THE RIGHT MICROSCOPE
FOR THE RIGHT SAMPLE
Eng. Filippo Piccinini, PhD
School of Engineering, Cesena, University of Bologna
2nd December 2015
Myself
First Name, Surname
Filippo Piccinini
Place of birth
Forlimpopoli, FC, Italy
Date of birth
April 20, 1985
Title
Biomedical Engineer, PhD
Doctorate
European Doctorate in Information Technology
Affiliation
Advanced Research Centre on Electronic Systems,
University of Bologna
Research group
Computer Vision Group (CVG),
University of Bologna
Supervisor
Prof. Alessandro Bevilacqua
Email
f.piccinini@unibo.it
Skype
filippo.piccinini85
Web site
www.filippopiccinini.it
Slides available at: www.filippopiccinini.it section “Teaching Activities”
1
ARCES
ARCES
2
ARCES
Computer Vision Group
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IMAGE PROCESSING AND ANALYSIS
Outdoor imaging
Aerospace imaging
Biomedical imaging
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5
COORDINATOR:
Dr. Spartaco Santi,
Istituto Ortopedico Rizzoli, Bologna
COORDINATOR:
Dr. Spartaco Santi,
Istituto Ortopedico Rizzoli, Bologna
Take home message
“The right
microscope
for the right
sample”
Courtesy of Prof. Ruth Kroschewski
The right microscope for the right sample
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The right microscope for the right sample
The right microscope for the right sample
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The right microscope for the right sample
The right microscope for the right sample
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The right microscope for the right sample
The right microscope for the right sample
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The right microscope for the right sample
The right microscope for the right sample
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Outline
Microscopy in general
Light microscopes
Fluorescence
Confocal microscopes
Theses and stages
Outline
Microscopy in general
Light microscopes
Fluorescence
Confocal microscopes
Theses and stages
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Microscopy
Courtesy of Prof. Ruth Kroschewski
Microscopy
Courtesy of Prof. Roger Wepf
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Microscopy
Courtesy of Prof. Roger Wepf
Microscopy
FEW MATHEMATICS' FORMULAS
d  0.6098

v
f
E  h* f

Abbe’s law
NA
d
ʎ
NA
v
f
E
h
M
= resolution
= wavelength
= Numerical Aperture
= light’s speed
= frequency
= Energy
= Planck constant
= magnification
Planck’s law
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Microscopy
NUMERICAL APERTURE MICROSCOPYµ TUTORIAL
http://www.microscopyu.com/tutorials/java/objectives/nuaperture/index.html
M  K  NA
Microscopy
THE FUNDAMENTAL COMPONENT
Courtesy of Dr. Cristiano Rumio
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QUESTIONS?
WHAT IS THE WAVELENGTH RANGE
OF THE VISIBLE REGION?
Outline
Microscopy in general
Light microscopes
Fluorescence microscopes
Confocal microscopes
Theses and stages
15
Light microscopes
ITALIANS ALSO IN THE STORY OF MICROSCOPY
The first developed microscope was the light (or optical) microscope. An early light microscope
was made in 1590 in Netherlands, but the original inventor is not easy to be identified. Giovanni
Faber in 1625 coined the name “microscope” for Galileo Galilei's magnification instrumentation.
ZEISS AXIOVERT 200
Lab. Cell Culture, IRST Meldola (FC)
NIKON ECLIPSE TE 2000-U
Cellular and Molecular Eng. Lab., Cesena
NIKON ECLIPSE TE 2000-U
Lab. Bone Regeneration, IOR Bologna
Light microscopes
UPRIGHT MICROSCOPE
From Optika B-353 Pli User’s Guide
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Light microscopes
From Optika B-353 Pli User’s Guide
Light microscopes
INVERTED
MICROSCOPE
OLYMPUS IX71
Lab. Cell Culture, IRST (FC)
From Olympus IX71 User’s Guide
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Light microscopes
MICROSCOPES USING REFLECTING LIGHT
for surface analysis
VEHO 400x
COMPACT DIGITAL MICROSCOPE
From www.veho-uk.com
Take home message
For live cell culture analysis
INVERTED
MICROSCOPE
For thin specimen analysis
UPRIGHT
MICROSCOPE
For thick samples analysis
MICROSCOPE USING
REFLECTING LIGHT
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Light microscopes
ANALYSIS TECHNIQUES:
BRIGHTFIELD
PHASE-CONTRAST
DIFFERENTIAL INTERFERENCE CONTRAST (DIC)
Brightfield
ADVANTAGES
Simplicity of setup with only basic equipment required.
LIMITATIONS
Very low contrast of most biological samples.
The sample often has to be stained before viewing.
Mesenchymal stem cell
Lung tissue
Blood cells
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Phase-contrast
Phase contrast microscopy, first described in 1934 by the Dutch physicist Frits Zernike, is a contrastenhancing 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
From www.microscopyu.com
Differential Interference Contrast (DIC)
DIC microscopy, also known as Nomarski Interference Contrast (NIC), is an optical microscopy illumination
technique used to enhance the contrast in unstained, transparent samples. DIC works on the principle of
interferometry. The images obtained are similar to those obtained by phase contrast microscopy but without the
bright diffraction halo. The DIC images have the appearance of a 3D physical relief corresponding to the
variation of optical density of the sample, emphasizing lines and edges though not providing a topographically
accurate image.
ADVANTAGES
Very high contrast of thin samples also not stained. Suitable for live imaging.
LIMITATIONS
Objectives specific for DIC analysis and not suitable for fluorescence analysis
HeLa cells
From www.microscopyu.com
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QUESTIONS?
WHAT IS THE BEST MICROSCOPE FOR LIVE
CELL CULTURE ANALYSIS?
For live cell culture analysis
INVERTED
MICROSCOPE
Outline
Microscopy in general
Light microscopes
Fluorescence
Confocal microscopes
Theses and stages
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Fluorescence
Fluorescence is the emission of light by a
substance that has absorbed light or other
electromagnetic radiation of a different
wavelength. In most cases, emitted light has a
longer wavelength, and therefore lower energy,
than the absorbed radiation.
From http://en.wikipedia.org
Fluorophore
A fluorophore (or fluorochrome) is a
fluorescent chemical compound that
can re-emit light upon light excitation.
Courtesy of Dr. Cristiano Rumio
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Fluorescence
Fluorescence
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Fluorescence
Fluorescence
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Fluorescence
QUESTIONS?
HOW IS THE ENERGY (HIGHER? LOWER?) OF THE
LIGHT EMITTED BY A FLUOROCHROME WITH RESPECT
TO ENERGY OF THE EXCITATION LIGHT?
d  0.6098

NA
v

f
E  h* f
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QUESTIONS?
HOW IS THE ENERGY (HIGHER? LOWER?) OF THE
LIGHT EMITTED BY A FLUOROCHROME WITH RESPECT
TO ENERGY OF THE EXCITATION LIGHT?
d  0.6098

NA
v

f
E  h* f
Outline
Microscopy in general
Light microscopes
Fluorescence
Confocal microscopes
Theses and stages
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Confocal Microscopy
THE PRINCIPLE OF CONFOCAL IMAGING WAS PATENTED IN 1957 BY MARVIN MINSKY
Characteristics
•
Fluorescence microscopy
•
Scanning in x, y and z
•
Thickness not so critical
•
Slow for large image area
•
Pixel by pixel images
From www.microscopyu.com
Confocal Microscopy
www.microscopyu.com/tutorials/java/virtual/confocal/index.html
WHAT CAN WE DO?
www.microscopyu.com/moviegallery/sweptfield/folu-egfp-eb3-sfc
 High resolution images
 Time lapse experiment
 Z analysis
From www.microscopyu.com
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Confocal Microscopy
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.
Light sheet microscope
Zeiss Light Sheet v2.1
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Light sheet microscope
Zeiss Light Sheet v2.1
Light sheet fluorescence microscopy (LSFM) is a
fluorescence microscopy technique with good optical
sectioning capabilities and high speed. In contrast to
epifluorescence microscopy only a thin slice of the
sample is illuminated perpendicularly to the direction of
observation. For illumination, a laser light-sheet is used.
As only the actually observed section is illuminated, this
method reduces the photodamage and stress induced
on a living sample. Also the good optical sectioning
capability reduces the background signal and thus
creates images with higher contrast, comparable to
confocal microscopy. Because LSFM scans samples by
using a plane of light instead of a point (as in confocal
microscopy), it can acquire images at speeds 100 to
1000 times faster than those offered by point-scanning
methods. LSFM combines good z-sectioning (as
confocal) and only illuminates the observed plane. This
method is used in cell biology and for microscopy of
whole living creatures, such as embryos.
Outline
Microscopy in general
Light microscopes
Fluorescence
Confocal microscopes
Theses and stages
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Computer Vision Group
To: ARCES, Bologna
Theses and stages in:
Image correction techniques: vignetting, depth-of-focus, mosaicing.
Analysis of images acquired by flying drones
Cellular and Molecular Engineering Laboratory
To: School of Engineering, Cesena
Theses and stages in:
Bacterial segmentation and tracking
Fluorescent signal normalization for analysis of synthetic biology circuits
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Radiobiology Laboratory
To: IRCCS-IRST, Meldola (FC)
Theses and stages in:
Multicellular cancer aggregates analysis
Radiotherapy and chemotherapy protocol analysis for lung cancer
Bone Regeneration Laboratory
To: Istituto Ortopedico Rizzoli, Bologna
Theses and stages in:
Mesenchymal Stem Cell characterization and differentiation
Regeneration Medicine for bone cancer
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Light Microscopy and Screening Centre
To: Swiss Federal Institute of Technology (ETH), Zurich, Switzerland
Theses and stages in:
Cellular screening analysis
Microscopic imagery tracking, detection, segmentation
Synthetic and Systems Biology Unit
To: Biological Research Centre, Szeged, Hungary
Theses and stages in:
Microscopic image segmentation and tracking
Machine learning methods
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THANK YOU!
Filippo Piccinini
f.piccinini@unibo.it
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