FLUORESCENCE (SMOC) - UAMbabia.cbm.uam.es/~smoc/Software_Manuales_SMOC/Fluorescence... ·...

FLUORESCENCE QUANTIFICATION WITH FIJI

Alfonsa Díaz Torres

Servicio Microscopía Óptica y ConfocalCentro Biología Molecular Severo Ochoa

Madrid 2016SERVICIO DE M

ICROSCOPÍA ÓPTICA O CONFOCAL (SMOC)


1. Image analysis

– Digital image

2. Sample preparation

3. Image acquisition

4. Corrections

5. Fluorescence intensity quantification

– Set Measurements

– Limit to Threshold

– Images with multiple objects

– Images with multiple planes

SERVICIO DE MICROSCOPÍA ÓPTICA O CONFOCAL (S

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IMAGE ANALYSIS

Techniques for getting information from images.

– Obtain quantitative data in numerical form

– Image capture and analysis software


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DIGITAL IMAGE

• Dot mosaic (pixels).

– Color or grayscale.


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DIGITAL IMAGE

• Dot mosaic (pixels).

– Color or grayscale.

8 bits = 28 = 256

0

255

pixel Color

Numero binario equivalente

Decimal number

12 bits = 212 = 4096

16 bits = 216 = 65536


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1. Image analysis

– Digital image



4. Corrections







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SAMPLE PREPARATION

Prepare samples the same day using exactly the sameprotocol


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SAMPLE PREPARATION

Controls

AUTOFLUORESCENCE: Identical protocol withoutprimary or secondary antibodies.

SECONDARY ANTIBODIES: Incubate the sample onlywith the secondary antibodies.


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SAMPLE PREPARATION

Controls

CROSSTALK OR CHANNEL INTERFERENCEStain samples with each primary/secondaryantibody combination separately and acquireimages for all the channels with the sameacquisition parameters as those used indouble or triple-stained preparations


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1. Image analysis

– Digital image



4. Corrections







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IMAGE ACQUISITION

CONFOCAL SYSTEM

• Same conditions and same day

• Láseres estables (1h antes encen

• No tener pixeles saturados

Set conditions according to the brightest sample


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IMAGE ACQUISITION

CONFOCAL SYSTEM


• Allow lasers to stabilize (switch on 1h before)




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IMAGE ACQUISITION

CONFOCAL SYSTEM



• No saturated pixels


Range Indicator paletteRed = saturation


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IMAGE ACQUISITION

CONFOCAL SYSTEM




• Check controls



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IMAGE ACQUISITION

CONFOCAL SYSTEM

• Avoid photobleaching

• Field centered

• 12 Bits


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IMAGE ACQUISITION

WIDE-FIELD SYSTEM


• Lámpara estable (1h antes encendida)


• Evitar photobleaching



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IMAGE ACQUISITION

SISTEMA CAMPO ANCHO

• Mismas condiciones y mismo día





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IMAGE ACQUISITION

SISTEMA CAMPO ANCHO

• Mismas condiciones y mismo día




If the camerahas variablesensitivity, thisvalue must alsobe the samebetweensamples


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IMAGE ACQUISITION

WIDE-FIELD SYSTEM


• Do not autoscale

• Allow lamp to stablize (Switch on 1h before use)



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IMAGE ACQUISITION


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IMAGE ACQUISITION

WIDE-FIELD SYSTEM


• Do not autoscale

• Allow lamp to stablize (Switch on 1h before use)


• Avoid photobleaching


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IMAGE ACQUISITION

WIDE-FIELD SYSTEM

• Check controls

• Use the highest bit depth allowed by the system

• Select the center quadrant

• Zoom 1


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IMAGE ACQUISITION


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IMAGE ACQUISITION

For quantification:

IMAGES ARE NOT BEAUTIFUL, THEY ARE DARK!!


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1. Image analysis

– Digital image



4. Corrections







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CORRECTIONS

WIDE-FIELD SYSTEM

• Background correction

• Shading correction


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Background correction METAMORPH

Select “Image”/“Keep Shutter Closed” andacquire image in “Acquire Background”.

Images will be corrected for camerabackground in the absence of light.

To save the background image: “DisplayBackground Image” and save that image.

Acquisition conditions must be identical forthe background image and the final one.Check that a green icon appears next to“Bkgd”.

CORRECTIONS


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Shading

CORRECTIONS


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BEFORE

AFTER

CORRECTIONS


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Shading correction METAMORPH

Corrects defects in field illumination.

To acquire a shading image:

Defocus the preparation enough to see auniformly illuminated background field.

CORRECTIONS


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Shading correction METAMORPH

Select “Image” and capture an image in“Acquire Shading Reference”.

To save the reference image select “DisplayShading Image” and save that image.

Acquisition conditions must be identical forthe shading image and the final one. Checkthat a green icon appears next to “Shd”.

CORRECTIONS


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CORRECTIONS

FIJI

• Background correction

• Shading correction


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Background correction Fiji: Substract Background

CORRECTIONS


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Background correction

• Process ▶ Subtract background Size of the largest object that is not part of the background

Rolling-ball algorithm

CORRECTIONS

NON-UNIFORM BACKGROUNDSERVICIO DE MICROSCOPÍA ÓPTICA O CONFOCAL (S

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• Process ▶ Subtract background

Size of the largest object that is not part of the background

CORRECTIONS


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Se

CORRECTIONS

Fiji : Math


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• Process ▶Math ▶ Subtract

This will subtract the mean of theROI from the image plus anadditional value equal to thestandard deviation of the ROImultiplied by the scaling factoryou enter. The default value is 3.

CORRECTIONS


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CORRECTIONS

We have to calculate:- The average background value

(usually using a ROI)- Its standard deviation


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• Drawing a ROI

CORRECTIONS




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• Analyze ▶ Set measurements ▶ Subtract

CORRECTIONS




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• Analyze ▶ Set measurements

CORRECTIONS




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• Analyze ▶measure

CORRECTIONS




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Select a ROI in the background and calculate its mean value and standard deviation


CORRECTIONS

UNIFORM BACKGROUNDMean +( StdDev x 3)


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CORRECTIONS

FIJI

• Shading


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Shading correction Fiji: Image calculator

CORRECTIONS


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Shading correction

• Process ▶ image calculator

Open the uncorrected image and the flat-field image (shading image).

Uncorrected image Shading-corrected image

CORRECTIONS


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Shading correction

If you do not have a reference shading image, you can use the FFT

Bandpass function as an alternative method of shading correction. It isless ideal but still produces acceptable results in most cases.

• Process ▶ FFT ▶ Bandpass Filter

CORRECTIONS

This tool removes highspatial frequencies(blurring the image)and low spatialfrequencies (similar tosubtracting a blurredimage).

It can also suppresshorizontal or verticalstripes that werecreated by scanning animage line by line.SERVICIO DE M


Shading correction

CORRECTIONS

Uncorrected image Shading corrected in ImageJ FFT Method


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FLUORESCENCE INTENSITY QUANTIFICATION WITH IMAGEJ-FIJI

1. Image analysis

– Digital image



4. Corrections







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FLUORESCENCE INTENSITY QUANTIFICATION

• 1) You can simply hover the cursor over a given area in the image and read out the pixel intensity at that pixel on the toolbar. – For RGB images, there will be three

numbers: red, green and blue.


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• 2) Analyze option

– Go to Analyze/Set Measurements.


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– Go to Analyze/Set Measurements.

Check the boxes for theinformation you want.

You can get information onarea, diameter, perimeter andother factors as well asintensity information.


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SET MEASUREMENTS OPTIONSArea in pixels squared or inmeasurement units of the selectedimage or area.


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SET MEASUREMENTS OPTIONSArea in pixels squared or inmeasurement units of the selectedimage or area.

To see if the image is calibrated andthe measurement units :

Image/properties


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SET MEASUREMENTS OPTIONSAverage gray values of theselection.

Sum of pixel gray levels from theselected zone divided by thenumber of pixels.

5 10

15 0

5+10+15+0

4= = 7,5

∑ pixel values

pixel number


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∑ pixel values

pixel number

Different ROIsizes can becomparedSERVICIO DE M


SET MEASUREMENTS OPTIONS

Standard deviation of thevalues used to generate the grayvalue mean.

Most frequent gray value in theselected area.


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SET MEASUREMENTS OPTIONSMinimum and maximum grayvalues in the selected area.

Median value.


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Provides two values:

IntDenThis is equivalent to the productof Area and Mean Gray Value.


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Provides two values:

IntDenThis is equivalent to the productof Area and Mean Gray Value.

RawIntDenThe sum of all pixel values in the image or selection.


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SET MEASUREMENTS OPTIONSIntDenThis is equivalent to the product of Area and Mean Gray Value.

RawIntDenThe sum of the values of the pixels in the image or selection.

15 2040 5

0 1

2 0

8 3

0 2

Area = 10Mean = 20Int Den = 200

Area = 100Mean = 2Int Den = 200

Area = 10Mean = 20Raw Int Den = 80

Area = 100Mean = 2Raw Int Den = 16

Different ROIsizes can becompared

Different ROIsizes can not becompared

0 1

2 0

8 3

0 2

15 2040 5


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SET MEASUREMENTS OPTIONSThe position (slice, channel and frame)in the stack or hyperstack of theselection.


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– Go to Analyze/SetMeasurements.

Mean grey value

• Then selecting Analyze/Measure,you will get information on theentire image.


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• 3) Limit your measured area– Draw a region of interest (ROI) around

your object of interest with the drawing tools .

• Analyze/Measure


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• 3) Limit your measured area– To copy/paste the shape or ROI to

another image in order to compare equivalent regions in different images

• Edit/Selection/RestoreSelection


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• 3) Limit your measured area– “Limit to Threshold”

• Analyze/Set Measurementscheck Limit to Threshold


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• 3) Limit your measured area– “Limit to Threshold”

• Image/Adjust/Threshold. To highlight the area you want to analyze.

• Analyze/Measure. Will give you intensity measurements only in your thresholded area.SERVICIO DE M



• 3.1) Using “Limit to Threshold”Histogram:represents thedistribution of pixelintensities in theimage.0 = black255 = white

Dragging the sliders selects different regions within the greyscale.

In this case, all the pixels between 76 (dark grey) and 182 (mid grey) are highlighted in red.

Maintains the same limits for all images

Select dark background ifthe background ishighlighted in red


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• 3.1) Using “Limit to Threshold”

There are many algorithms you can use to calculate the threshold without introducing user-bias.


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• 3.1) Using “Limit to Threshold”

• We must choose the most appropriate method or algorithm to segmentour image

Original image

Test algorithms with several of ourimages to decide which is the bestSERVICIO DE M



• 3.2) Combine “Threshold” and ROI

Use a selection tool to mark your ROI.Measurements will now be limited to pixels whichfall within the selected area and are within theselected threshold intensity range.


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• 3.2) Combining “Threshold” and multiple ROIs•Analyse / Tools / ROI Manager

•Select a ROI and add it to ROI Manager: “Add” Button

•Repeat as required•Click the measure button to see the measurements

It gives us the measurements we have

selected in Set Measurements


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• 4) To create a plot ofintensity values acrossfeatures in your image.

– The plot gives intensity values along the line drawn across the image.

• Analyze/Plot profile

– To obtain a similar plot for intensity values through a z or time stack, or within an ROI drawn on a stack.

• Image/Stacks/Plot z-axis profileSERVICIO DE MICROSCOPÍA ÓPTICA O CONFOCAL (S

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• Draw a line inthe area tobe analyzedwith thedrawingtools.

• Analyze/Plotprofile


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List buttonGives a list of the intensityvalues used to create thegraph.


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• Draw a line inthe area to beanalyzed withthe drawingtools.

• Analyze/Plotprofile

• Image/Stacks/Plot z-axisprofileSERVICIO DE M


FLUORESCENCE INTENSITY QUANTIFICATION FOR EACH OBJECT IN IMAGES WITH MULTIPLE

OBJECTS

– Make a copy of your image

• Image/Duplicate

– Threshold to highlight all thestructures you want to measure• Image/Adjust/Threshold

- Manually- Using algorithmsSERVICIO DE M



OBJECTS

– If you have particles that have mergedtogether

• Apply (This will create a binary versionof the image )

- Two pixel intensities: black (=0) andwhite (=255).SERVICIO DE M



OBJECTS

– If you have particles that have mergedtogether

• Process/Binary/Watershed

Watershed can often accurately separate particlesby adding a 1 pixel thick line where it calculatesthe division should be.


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OBJECTS

• Analyze/Set measurements

• Set the “Redirect to” line to the name ofthe copy of the image that is still ingrayscale.

• If you don’t do this, your intensity valueswill be read from the binary image, andthey will all be 255!

• Checking “display label” will label yourdata table with the image name andparticle number.

• Use the checkboxes to select whichstatistics you want from your image.


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OBJECTS

– Click on the binary or thresholdedimage to select it, then go to:• Analyze/Analyze Particles

-s

Poner el mayor tamaño de lasparticulas a contar.

Debe estar en pixels o una unidadde medida si la imágen estácalibrada (measurement^2)

• Para ver si la imágen esta calibrada:Image/PropertiesSERVICIO DE M



OBJECTS

– Click on the binary or thresholdedimage to select it, then go to:• Analyze/Analyze Particles

-Size

Particles smaller than that valueare ignored

It will either be in pixels, or, ifyour image is calibrated, in a unitof measurement^2

• To check if your image is calibrated:Image/PropertiesSERVICIO DE M



OBJECTS


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OBJECTS

• File/Save as text

To save the results window


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OBJECTS

• Image/Overlay/Flatten

• File/Save as/Tiff

To save the image with the numbers


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FLUORESCENCE INTENSITY QUANTIFICATION FOR Z STACK

IMAGES

• Image/stack/Z-Project

Z stack images

Z Project is a method of analyzing a stackby applying different projection methodsto the pixels within the stack


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IMAGES


Z stack images

There are six different projection types to choose from

will determine therange of the stackthat will beincluded in the zprojection


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IMAGES


Z stack images

Maximum Intensity projection createsan output image whose pixelscorrespond to the maximum value ofeach pixel position (in xy) across all thestack images (z).

Sum Slices projection creates an imagethat is the sum of the selected slices inthe stack.

Proceed for projection as for one plane imagesSERVICIO DE MICROSCOPÍA ÓPTICA O CONFOCAL (S

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NEVER SAVE YOUR IMAGES AS JPG

TIFF JPG


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ACKNOWLEDGEMENTS

Javier Díez-Guerra

Ángeles Muñoz

Teresa Villalba

Carmen Sánchez

Alejandro Molina

Laboratorio 310: 91 196 4643E-mail: [email protected] DE M


mailto:[email protected]

FLUORESCENCE (SMOC) - UAMbabia.cbm.uam.es/~smoc/Software_Manuales_SMOC/Fluorescence... ·...

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