microscope limits and 3d cell...

MICROSCOPE LIMITS

and

3D CELL CULTURES

21st May 2015

Eng. Filippo Piccinini, PhD

University of Bologna, Italy

Outline

Brief bibliography

Exceeding some microscope limits

Three-dimensional cell cultures

Literature and contacts

First Name, Surname Filippo Piccinini

Place of birth Forlimpopoli, FC, Italy

Date of birth April 20, 1985

Master degree Biomedical Engineer

PhD degree European Doctorate in Information Technology

Email [email protected]

Mobile +39 3495000398

Website www.filippopiccinini.it

Current position

Post Doc Research Fellow, Advanced Research Centre on Electronic Systems (ARCES), Computer Vision Group (CVG), University of Bologna

Supervisor Prof. Alessandro Bevilacqua

Associations Founder member of the “Mesenchymal Stem Cells Italian Group (GISM)” (www.gismonline.it)

Research keywords Quantitative microscopy, 3D cell cultures, Mesenchymal stem cell applications

Where people typically think Italian researchers work

Where my mother thinks I typically work

University of Bologna - Where I work

BOLOGNA

MELDOLA

FAENZA

Connections with Prof. P. Horvath

CIMST 2010 Interdisciplinary Summer School on

Bio-medical Imaging

ETH Zurich, Switzerland, September 6-17, 2010

3-month stay, Light Microscopy and Screening Center

ETH Zurich, Switzerland, May 9, 2011 – August 26, 2011

2010

2011

3-month stay, Light Microscopy and Screening Center

ETH Zurich, Switzerland, May 7, 2011 – August 8, 2012

2012

University seminar and PhD defence

University of Bologna, Italy, April 17, 2013 – April 22, 2013

2013

2-month stay, BIOMAG Peter Horvath Laboratory

Szeged, Hungary, May 17, 2015 – July 24, 2015

2015

Outline

Brief bibliography




Which microscope limits?

Limited x-y field of view

Multi-focus planes in objects with large depth

Inhomogeneous illumination and intensity





Dimension of a single image





MOSAICING





MOSAICING

VIGNETTING CORRECTION





MOSAICING


DEPTH-OF-FOCUS EXTENSION

Mosaicing

MOSAICING



Mosaic

A large image obtained stitching together two or more images without losing resolution

Mosaicing – OnLine method

WARPING

MODEL

ESTIMATION

IMAGE WARPING

AND STITCHING

FEATURES

DETECTION AND

MATCHING


WARPING

MODEL

ESTIMATION

IMAGE WARPING

AND STITCHING

FEATURES

DETECTION AND

MATCHING

Shi-Tomasi corners, Lucas-Kanade tracker


WARPING

MODEL

ESTIMATION

IMAGE WARPING

AND STITCHING

FEATURES

DETECTION AND

MATCHING


WARPING

MODEL

ESTIMATION

IMAGE WARPING

AND STITCHING

FEATURES

DETECTION AND

MATCHING

GEOMETRIC

REGISTRATION


WARPING

MODEL

ESTIMATION

IMAGE WARPING

AND STITCHING

FEATURES

DETECTION AND

MATCHING

TONAL

REGISTRATION

GEOMETRIC

REGISTRATION


WARPING

MODEL

ESTIMATION

IMAGE WARPING

AND STITCHING

FEATURES

DETECTION AND

MATCHING

VIGNETTING

CORRECTION

TONAL

REGISTRATION

GEOMETRIC

REGISTRATION


WARPING

MODEL

ESTIMATION

IMAGE WARPING

AND STITCHING

FEATURES

DETECTION AND

MATCHING

VIGNETTING

CORRECTION

30 Mesenchymal Stem Cells images

Mosaicing – Different approaches

No tonal registration

TONAL REGISTRATION

Vignetting correction

Translative

WARPING MODEL

Affine Projective

F2F

GEOMETRIC REGISTRATION

F2M

Mosaicing

CONCLUSIONS

The tonal registration always provides improvements

Generally, in widefield microscopy the best mosaicing warping model is the

projective one, although the translative model is often used

We developed a user-friendly tool to obtain on-line mosaics of images acquired

using a widefield microscope, also testing different registration configurations

We developed a user-friendly tool to obtain on-line mosaics of images acquired

using a widefield microscope, also testing different registration configurations

The tonal registration always provides improvements

Generally, in widefield microscopy the best mosaicing warping model is the

projective one, although the translative model is often used

F. Piccinini, A. Bevilacqua, E. Lucarelli, “Automated image mosaics by non-automated light

microscopes: the MicroMos software tool”, Journal of Microscopy, 252(3):226-250, 2013.

Vignetting

MOSAICING



Vignetting

Fall-off of brightness intensity from the principal point towards the

image borders due to an uneven illumination field, lens, etc

EARLY PROBLEM THAT AFFECTS DIGITAL IMAGING

IF NO CORRECTION IS ACCOMPLISHED,

THE IMAGES COULD BE NOT COMPARABLE

Simplified flat-field correction

IFFC = IORI

V

=

Simplified flat-field correction

HOMOGENEOUS REFERENCE FREE OF OBJECTS

Vignetting function estimated using

SEVERAL REASONS COULD MAKE THIS

APPROACH DIFFICULT TO BE APPLIED

Empty field (in light microscopy)

Fluorescence calibration slide (in fluorescence microscopy)

The nice story of the happy end of a master thesis

This is a nice story regarding the topic “Illumination Correction in Microscopy”, started precisely in 2007 with my master thesis:

“F. Piccinini, A. Bevilacqua, M. Ursino, E. Lucarelli, Algorithm for the building mosaics of images regarding

adherent live stem cells, 14th October 2009, Biomedical Engineering, University of Bologna”,

That after two years brought to a first contribution in a conference:

“A. Bevilacqua, F. Piccinini, A. Gherardi, Vignetting correction by exploiting an optical microscopy image

sequence. In Proc. of the 33rd Annual International Conference of the IEEE Engineering in Medicine and Biology

Society (EMBS), Boston, USA, August 30 – September 3, 2011”,

Then an article in Journal of Microscopy:

“F. Piccinini, E. Lucarelli, A. Gherardi and A. Bevilacqua, Multi-image based method to correct vignetting effect in

light microscopy images. Journal of Microscopy, October 2012.”,

In 2013 a second conference contribution:

“F. Piccinini, A. Bevilacqua, K. Smith, P. Horvath, Vignetting and photo-bleaching correction in automated

fluorescence microscopy from an array of overlapping images. In Proc. of the 10th IEEE International Symposium

on Biomedical Imaging (ISBI), San Francisco, CA, USA, April 7-11, 2013”

And, at the end:

“K. Smith, Y. Li, F. Piccinini, G. Csucs, Cs. Balazs, A. Bevilacqua, P. Horvath, CIDRE: An illumination correction

method for optical microscopy. Nature Methods, May 2015”!

CIDRE general vignetting correction method

“K. Smith, Y. Li, F. Piccinini, G. Csucs, Cs. Balazs, A. Bevilacqua, P. Horvath, CIDRE: An illumination correction

method for optical microscopy. Nature Methods, May 2015.”

CIDRE is available as

MATLAB and IMAGEJ

open source software

Vignetting: cell in a stitching region of a mosaic

Depth of focus

MOSAICING



Depth of focus

FOCUS PLANE 1:

BORDER IN-FOCUS

FOCUS PLANE 2:

DEBRIS IN-FOCUS

2D RECONSTRUCTION

COMPLETELY IN-FOCUS

Depth of focus

Starting from a set of images acquired at different depths (z-plane), if the z distance between

two subsequent images is less than DF, it is possible to reconstruct a single 2D image

completely sharp where each part of the objects is in-focus

OBJECT RECONSTRUCTION

2D RECONSTRUCTED IMAGE

Depth of focus

INPUT

IMAGES STACK

OUTPUT

DOFE

Outline

Brief bibliography




SPHEROID

“3D multicellular aggregate built in

vitro used as a model for testing

drugs and cancer treatments”

Courtesy by 3DBiomatrix Courtesy by Synthecon Inc.

Common systems to build spheroids

Antigravity bioreactor Hanging drop plates

Spheroid placed in low-attachment multi-well plates

to test drug dosages or radiotherapy treatments

Plate preparation

Phantoms for linear accelerator

Round-bottomed

96-well low attachment

3D cell culture Lab equipment (p. 1/6)

LIGHTSHEET MICROSCOPE

Zeiss LightSheet 2.1 Analysis of 3D permeability of drugs


CONFOCAL MICROSCOPE

Nikon Eclipse Ti microscope equipped with an A1R confocal laser scanner

Time-lapse analysis of surface reorganization of microtissues


UPRIGHT AND INVERTED WIDEFIELD MICROSCOPES

Nikon Eclipse Te 2000-U Zeiss AxioScope for simultaneous observation of 3 operators

Morphology features evolution


HISTOLOGY ANALYSIS STATION

Cryostat Leica CM 1900 Internal tissue diversification and cell organization


MULTI-WELL PLATE READERS (FLOW CYTOMETER, LUMINOMETER)

Flow cytometer Becton Dickinson (BD) FACSCanto

Luminometer Promega Glomax

Cell viability analysis


LINEAR ACCELERATOR and PLATE HOLDER for RADIOBIOLOGY EXPERIMENTS

Elekta Synergy LINAC

PMMA plate holder

Microtissue structural damages

caused by radiations

Bone substitutes covered by mesenchymal stromal cells

AUTOMATIC CELL SEGMENTATION ANALYSIS OF REJECTION

F. Piccinini, M. Pierini, E. Lucarelli, A. Bevilacqua, Semi-quantitative monitoring of confluence of adherent mesenchymal stromal cells on calcium-

phosphate granules by using widefield microscopy images. Journal of Materials Science: Materials in Medicine, 25(10):2395-2410, 2014

“Tumour shape and volume are

among the most relevant features

for care treatment evaluation”

State-of-the-art approaches for spheroids

MODEL ASSUMPTIONS

Local spherical symmetry

Force of gravity

FORMULAS TYPICALLY USED

State-of-the-art approaches for spheroids

MODEL ASSUMPTIONS

Local spherical symmetry

Force of gravity

FORMULAS TYPICALLY USED

SPHERE method

ELLIPSOID method

Our approach

3D RECONSTRUCTION METHOD

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Our approach


DEPTH-OF-FOCUS RECONSTRUCTION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Input Output Input

Output


Our approach


SPHEROID SEGMENTATION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Our approach


PROTUBERANCE SEGMENTATION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Our approach


3D MAP CREATION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Our approach


INTERCONNECTING THE 3D PARTS

Single connection Parts interconnected with cylinders

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

Our approach


SURFACE QUANTIZATION

Spheroid

segmentation

3D map

creation

Protuberance

segmentation

Surface

visualization

Surface

quantization

Image

preprocessing

ReViSP software tool

Reconstruction and Visualization

from a Single Projection (ReViSP)

Microscope image GUI of ReViSP 3D mesh of the spheroid

ReViSP software tool

Reconstruction and Visualization

from a Single Projection (ReViSP)

Microscope image GUI of ReViSP 3D mesh of the spheroid

ReViSP does not require any prior information except the

binary mask of the spheroid to be reconstructed

ReViSP live demo

Software freely available at:

http://sourceforge.net/p/revisp

AnaSP: software suite to analyse several features

Rembrandt, 1629 Rembrandt, 1634 Rembrandt, 1640 Rembrandt, 1660

DAY08 DAY15 DAY22 DAY35 DAY01

DAY01

DAY08

AnaSP: software suite to analyse several features

AnaSP GUI

SPHEROID SEGMENTATION

MORPHOLOGICAL FEATURES COMPUTATION

Software freely available at:

http://sourceforge.net/p/anasp

Outline

Brief bibliography




Journal articles

F. Piccinini, E. Lucarelli, A. Gherardi, A. Bevilacqua, Multi-image based method to correct vignetting effect in light microscopy images. Journal of

Microscopy, 248(1):6-22, 2012.

K. Smith, Y. Li, F. Piccinini, G. Csucs, C. Balazs, A. Bevilacqua, P. Horvath, CIDRE: an illumination-correction method for optical microscopy. Nature

Methods, 12(5): 404–406, 2015.

EXTENSION OF THE MICROSCOPE LIMITS

F. Piccinini, A. Bevilacqua, E. Lucarelli, Automated image mosaics by non-automated light microscopes: the MicroMos software tool. Journal of

Microscopy, 252(3):226-250, 2013.

F. Piccinini, A. Tesei, W. Zoli, A. Bevilacqua, Extending the Universal Quality Index to assess N-image fusion in light microscopy. International

Journal of Bioelectromagnetism, 14(4):217-222, 2012.

F. Piccinini, A. Tesei, W. Zoli, A. Bevilacqua, Extended depth of focus in optical microscopy: assessment of existing methods and a new proposal.

Microscopy Research and Technique, 15(11):1582-1592, 2012.

F. Piccinini, AnaSP: a software suite for automatic image analysis of multicellular spheroids. Computer Methods and Programs in Biomedicine,

119(1): 43–52, 2015.

THREE-DIMENSIONAL CELL CULTURES

F. Piccinini, A. Tesei, C. Arienti, A. Bevilacqua, Cancer multicellular spheroids: Volume assessment from a single 2D projection. Computer Methods

and Programs in Biomedicine, 118(2):95–106, 2015.

F. Piccinini, A. Tesei, G. Paganelli, W. Zoli, A. Bevilacqua, Improving reliability of live/dead cell counting through automated image mosaicing.

Computer Methods and Programs in Biomedicine, 117(3):448-463, 2014



Contacts

Post-doc positions available for computer scientists

Positions available for students interested in Theses

Collaborators? Always welcome!!!

Prof. Alessandro Bevilacqua,

[email protected]

Eng. Filippo Piccinini,

[email protected]

mailto:[email protected]

mailto:[email protected]

THANK YOU

Eng. Filippo Piccinini PhD,

www.filippopiccinini.it

Email: [email protected]

Mobile: +39 3495000398

Skype: filippo.piccinini85

Cell culture: 3D VS 2D

3D CELL CULTURE 2D CELL CULTURE

Physiologic cell-cell contact Cell-cell contact only on the cell edges,

and cells mostly in contact with plastic

Cells interact with extracellular matrix Cells contact extracellular matrix mostly

on one surface

Diffusion gradient of drugs, gases,

nutrients, and waste No gradients present

Co-culture of multiple cells mimics

microenvironment

Co-cultures unable to establish a

microenvironment

MORE SIMILAR TO

IN VIVO TUMOURS



Zeiss LightSheet 2.1 Analysis of 3D permeability of drugs


CONFOCAL MICROSCOPE

Nikon Eclipse Ti microscope equipped with an A1R confocal laser scanner

Time-lapse analysis of surface reorganization of microtissues


UPRIGHT AND INVERTED WIDEFIELD MICROSCOPES

Nikon Eclipse Te 2000-U Zeiss AxioScope for simultaneous observation of 3 operators

Morphology features evolution


HISTOLOGY ANALYSIS STATION

Cryostat Leica CM 1900 Internal tissue diversification and cell organization


MULTI-WELL PLATE READERS (FLOW CYTOMETER, LUMINOMETER)

Flow cytometer Becton Dickinson (BD) FACSCanto

Luminometer Promega Glomax

Cell viability analysis


LINEAR ACCELERATOR and PLATE HOLDER for RADIOBIOLOGY EXPERIMENTS

Elekta Synergy LINAC

PMMA plate holder

Microtissue structural damages

caused by radiations

Current topics


Permeability of cell viability kits designed for 3D multicellular aggregates

Structural composition of “protuberances” formed over time by the cancer spheroids

CONFOCAL MICROSCOPE

Membrane internalization and over time co-localization of drugs

Mesenchymal stromal cells migration in co-culture 3D microtissues

WIDEFIELD MICROSCOPE

Confluence and proliferation of mesenchymal stromal cells covering bone substitute granules

Spheroid morphology evolution after radiation exposition

LUMINOMETER AND FLOW CYTOMETER

Differentiation and metabolism activity of cells growing in 3D

Viability analysis of substrates of cells at different depth positions from the spheroid’s border

LINEAR ACCELERATOR

Analysis of volume, sphericity, and necrosis after radiation exposition

Effect of radiation in cultures of cells growing in hypoxia and hypoxemia conditions

Spheroid reduction after exposure to radiation

EXPERIMENT DURATION 22 days – 4 weeks

PROTOCOL Radiological treatments in the first week

Images acquired at least 3 times a week

Vitality test performed once a week

A000 Control plate: no treated

B205 2.0 Gray for 5 treatments. Total Gray: 10.0

C755 7.5 Gray for 5 treatments. Total Gray: 37.5

D655 6.5 Gray for 5 treatments. Total Gray: 32.5





PLATES CHARACTERISTIC

SURVIVED SPHEROIDS

DAY

0 1 2 3 4 7 8 11 14 16 18 21

PLA

TE

A000 16 16 14 14 14 14 14 14 14 12 11 9

B205 16 16 9 9 9 9 6 6 6 4 4 3

C705 15 15 12 12 12 12 12 12 11 7 5 5

D655 16 16 14 14 14 14 11 10 9 5 4 4

D654 16 16 14 14 14 14 14 14 13 10 9 6

D653 16 16 14 14 14 14 13 13 12 8 8 8

D652 16 16 14 14 14 14 12 12 12 10 8 8

D651 16 16 14 14 14 14 13 12 11 8 8 7

Spheroid reduction after exposure to radiation

Growth-curves of plates treated always at

6.5 Gray for different number of treatments

Growth-curves of plates treated 5 times at

different Gray levels

20

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aliz

ed m

edia

n v

olu

me

Norm

aliz

ed m

edia

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Bone substitutes covered by mesenchymal stromal cells

AUTOMATIC CELL SEGMENTATION ANALYSIS OF REJECTION



Mesenchymal stromal cells as light-activated local killer

Cancer cell

Light-activated MSC

carrying a cytotoxic drug

Spheroid co-culture

cancer cells – engineered MSC

Light activation of MSC and

release of cytotoxic drug Cancer cells

destruction

SP

HE

RO

ID C

O-

CU

LT

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UIL

T B

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US

ING

TH

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ELLE

T

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LT

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Courtesy by Dr. Serena Duchi, Rizzoli, Bologna

Metastasis - Tissue contamination analysis

Courtesy by www.ivtech.it

IVTech Perfusion system for spheroids

Fresh culture medium

Peristaltic pump

Cancer lung spheroid Spheroid of healthy hepatic cells

Reservoir

microscope limits and 3d cell...

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