree) gloves and/or clean the glassyour imaging device
the gel
permanent markers for gel labelingsometimes interferes with the scan. To simplify thesubsequent analysis please make sure that the label
overlap with parts of the gel or spot
• Gel ruptures and gel pieces
Work carefully and use gel strengthenernecessary try to assemble the gel piecesthem before imaging.
Spot ruptures cause problems with spot detectionediting.
• Fluorescent sprinkles
Rinse the gel with destainer or water andgloves, dust free experimental equipment
Fluorescent sprinkles can disturb the spot detection
strengthener to give the gel more stability. Ifpieces to minimize the distance between
detection. This may be corrected by manual spot
and avoid evaporation. Use powder freeequipment in a dust free environment.
detection.
Signal intensitiesimage file. Ineach pixel,each color channelRGB color spaceand blue).
Make sure you
Bit Depth
Make sure youthe extra colorfor the subsequent
When your imagefile this meansbits, i.e. representsintensity levelsincludes 256number of bits(or color depth)
bit depth
1 bit
8 bit
12 bit
16 bit
Typical bit depths.
intensities are encoded as numbers in theIn grayscale images this is one number for
while in color images one number forchannel has to be stored per pixel (e.g. inspace one intensity value for red, green,
you use grayscale instead of color images:
Bit Depth
you use grayscale instead of color images:color information does not provide benefit
subsequent analysis of 2D gels.
image file is, for example, a "16-bit TIFF"means that each number is encoded with 16
represents on of 65,536 (= 216) differentlevels. In contrast, an 8-bit image file only256 (= 28) different intensity levels. The
bits per color is also called the bit depthdepth).
intensity levels example
2 black and white FAX image
256 GIF image
4,096 TIFF image
65,536 TIFF image
Typical bit depths.
TIFF images can have different bit depthsa widely used image file format.
Generally, higher bit depth is better for quantitativeexists between accuracy and storage spaceas the respective 8-bit file, but comprisescommon image processing programs are8 bit.8 bit.
Decreasing bit depth in an existing imageaccuracy. Conversely, it is not recommendedimaging process: transforming an 8-bit imageagain 256 out of 65,536 possible gray values
You can find more information about bitWikipedia Category: Color Depth1.
Although high bit depth enhances imagequantitation, it is often not visible on thethe respective technologies.
1 - http://en.wikipedia.org/wiki/Color_depth
depths. This is one of the reasons why TIFF is
quantitative image analysis. A tradeoffspace: a 16 bit TIFF file two times larger
256 times more intensity levels. Manynot able to deal with bit depths above
image file normally results in loss ofrecommended to increase bit depth after the
image to a 16-bit image will result invalues.
bit depth in image file formats in the
image analysis, particularly protein spotscreen or in print due to restrictions of
Compressionalgorithms thatwhile retainingIf you usecompressionfile formats do
Image compression
Image File Formats:Image Compression
Image compressionlossy or losslessthat informationyielding a highersizes than losslessbe saved incompression,Often compressionpossible. Thecompressionheavily changecompressioninfluence of
We recommendimage analysisimage analysis
Compression of image data is achieved withthat reduce the size of a gel image file
retaining all or most of the image information.calibrated image file formats, image
compression is not an option, because compresseddo not store the calibration information.
compression algorithms can be classified as
Image File Formats:Image Compression
compression algorithms can be classified aslossless methods. Lossy compression means
information can be lost in the image details,higher compression ratio and smaller filelossless methods. Uncalibrated files mayin file formats that use lossless data
compression, e.g. subformats of TIFF / TIF or PNG.compression to 50 % of the original size is
The commonly used JPG format uses a lossycompression method. Large compression ratios may
change or destroy your data. Even for lowcompression ratios, there is still an unknown
compression on spot quantities.
recommend avoiding the JPG file format foranalysis purposes.analysis purposes.
Image compression only saves disc spaceworking memory (RAM) needed for imageare also completely extracted before starting
The following table summarizes the propertiesformats.
format compression gray levels
GIF lossless 256
BMP no 2; 256
PNG lossless 256
JPG lossy 256
TIF(F) no (lossless) 2;256;1024;4096;65536
GEL no 1024;4096;65536
IMG/INF no 1024;4096;65536
Commonly used image formats. Avoid formats like
You can find more information aboutCategory: Image File Formats1
1 - http://en.wikipedia.org/wiki/Image_file_formats
Category: Image File Formats1
space but does not affect the amount ofimage analysis because compressed files
starting the analysis.
properties for commonly used image file
use for quantitative
analysiscalibration
no no
(yes) no
yes no
no no
2;256;1024;4096;65536 yes no
1024;4096;65536 yes yes
1024;4096;65536 yes yes
like JPG.
image file formats in the Wikipedia
org/wiki/Image_file_formats
Some imagingvalues thanway to dealintensities linearlyimage file format(A/D) signalintensity levels,256 levelsExample of an
Data Reduction and Image Calibration
256 levelscondenses fourlevel in theprocess causes
Especially ifcondensationdynamic rangeby the A/D converterdeliver 1,024scanning only920, only theseencoded inlevels are condensedComparedincreased by
Example of animage calibration curve.
increased by
imaging devices can measure more intensityfit into the available image formats. One
deal with this is to convert the signallinearly into the bit depth offered by theformat. Example: The Analog to Digital
signal converter can deliver 1,024 differentlevels, but the image file format is limited to
(8-bit depth). Linear transformation
Data Reduction and Image Calibration
(8-bit depth). Linear transformationfour A/D converter levels to one intensity
the image. Obviously, this condensationcauses loss of accuracy.
images are captured with light scannerscondensation can be improved by using only the real
range of the measured signals as deliveredconverter. Example: The A/D converter can
024 different intensity levels. If the gelonly renders intensities between 128 and
these 792 intensity levels have to bethe image file: thus three A/D converter
condensed to one image intensity level.to the example above, accuracy is
by 25%.by 25%.
There are instruments that can measureintensity levels, far more than can be encodedorder to save as much information asintensities are transformed to pixel graycalibration curve during scanning. Typically,curve ensures that lower intensities arehigher intensities.
Calibration curves are vendor specific andformats like TIFF. For this reason vendor specificGEL or IMG/INF which include specific parameters
On the basis of the calibration curve theoriginally measured signal intensities. A wrongincorrect quantitative values. If your imagethe vendor specific file format, make surestandard image file format like TIFF.
Commonly used image processing packagescalibration. It is even possible that calibrationimages with these packages.
measure 100,000 and more different signalencoded in a TIFF or similar file format. In
as possible in these files, measuredgray values according to a nonlinear
Typically, the respective nonlinear calibrationare encoded with higher accuracy than
and not saved in standard image filespecific formats have been created like
parameters of the calibration curve.
the image analysis software decodes thewrong or no calibration curve results in
image analysis software cannot interpretsure that you save your images in a
packages (e.g. Photoshop) ignore grayscalecalibration information is lost when saving
Notes
Copyright and TradmarksAll material in this brochure is Copyright
Reserved. DECODON, DECODON logo, Delta2D,
or registered trademarks of DECODON GmbH in Germany
other countries all over the world. All other
trademarks or registered trademarks of their
DECODON GmbHWalther-Rathenau-Str. 49a
17489 Greifswald, Germany
www.decodon.cominfo@decodon.com
phone: +49(0)3834 515230fax: +49(0)3834 515239
Copyright © DECODON GmbH. All Rights
. DECODON, DECODON logo, Delta2D, and Protecs are trademarks
DECODON GmbH in Germany and in several
other products mentioned are
their respective companies.
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