2011 FP Microscopes -...

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UNIVERSITY OF BOLOGNA – ARCES

FILIPPO PICCININI

WHAT KIND OF MICROSCOPE?

March 25, 2011

Filippo Piccinini

[email protected]

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 [email protected]

Web site www.filippopiccinini.altervista.org

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Filippo Piccinini

[email protected]

Electron microscopy

Imaging in life sciences

Light microscopy

Thesis & Stage

Filippo Piccinini

[email protected]

Electron microscopy


Light microscopy

Thesis & Stage

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Filippo Piccinini

[email protected] in life sciences

Courtesy of Prof. Ruth Kroschewski

Filippo Piccinini



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Filippo Piccinini


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



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Filippo Piccinini



Filippo Piccinini

[email protected]

Electron microscopy


Light microscopy

Thesis & Stage

Bright field, Phase contrast, Fluorescence, Confocal

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Filippo Piccinini

[email protected] 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


From Optika B-353 Pli User’s Guide

UPRIGHT MICROSCOPE

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Filippo Piccinini


From Optika B-353 Pli User’s Guide

Filippo Piccinini


From Olympus IX71 User’s Guide

INVERTED

MICROSCOPE

OLYMPUS IX71Lab. Cell Culture, IRST (FC)

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Filippo Piccinini


VEHO 400x

MICROSCOPE USB

From www.veho-uk.com

Filippo Piccinini


Data 08/02/2011

Patient ************

Microscope Zeiss Axioscope

Acquisition Mode Bright Field

Objective 10x

Description Lung tumor tissue

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Filippo Piccinini

[email protected] 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



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


Data 18/08/2009

Patient ************

Microscope Nikon Eclipse TE2000-U

Acquisition Mode Phase Contrast

Objective 10x

Description Mesenchymal Stem Cells

Filippo Piccinini

[email protected]

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

[email protected]


Filippo Piccinini

[email protected]

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Filippo Piccinini

[email protected]

Filippo Piccinini

[email protected]

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Filippo Piccinini

[email protected]

Filippo Piccinini

[email protected]

• 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


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

[email protected]


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

[email protected]


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

[email protected]

Electron microscopy


Light microscopy

Thesis & Stage

SEM-SE, SEM-BSE, TEM

Filippo Piccinini


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.


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Filippo Piccinini


SEM – Scanning Electron Microscopy


Filippo Piccinini


SEM – Scanning Electron Microscopy

Back Scattered Electron Secondary Electron


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Filippo Piccinini


... 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


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.


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Filippo Piccinini


TEM – Transmission Electron Microscopy


A TEM image of the polio virus.

The polio virus is 30 nm in size

Filippo Piccinini

[email protected]

Electron microscopy


Light microscopy

Thesis & Stage

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Filippo Piccinini

[email protected] 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

[email protected]

[email protected]

[email protected]

PER INFORMAZIONI:

Filippo Piccinini

[email protected] & 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


TITLES

- Development of a mosaicing algorithm to increase the field of view of a microscope

- Multichannel image mosaicing for high content analysis

Filippo Piccinini


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


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?

[email protected]

[email protected]

THANK YOU!

2011 FP Microscopes -...

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