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

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pix

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of

a

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]

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dia

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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

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spo

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are

a [

pix

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of

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spo

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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

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ls

dia

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ter

[pix

el]

of

a

5m

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iam

ete

r

are

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el]

of

a

circ

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r5m

m-d

iam

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f a

20cm

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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

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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

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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.