Quality Image Criteria in Photonic Microscopy

DDaQuaTa International Workshopthomas.guilbert@inserm.fr

Lyon, December 12 2016

IMAG’IC facility, photonic imaging :• From cell to entire organs – intra vital microscopy• 13 photonic microscopes: macroscope – widefields – confocals – multiphotonic –

SR – flow imaging generate more than 5Tb/year• 5 image processing computers• 1 database server: images and research data storage

Combine image informations from

database with quantitative parameters to defineData Quality Criteria.

Institut Cochin : • Biomedical research center based in Paris (14ème)• 3 departments, 38 teams, 10 technologic facilities, 600 researchers

What is a simple microscope? From light source to final image

camera

What is a microscope? The device quality

• wavelength – source / associated filters

• magnification / numerical aperture – objective

• staining / fluorophore used – sample preparation

• pixel size / sensitivity – detector

• global system resolution – wavelength / numerical aperture

All microscope devices have an intrinsic quality criterion. All of them lead to metrology criteria which have to be taken into account.

More subjective criteria: • User experience on each given microscope• Sample quality• Staining quality• Sample preparation quality

One purpose: good images.

What is an image and how to define a good quality image?

4096

Human eye 20-50 levels

PMTs

65536

1 1

• Spatial resolution• Dynamic• SNR

cameras

Number of intensity levels that the sensor can read

Depends mainly on exposure time and excitation intensity

under

over

OK

Depends on camera pixel size (6,45 to 30μm) and the imaging system resolution (numericalaperture and wavelength).

What is an image and how to define good quality image?

SNR =I.Q E.t

I.Q E.t + Nd.t + N r ²

I : photon flow N d : dark noiseQ E : quantum efficiency N r : read-out noiset : integration time

SNR : Signal to Noise Ratio

SNR is an important parameter at low signal intensity and low acquisition time- Increasing SNR leads to increase the exposure time or the excitation intensity

How to link microscope devices and image quality?

What is the image of a point through a conventional microscope?

The Point Spread Function is the response of an imaging system to a point source.

bead (point source) PSF bead’s image (pure PSF)

Knowing the theorical PSF of the optical system, there are mathematic iterationalgorithms enabling to deconvoluate images with a quality criterion based on SNRimprovement (Softwares: Huygens, ImageJ, MetaMorph…)

Blind deconvolution: deconvolution without explicit knowledge of the theorical PSFfind the experimental PSF

What is an image of an objet compared to the real object?

DECONVOLUTION

How to define quality image criteria in photonic microscopy?

• Starting from the qualitative information of an imaging system...

Metadata: • wavelength – source / associated filters = time of use• magnification / numerical aperture – objective = PSF• staining / fluorophore used – sample preparation = control sample• pixel size – detector = sensitivity (QE) and intrinsic noise• global system resolution – wavelength / numerical aperture

METROLOGY CRITERIA

(ISO 9001)

• …to the quantitative information of actual images.

Numerical data: • spatial resolution• dynamic = PixMax - PixMin• native SNR• improved SNR from deconvolution algorithm

PROCESSED CRITERIA

How to go further using a database? (Sis’n Com company)

How to go further using a database? (Sis’n Com company)

Cochin Image Database (Sis’n Com): Online data server (photon microscopy images, image processing data…) Provides smart storage with image labelling – tags, online image processing (deconvolution, segmentation, tracking...), [implement a system to automatically find quality criteria of stored data]

How to go further using a database? (Sis’n Com company)

Using systematic blind deconvolution, it seems possible to get back to the experimental PSF.We could then imagine using our server to follow the evolution of the PSF of one given systemfor one given objective…

Cochin Image Database (Sis’n Com): Online data server (photon microscopy images, image processing data…) Provides smart storage with: image labelling – tags (IDV consortium project), online image processing (deconvolution, segmentation, tracking...), [implement a system to automatically find quality criteria of stored data]

How to go further using a database? (Sis’n Com company)

CCochhiin IImage DDatabbase ((SSiis’’n CCom)):

Global Data Quality Criteria remain to be defined

Multiphoton microscopy of lung slices(endogeneous signals)

A reference image is needed to distinguish healthy ECM from fibrotic tissue: how to define this reference in

term of quality?

Stochastic localisation microscopyof microtubules (tubulin)

The temporal dimension is needed to localise single molecules. A final

image is created using barycenters of these molecules: how to estimate the

localisation errors and the false positives?