bioimaging &optics platformbiop.epfl.ch/pdf/pt-biopii_quantitativeimaging_2011.pdf · dr. arne...

Dr. Arne SeitzPT-BIOP Course, Confocal Microscopy 2011, EPFL

BioImaging &Optics Platform

Quantitative imaging



A typical image



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What is a quantitative imaging• Getting quantitative information about the specimen from the image• It is necessary to avoid subjective bias and to present the overall pattern of the data• Relative quantification – measure relative intensity• Absolute quantification – measure the number of photons• Thick specimens are difficult to quantify due to scattering and absorption• Important for the most of light microscopy methods which are intensity based (e.g. Colocalisation, FRET)



Concentration of fluorophoresAbsorption: Calculation of absolute concentration of moleculesis easily possible

I0 IT

)lg()(0

TdcEIIT T

−=⋅⋅=

=

λε



Concentration of fluorophores

Fluorescence: Calculation of concentration of molecules is difficult

I0(λ1) IF

),,( 10 λIcfIF =

Absolute measurements of fluorophore concentration is almost impossible but relative measurements are possible. Comparable and reproducible imaging conditions are needed.



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Overview

• System calibration •Noise issues• Image processing



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System calibration

• AOTF calibration• Detector linearity• Gain calibration



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AOTF calibration

488 nm, PE RS• AOTF transmission is not linear • In single spot confocal it is normally more linear



Detection DevicesArray detector Point detector

Photonselectronspixel values



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Signal linearity

• Normally no fluorophore saturation for spinning disc• More pronounced in single beam confocal• Depends on fluorophore coefficient of extinction

PE RS, 1 µm beads , 488 nm excitation



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CCD linearity

1 µm beads , 488 nm excitation

• (EM)CCD is very linear



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Analog (amplifier) gain

• Normally linear in whole gain range• Sometimes used in confocals as well

Hamamatsu Orca ERG CDD camera



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Electron multiplier gain

• For PMT and EMCCD gain is exponential• Typical max gain values: EMCCD ~103, PMT ~ 106

Hamamatsu C9100-50 EMCCD camera



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PMT gain

Hamamatsu C9100-50 EMCCD camera600 700 800 900 1000

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Confocal microscopeDwell time: 50 µs Dwell time: 6 µs Dwell time: 1.6 µs

Pixel intensity is not proportional to the number of collected photons !!!



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System calibration: summary

• System calibration is necessary for comparison between different images• Image with the same microscope settings if possible• Direct monitoring of excitation power is necessary for comparison of different systems• Check for absence of fluorophore saturation• Measuring system PSF is a good way to monitor the system performance • Calibration should be repeated regularly to monitor stability of the system



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Noise issues

• Role of noise• Noise and resolution• Main sources of noise•How to control noise



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Role of noise in quantification

• Detectable difference in intensity is approximately two times bigger than noise level • Example: SNR 5 (noise is 20% of signal). Only 40% difference in signal can be reliably detected.

1 µm beads , 488 nm excitation

SNR 10 SNR 5 SNR 3



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Noise decreases resolution

OTF for PlanApo 63x/1.4 objective, 500 nm emission

• SNR 5 results in about 30% decrease of resolution



Image segmentation

Image based analysis:•Quick, easy•Prone to errors

Object based analysis:•Segmentation is needed•Time consuming



PMT Gain reduces SNR

400 500 600 700 800 900

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SNR

gain value400 500 600 700 800 900 1000 1100 1200 1300

-400-200

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Dark image



Noise reduction

1.0 1.5 2.0 2.5 3.0 3.5 4.0

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sqrt(number of averages)0 2 4 6 8 10 12 14 16 18

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number of averages

Mean



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How to control noise

• More optimal filters• Better objectives• Accurate specimen preparation• Better detectors• Brighter fluorophores• Longer exposure/dwell time• Larger pixel size • Higher excitation power

Image quality depends not only on noise, but also on signal and background



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Noise issues: summary

• Noise effects both contrast and resolution• Average over several frames (e.g. time laps) or several pixels (e.g. roi in FRAP)• Get imaging system optimised• For noisy stacks use average instead of maximum intensity projection• Confocal images more noisy than brightfield• Average or accumulate noisy signal. This also helps to avoid saturation



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Image processing

• Background subtraction• Flat field correction• Photobleaching correction• Correct sampling• Filtering /deconvolution



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Background subtraction

Leica AF600063 x/1.3 Imm objective

original background background corrected

• Useful especially in transmission mode• Removes of dust particles, spots from the image



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Flat field correction

• Useful especially in fluorescence mode• Flat field can be measured with calibration slide• Subtract dark image before flat field correction

PerkinElmer ERS100 x/1.3 Oil objective



Flat field confocal microscope

Zoom: 0.6 Zoom: 1.0

Zoom: 2.0 Zoom: 4.00 300 600 900

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Bit depth

Bit Depth GrayscaleLevels

Dynamic Range(Decibels)

1 2 6 dB2 4 12 dB3 8 18 dB4 16 24 dB5 32 30 dB6 64 36 dB7 128 42 dB8 256 48 dB9 512 54 dB

10 1,024 60 dB11 2,048 66 dB12 4,096 72 dB13 8,192 78 dB14 16,384 84 dB16 65,536 96 dB18 262,144 108 dB

20 1,048,576 120 dB



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Photobleaching correction

• Max bleaching: fixed sample - 50%, live sample - 20%• For strong bleaching SNR is different at the beginning and at the end of the series• Fluctuations of laser or lamp power are corrected similarly



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Image processing: summary

• Flatfield correction and background subtraction are already necessary for analysis within single image• For quantitation use images with at least 12 bit grey values• Use full dynamic range of detector with offset and gain settings preventing clipping or saturation• Use correct image sampling with pixel size 2 to 3 times smaller than resolution• To remove noise use Gaussian or mean filter with kernel size close to the resolution of the system



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Quantitative imaging: summary

• Quantitative imaging is a powerful tool to get unbiased/reproducable results•Quantitative imaging requires proper calibration of

•PMT•AOTF

• Image processing can be used to correct for:•Uneven illumination (flatfield correction)•Photobleaching•Background artefacts

bioimaging &optics platformbiop.epfl.ch/pdf/pt-biopii_quantitativeimaging_2011.pdf · dr. arne...

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