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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
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
Exceeding some microscope limits
Three-dimensional cell cultures
Literature and contacts
Which microscope limits?
Limited x-y field of view
Multi-focus planes in objects with large depth
Inhomogeneous illumination and intensity
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
Which microscope limits?
Limited x-y field of view
Multi-focus planes in objects with large depth
Inhomogeneous illumination and intensity
MOSAICING
Which microscope limits?
Limited x-y field of view
Multi-focus planes in objects with large depth
Inhomogeneous illumination and intensity
MOSAICING
Which microscope limits?
Limited x-y field of view
Multi-focus planes in objects with large depth
Inhomogeneous illumination and intensity
MOSAICING
VIGNETTING CORRECTION
Which microscope limits?
Limited x-y field of view
Multi-focus planes in objects with large depth
Inhomogeneous illumination and intensity
MOSAICING
VIGNETTING CORRECTION
DEPTH-OF-FOCUS EXTENSION
Mosaicing
MOSAICING
VIGNETTING CORRECTION
DEPTH-OF-FOCUS EXTENSION
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
Mosaicing – OnLine method
WARPING
MODEL
ESTIMATION
IMAGE WARPING
AND STITCHING
FEATURES
DETECTION AND
MATCHING
Shi-Tomasi corners, Lucas-Kanade tracker
Mosaicing – OnLine method
WARPING
MODEL
ESTIMATION
IMAGE WARPING
AND STITCHING
FEATURES
DETECTION AND
MATCHING
Mosaicing – OnLine method
WARPING
MODEL
ESTIMATION
IMAGE WARPING
AND STITCHING
FEATURES
DETECTION AND
MATCHING
GEOMETRIC
REGISTRATION
Mosaicing – OnLine method
WARPING
MODEL
ESTIMATION
IMAGE WARPING
AND STITCHING
FEATURES
DETECTION AND
MATCHING
TONAL
REGISTRATION
GEOMETRIC
REGISTRATION
Mosaicing – OnLine method
WARPING
MODEL
ESTIMATION
IMAGE WARPING
AND STITCHING
FEATURES
DETECTION AND
MATCHING
VIGNETTING
CORRECTION
TONAL
REGISTRATION
GEOMETRIC
REGISTRATION
Mosaicing – OnLine method
WARPING
MODEL
ESTIMATION
IMAGE WARPING
AND STITCHING
FEATURES
DETECTION AND
MATCHING
VIGNETTING
CORRECTION
30 Mesenchymal Stem Cells images
Mosaicing – OnLine method
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 CORRECTION
DEPTH-OF-FOCUS EXTENSION
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
Vignetting: cell in a stitching region of a mosaic
Depth of focus
MOSAICING
VIGNETTING CORRECTION
DEPTH-OF-FOCUS EXTENSION
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
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
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
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
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
Exceeding some microscope limits
Three-dimensional cell cultures
Literature and contacts
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
3D cell culture Lab equipment (p. 2/6)
CONFOCAL MICROSCOPE
Nikon Eclipse Ti microscope equipped with an A1R confocal laser scanner
Time-lapse analysis of surface reorganization of microtissues
3D cell culture Lab equipment (p. 3/6)
UPRIGHT AND INVERTED WIDEFIELD MICROSCOPES
Nikon Eclipse Te 2000-U Zeiss AxioScope for simultaneous observation of 3 operators
Morphology features evolution
3D cell culture Lab equipment (p. 4/6)
HISTOLOGY ANALYSIS STATION
Cryostat Leica CM 1900 Internal tissue diversification and cell organization
3D cell culture Lab equipment (p. 5/6)
MULTI-WELL PLATE READERS (FLOW CYTOMETER, LUMINOMETER)
Flow cytometer Becton Dickinson (BD) FACSCanto
Luminometer Promega Glomax
Cell viability analysis
3D cell culture Lab equipment (p. 6/6)
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
3D RECONSTRUCTION METHOD
DEPTH-OF-FOCUS RECONSTRUCTION
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Input Output Input
Output
VIGNETTING CORRECTION
Our approach
3D RECONSTRUCTION METHOD
SPHEROID SEGMENTATION
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Our approach
3D RECONSTRUCTION METHOD
PROTUBERANCE SEGMENTATION
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Our approach
3D RECONSTRUCTION METHOD
3D MAP CREATION
Spheroid
segmentation
3D map
creation
Protuberance
segmentation
Surface
visualization
Surface
quantization
Image
preprocessing
Our approach
3D RECONSTRUCTION METHOD
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
3D RECONSTRUCTION METHOD
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
Exceeding some microscope limits
Three-dimensional cell cultures
Literature and contacts
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
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
Contacts
Post-doc positions available for computer scientists
Positions available for students interested in Theses
Collaborators? Always welcome!!!
Prof. Alessandro Bevilacqua,
Eng. Filippo Piccinini,
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
3D cell culture Lab equipment (p. 1/6)
LIGHTSHEET MICROSCOPE
Zeiss LightSheet 2.1 Analysis of 3D permeability of drugs
3D cell culture Lab equipment (p. 2/6)
CONFOCAL MICROSCOPE
Nikon Eclipse Ti microscope equipped with an A1R confocal laser scanner
Time-lapse analysis of surface reorganization of microtissues
3D cell culture Lab equipment (p. 3/6)
UPRIGHT AND INVERTED WIDEFIELD MICROSCOPES
Nikon Eclipse Te 2000-U Zeiss AxioScope for simultaneous observation of 3 operators
Morphology features evolution
3D cell culture Lab equipment (p. 4/6)
HISTOLOGY ANALYSIS STATION
Cryostat Leica CM 1900 Internal tissue diversification and cell organization
3D cell culture Lab equipment (p. 5/6)
MULTI-WELL PLATE READERS (FLOW CYTOMETER, LUMINOMETER)
Flow cytometer Becton Dickinson (BD) FACSCanto
Luminometer Promega Glomax
Cell viability analysis
3D cell culture Lab equipment (p. 6/6)
LINEAR ACCELERATOR and PLATE HOLDER for RADIOBIOLOGY EXPERIMENTS
Elekta Synergy LINAC
PMMA plate holder
Microtissue structural damages
caused by radiations
Current topics
LIGHTSHEET MICROSCOPE
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
D654 6.5 Gray for 4 treatments. Total Gray: 26.0
D653 6.5 Gray for 3 treatments. Total Gray: 19.5
D652 6.5 Gray for 2 treatments. Total Gray: 13.0
D651 6.5 Gray for 1 treatments. Total Gray: 6.5
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
Norm
aliz
ed m
edia
n v
olu
me
Norm
aliz
ed m
edia
n v
olu
me
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
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
UR
E B
UIL
T B
Y
US
ING
TH
E P
ELLE
T
CU
LT
UR
E M
ET
HO
D
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