2011 fp microscopes -...
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UNIVERSITY OF BOLOGNA – ARCES
FILIPPO PICCININI
WHAT KIND OF MICROSCOPE?
March 25, 2011
Filippo Piccinini
f.piccinini@unibo.it
PH.D STUDENTEuropean Doctorate in Information Technology by
ARCESAdvanced Research Centre on Electronic Systems,
University of Bologna
SUPERVISORProf. Alessandro Bevilacqua
Prof. Mauro UrsinoCO-TUTOR
Who am I?
MAIN RESEARCH TOPIC
Biomedical Image Elaboration
First Name, Surname Filippo Piccinini
Place of birth Forlimpopoli, FC, Italy
Date of birth April 20, 1985
Title Biomedical Engineer
Email f.piccinini@unibo.it
Web site www.filippopiccinini.altervista.org
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Filippo Piccinini
f.piccinini@unibo.itOutline
Electron microscopy
Imaging in life sciences
Light microscopy
Thesis & Stage
Filippo Piccinini
f.piccinini@unibo.itOutline
Electron microscopy
Imaging in life sciences
Light microscopy
Thesis & Stage
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Filippo Piccinini
f.piccinini@unibo.itImaging in life sciences
Courtesy of Prof. Ruth Kroschewski
Filippo Piccinini
f.piccinini@unibo.itImaging in life sciences
Courtesy of Prof. Ruth Kroschewski
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Filippo Piccinini
f.piccinini@unibo.itImaging in life sciences
f
v=λ
fhE *=
v = speed of light, about 3*10^8 m/s; for sound waves in air speed of sound is 343 m/s
h = Planck constant
Courtesy of Prof. Roger Wepf
Filippo Piccinini
f.piccinini@unibo.itImaging in life sciences
Courtesy of Prof. Roger Wepf
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Filippo Piccinini
f.piccinini@unibo.itImaging in life sciences
Courtesy of Prof. Ruth Kroschewski
Filippo Piccinini
f.piccinini@unibo.itOutline
Electron microscopy
Imaging in life sciences
Light microscopy
Thesis & Stage
Bright field, Phase contrast, Fluorescence, Confocal
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Filippo Piccinini
f.piccinini@unibo.itLight microscopy
ZEISS AXIOVERT 200Lab. Cell Culture, IRST Meldola (FC)
NICON ECLIPSE TE 2000-ULab. Bone Regeneration, IOR Bologna
HISTORY: The first microscope to be developed was the optical (or light) microscope, althoughthe original inventor is not easy to identify. An early microscope was made in 1590 inNetherlands. Giovanni Faber coined the name microscope for Galileo Galilei's compoundmicroscope in 1625.
Filippo Piccinini
f.piccinini@unibo.itLight microscopy
From Optika B-353 Pli User’s Guide
UPRIGHT MICROSCOPE
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Filippo Piccinini
f.piccinini@unibo.itLight microscopy
From Optika B-353 Pli User’s Guide
Filippo Piccinini
f.piccinini@unibo.itLight microscopy
From Olympus IX71 User’s Guide
INVERTED
MICROSCOPE
OLYMPUS IX71Lab. Cell Culture, IRST (FC)
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Filippo Piccinini
f.piccinini@unibo.itLight microscopy
VEHO 400x
MICROSCOPE USB
From www.veho-uk.com
Filippo Piccinini
f.piccinini@unibo.itLight microscopy
Data 08/02/2011
Patient ************
Microscope Zeiss Axioscope
Acquisition Mode Bright Field
Objective 10x
Description Lung tumor tissue
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Filippo Piccinini
f.piccinini@unibo.itPhase contrast
From www.microscopyu.com
Phase contrast microscopy, first described in 1934 by Dutch physicist Frits Zernike, is a contrast-
enhancing optical technique that can be utilized to produce high-contrast images of transparent
specimens, such as living cells and thin tissue slices. The phase contrast technique employs an optical
mechanism to translate minute variations in phase into corresponding changes in amplitude, which can
be visualized as differences in image contrast.
http://www.microscopyu.com/tutorials/java/kohler/index.html
Filippo Piccinini
f.piccinini@unibo.itPhase contrast
From www.microscopyu.com
A phase plate is mounted in or near the objective rear focal plane in order to selectively alter the phase and amplitude of
the surround (or undeviated) light passing through the specimen. Often, the ring is also coated with a partially absorbing
metallic film to reduce the surround light amplitude by 60-90 percent.
The condenser annulus is typically constructed as an opaque flat-black (light absorbing) plate with a transparent annular
ring, which is positioned in the front focal plane (aperture) of the condenser so the specimen can be illuminated by
defocused, parallel light wavefronts emanating from the ring.
P = S + DP = resultant particle wave
S = surround or undeviated wave
D = diffracted wave
http://www.microscopyu.com/tutorials/java/phasecontrast/positivenegative/index.htmlhttp://www.microscopyu.com/tutorials/java/phasecontrast/microscopealignment/index.html
http://www.microscopyu.com/galleries/phasecontrast/index.html
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Filippo Piccinini
f.piccinini@unibo.itPhase contrast
Data 18/08/2009
Patient ************
Microscope Nikon Eclipse TE2000-U
Acquisition Mode Phase Contrast
Objective 10x
Description Mesenchymal Stem Cells
Filippo Piccinini
f.piccinini@unibo.itFluorescence
From http://en.wikipedia.org
Fluorescence is the emission of light by asubstance that has absorbed light or otherelectromagnetic radiation of a differentwavelength. In most cases, emitted light has alonger wavelength, and therefore lower energy,than the absorbed radiation.
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Filippo Piccinini
f.piccinini@unibo.itFluorescence
From http://en.wikipedia.org
Filippo Piccinini
f.piccinini@unibo.itFluorescence
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Filippo Piccinini
f.piccinini@unibo.itFluorescence
Filippo Piccinini
f.piccinini@unibo.itFluorescence
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Filippo Piccinini
f.piccinini@unibo.itFluorescence
Filippo Piccinini
f.piccinini@unibo.itConfocal
• Advanced fluorescence microscopy
• Focused light spot is scanned through the specimen
• Scanning in x, y and z possible
• Thickness not so critical
• Slow for large image area
• Pixel by pixel images
Characteristics
From www.microscopyu.com
Confocal microscopy is an optical imaging technique used
to increase optical resolution and contrast of a micrograph
by using point illumination and a spatial pinhole to eliminate
out-of-focus light in specimens that are thicker than the
focal plane.
The principle of confocal imaging was patented in 1957 by
Marvin Minsky
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Filippo Piccinini
f.piccinini@unibo.itConfocal
From www.microscopyu.com
www.microscopyu.com/tutorials/java/virtual/confocal/index.html
www.microscopyu.com/moviegallery/sweptfield/folu-egfp-eb3-sfc
WHAT CAN WE DO?
� High resolution images
� Time lapse experiment
� Z analysis
Filippo Piccinini
f.piccinini@unibo.itConfocal
From http://en.wikipedia.org
Bovine pulmonary arthery
endothelial cells.
Nuclei are stained blue with DAPI,
microtubles are marked green by
an antibody bound to FITC,
actin filaments are labelled red with
phalloidin bound to TRITC.
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Filippo Piccinini
f.piccinini@unibo.itOutline
Electron microscopy
Imaging in life sciences
Light microscopy
Thesis & Stage
SEM-SE, SEM-BSE, TEM
Filippo Piccinini
f.piccinini@unibo.itElectron microscopy
Electron Gun (Cathode Ray)
SEM (SE or BSE) – Scanning Electron Microscopy
TEM – Transmission Electron Microscopy
STEM – Scanning Transmission Electron Microscopy
It was invented by the German Ernst Ruska and Max
Knoll in 1931. In electron microscope is not use uses
photons as source of radiation but a beam of
electrons. The photons that make up a beam of light
have a wavelength much greater than that of
electrons, and the resolving power of a microscope is
inversely proportional to the wavelength of the
radiation used. Using electrons is possible reach a
resolution several orders of magnitude higher.
Courtesy of Prof. Roger Wepf
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Filippo Piccinini
f.piccinini@unibo.itElectron microscopy
SEM – Scanning Electron Microscopy
Courtesy of Prof. Roger Wepf
Filippo Piccinini
f.piccinini@unibo.itElectron microscopy
SEM – Scanning Electron Microscopy
Back Scattered Electron Secondary Electron
Courtesy of Prof. Roger Wepf
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Filippo Piccinini
f.piccinini@unibo.itElectron microscopy
... The difference between Backscattered electrons and secondary is physically the energy they have.
Backscattered electron are in our energy regime we use 1-30kV on an SEM – elastic scattered electron very close to one or
several nucleus from the sample atoms - since it is an elastic scattering event they do not loose energy - so their energy is
between 1-30kV depending which energy you use on an SEM - most of them are close to the primary energy of the beam –
the thicker the sample the more Backscattered electrons have the may have experienced multiple scattering event and
therefore the energy can also be lower than the primary energy....
Whereas the secondary electrons come from a direct interaction of the primary electron in the electron shells of an atom -
and hence the knock-out electron is called a "Secondary Electron - SE"... Since knocking out an electron from the electron
shell need less energy as the primary beam has - (they usually have something between 1-50 eV (Elektronvolt not kV)... The
are very slow - their mean free pathway is only about 5-7nm, and then they lost all energy in the sample... This is the reason
why you can only detect SE-electrons from the upper 5-7nm .... So SE will give you a good surface signal ....in addition since
they are so slow and low energy one can attract them towards an SE-detector even outside the beam path...
Whereas BSE have a higher energy and may fly undisturbed a longer distance in the sample - ad mainly in vacuum in
straight lines.
They are less sensitive to stray fields and attraction fields and will travel only in linear path towards a detector sitting opposite
to their take off angle - so they come from deeper areas of the sample (BSE scattering volume) and are strongly proportional
to the atomic number Z (SE only very weakly depend on the )... And hence one can get a material contrast in addition to the
deeper levels....
SE - surface details topology (not correlated to the material variation in a first estimate)
BSE - deeper layers (depending on the sample...) and directly Z-proportional
Roger Wepf
Filippo Piccinini
f.piccinini@unibo.itElectron microscopy
In SEM images are represented particular of the surface of 3D objects
Pollen from a variety of
common plants: sunflower
The image is magnified some
x500, so the bean shaped
grain in the bottom left corner
is about 50 µm long.
From http://en.wikipedia.org
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Filippo Piccinini
f.piccinini@unibo.itElectron microscopy
TEM – Transmission Electron Microscopy
From http://en.wikipedia.org
A TEM image of the polio virus.
The polio virus is 30 nm in size
Filippo Piccinini
f.piccinini@unibo.itOutline
Electron microscopy
Imaging in life sciences
Light microscopy
Thesis & Stage
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Filippo Piccinini
f.piccinini@unibo.itCVG LAB
Computer Vision LAB
ALMA MATER STUDIORUM
UNIVERSITA’ DI BOLOGNA
Responsabile:
Responsabile tecnico:
Prof. Alessandro Bevilacqua
Dr. Alessandro Gherardi
http://cvg.deis.unibo.it
Referente: Filippo Piccinini
alessandro.bevilacqua@unibo.it
alessandro.gherardi@unibo.it
f.piccinini@unibo.it
PER INFORMAZIONI:
Filippo Piccinini
f.piccinini@unibo.itTHESIS & STAGE
- Real time evaluation of cell culture confluence
- To count live cells from phase contrast images
- Segmentation and characterization of mesenchymal stem cells
TITLES
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Filippo Piccinini
f.piccinini@unibo.itTHESIS & STAGE
TITLES
- Development of a mosaicing algorithm to increase the field of view of a microscope
- Multichannel image mosaicing for high content analysis
Filippo Piccinini
f.piccinini@unibo.itTHESIS & STAGE
TITLES
- Localization and characterization for tumor cell in histological tissues
- Assessment of the level of expression of a fluorescent protein in single cell
Courtesy of Dr. Sara Bravaccini
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Filippo Piccinini
f.piccinini@unibo.itTHESIS & STAGE
THESES
http://cvg.deis.unibo.it/theses_it.html
STAGE TRAINING
CVG Computer Vision LAB – Via Genova 181 - Cesena
Bone Regeneration LAB – IOR - Bologna
Cell Culture LAB – IRST – Meldola (FC)
Microscopy LAB– IRST – Meldola (FC)
Laboratorio Ingegneria Cellulare e Molecolare – Via Venezia 52 – Cesena
Etc, etc, etc …
Etc, etc, etc …
Need information?
alessandro.bevilacqua@unibo.it
f.piccinini@unibo.it
THANK YOU!
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