Beruflich Dokumente
Kultur Dokumente
Table of Contents
1. What is Turku BioImaging?
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2. A brief history of Turku BioImaging
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3. Turku BioImaging facilities
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4. Light microscopy
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5. Confocal microscopy
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6. High content analysis
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7. STED microscopy
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8. FRET microscopy
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9. FCS and RICS
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10. Two-photon microscopy
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11. Spinning disk confocal microscopy
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12. TIRF microscopy
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13. ScanR automated fluorescence microscopy
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14. Stereo microscopy with microinjection
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15. Time-resolved fluorescence microscopy
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16. Live cell imaging
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17. Image analysis
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18. Flow cytometry
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19. Cell sorting
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20. Flow cytometry data analysis
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21. Atomic force microscopy
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22. Electron microscopy
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23. PET imaging
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24. Small animal ultrasound imaging
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25. Micro-CT imaging
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26. Imaging mass spectrometry
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27. Macroscopic in vivo imaging
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28. Laser-capture microdissection
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29. Masters Degree Programme in Biomedical Imaging72
30. Turku as an imaging stronghold
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Turku BioImaging email addresses
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Turku BioImaging embraces state-of-the-art imaging technologies used by the bioscience community of Turku. The organization is highly interdisciplinary, encompassing a wide range
of imaging modalities from molecular to cellular, from single
molecule analysis to whole animal imaging, and from analysis
of events at the single cell level to high-throughput screening.
The initiative also includes proteomics, systems biology, and
computational modeling of cellular processes, each of which is
associated with specific bioimaging modalities. Further, Turku
BioImaging also offers software tools for image data processing
and analysis. Turku BioImaging is closely integrated with the
Biocenter Finland National Imaging Infrastructure Network
(http://www.biocenter.fi/index3_biologicalimaging.html), as well as with
the pan-European imaging infrastructure EuroBioimaging (www.
eurobioimaging.eu).
MSc Programme in Biomedical Imaging
The various university departments and core facilities included
in Turku BioImaging have a great deal of experience in providing training in imaging and in facilitating access to modern
equipment. In this regard, the latest development is a novel international Master of Science Degree Programme in Biomedical
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Imaging. The MSc programme, jointly administered by the University of Turku and bo Akademi University (please see chapter
29), spans all imaging modalities and features lectures from local
and international experts in bioscience, medicine, physics, computer science, proteomics, and the visual arts.
In early 2009, the Ministry of Education selected the Turku BioImaging consortium as one of the 24 most significant nationallevel research infrastructures in Finland (Finnish research infrastructure roadmap: http://www.minedu.fi/export/sites/default/OPM/
Julkaisut/2009/liitteet/opm02.pdf?lang=fi).
Also in 2009, the key stakeholders signed an agreement on the
future of Turku BioImaging, with the aim of creating a leading bioimaging technology platform in Northern Europe in the
coming years. The partners of the initiative are the University of
Turku; bo Akademi University; Turku Science Park; the Hospital District of Southwest Finland; the Centre for Economic Development, Transport, and the Environment of Southwest Finland (the ELY Centre); and the Regional Council of Southwest
Finland.
At present, Turku BioImaging is extensively involved in coordination of the National Imaging Research Infrastructure Network
of Biocenter Finland, and participates actively in the EuroBioimaging initiative for the development of biomedical imaging
infrastructure in Europe.
EM Core Facility
The Laboratory of Electron Microscopy serves as a core facility
for nanolevel imaging and offers transmission electron microscopy, scanning electron microscopy, atomic force microscopy,
scanning tunneling microscopy, immunocytochemistry, and
elemental analysis and localization by combination X-ray spectrometry. Samples can be prepared from tissues, cells, bacteria,
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4. Light microscopy
Light microscopy includes both basic and advanced methods
whereby light is used to visualize objects. The range of applications
varies from single molecules and cells to animals (e.g., worms or
zebrafish). The use of fluorescence in addition to normal light allows visualization of elements that can be labeled with antibodies
or fluorescent tracers. Light microscopy provides numerous critical
tools for life science studies.
How does it work?
Light microscopy uses ultraviolet, visible, or infrared light to detect objects. Because light is highly versatile, photon detection,
used as an imaging modality, remains clearly separate from specific imaging modalities employing electrons (EM), X-rays (CT),
radioactive decay (PET), and molecular resonance in a magnetic
field (MRI).
What can be studied with this technique?
In all imaging, contrast is needed for visualization. Light microscopy techniques include bright-field, phase contrast, and
differential interference contrast (DIC) microscopy, wherein
contrast is obtained either by using a phase shift, polarization, or
staining with chemicals yielding visible colors. Fluorescence is
not necessary. However, fluorescence is a very efficient method
for production of contrast. For fluorescence imaging, Turku BioImaging hosts several fluorescence and confocal microscopes
(chapters 5, 6, 10, 11, 15, 16, 28, a total internal reflection microscope (chapter 12), and a super-resolution microscope (STED;
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5. Confocal microscopy
Confocal microscopy is a light microscopy technique that can produce true three-dimensional images of biological samples without
harming the cells, tissue or organism. Combined with advanced
software, confocal microscopy is one of the most popular and versatile biomedical imaging methods available.
How does it work?
Confocal microscopy is a fluorescence light microscopy technique whereby image detection is achieved through a small pinhole. The image is illuminated point-by-point with a laser that
excites fluorescence in the sample, and the detected light passes
through a pinhole located on a confocal plane on the other side
of the lens, defined by reference to the focal plane of the sample.
The pinhole excludes most of the light coming from either below or above the focal plane. Thus, an optical section is created
through the sample. After acquiring the image from one focal
plane, the focus can be changed and a new image acquired. In
this way a stack of optical sections covering the entire sample is
obtained. This stack forms a three-dimensional image of the entire sample, as opposed to the two-dimensional images obtained
using regular light microscopes. Because sectioning is optical,
non-invasive and relatively fast, living cells can be imaged and
four-dimensional images (three spatial dimensions plus time)
can be successfully acquired. Specialized software can be used
to create both three-dimensional images and four-dimensional
movies of imaged material, as well as to perform many types of
quantitative analysis.
The principle of confocal microscopy (simplified). The pinhole allows only light
emanating from the focal plane (yellow) to pass through to the detector. Light
coming from below (purple) or above (cyan) the focal plane is excluded.
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What is required?
Fluorescent samples prepared using suitable high-quality cover
glass (or appropriate live cell containers) are required. The microscopes are more complicated than basic light or fluorescence
microscopes, but basic training will overcome this problem.
Special software, such as BioImageXD developed by the University of Turku (please see chapter 17), can be utilized to generate
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Professor John Eriksson and senior researcher Pasi Kankaanp beside a Leica
TCS SP5 confocal microscope in BioCity.
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7. STED microscopy
Stimulated Emission Depletion (STED) microscopy is a confocallike microscopy technique, in which the traditional diffraction
resolution limit of optical microscopy can be effectively surpassed.
With a STED instrument, it is possible to acquire purely optical
super-resolution images, with details visible at the level of tens of
nanometers.
How does it work?
As first realized by Ernst Abbe in the late 19th century, the resolution of a standard far-field light microscope is limited by diffraction to about one-third of the wavelength used, which is approximately 180 nm for visible light. Recent research, pioneered
in the mid-1990s by Stefan Hell, has shown that the fundamental diffraction barrier can be broken in fluorescence far-field
microscopy, making it possible to achieve a resolution close to
the scale of individual molecules. The basic principle of STED
microscopy is the use of two lasers working in a synchronized
manner, in a setting that is otherwise rather similar to that of
scanning confocal microscopy. One of the lasers is employed
for normal fluorescence excitation, whereas the other forms a
red-shifted donut-shaped beam (of zero intensity in the center),
which is overlaid on the diffraction-limited fluorescence spot,
and then used to deplete all fluorescence at that spot, except for
the immediate center. This is achieved by exploiting an optical
process termed stimulated emission depletion. By scanning the
sample using a series of sub-diffraction spots, a purely optical
super-resolution image can be constructed.
What is required?
Samples for the STED instrument are prepared in the same manner as for normal fluorescence microscopy. However, two considerations arise:
The Leica TCS STED currently has two super-resolution channels. For fluorescence labeling ATTO647N and Chromeo494
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8. FRET microscopy
Frster or Fluorescence Resonance Energy Transfer (FRET) has
advanced to become one of the most popular applications in microscopy and fluorescence spectroscopy. It allows the visualization
of molecular interactions and dynamics in living cells. This spatiotemporal mapping of molecular activity will continue to attract
major attention in the post-genomic area. Developments with this
methodology to meet the demands of the post-genomic era will
have a significant potential in the future.
How does it work?
With Frster Resonance Energy Transfer (FRET), named after
Theodor Frster, a German physical chemist, nanometer-range
molecular proximities between donor and acceptor fluorophores
can be detected. These are typically attached to two bio-molecules to detect their interaction or co-clustering. If integrated
into one biomolecule they can report on its conformational state.
FRET can be applied in vitro, ex vivo and in vivo. Two key requirements for FRET are that the donor emission and the acceptor excitation spectra overlap and that the fluorophores are only
approximately 1-10 nm apart. The extent to which FRET occurs
is expressed by the energy transfer efficiency, which ranges from
0 (no FRET) to 1. However, in practice various (instrument dependent) FRET measures or indices are used.
What can be studied with this technique?
FRET is widely used in biochemical assays for high-throughput
screening under the name of HTRF or in the TaqMan probes
for RNA quantification. In cell biology FRET probes have provided unprecedented insight into where molecules are active and
where they form complexes.
When is this technique the right choice?
There are numerous applications for FRET in biology. Most
prominently, scientists are interested in quantifying molecular
interactions. Similarly, learning about conformational changes
can be achieved with FRET. However, the distance constraints
need to be kept in mind. For large complexes, alternative approaches such as FCCS (Fluorescence Cross Correlation Spectroscopy) are a better choice when assessing interactions.
What is required?
FRET can be determined by measuring the change in the fluorescence properties of the donor or the acceptor fluorophore, such
as their intensity or their fluorescence lifetime. The fluorescence
lifetime is the average time a fluorophore spends in its excited
state. FRET can be a process that leads to de-excitation, thus
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FRET reveals association between PTRF (a novel coat protein of caveolae, left)
and the caveolar component caveolin1 (middle) in vesicular structures on the
plasma membrane (arrowheads) and within (arrows) BHK cells. FRET was
calculated per pixel as a sensitised acceptor emission index FR, ranging from
1 (no FRET) to 10 and above as displayed on the yellow-hot look-up-table in.
Scale bar 2 m.
lowering the average fluorescence lifetime. Also the local environment of a fluorophore can affect its fluorescence lifetime. To
measure FRET, more or less specific instrumentation is required.
While intensity changes can be readily monitored with fluorescence plate readers, flow cytometers and microscopes, the fluorescence lifetime measurement requires hardware adaptations
of these instruments. In cell biology this is commonly realized
with Fluorescence Lifetime Imaging Microscopy (FLIM). FLIM
is very sensitive and produces images that reflect the changes of
the fluorophore environment, such as pH and polarity. A third
way of assessing FRET is to monitor the depolarisation (anisotropy) of polarized light due to FRET. This method is also the
only one to measure homo-FRET, which is the energy transfer
between identical fluorophores.
How to get started?
Setting up FRET methods and instrumentation is a time consuming process. Furthermore, often tailoring the biological experiment such that FRET can be used effectively is much more
demanding and expert advice should be sought whenever possible (see contact information below).
Availability within Turku Bioimaging
FRET can be done on all of the Cell Imaging Cores confocal
microscopes and the LSRII flow cytometer (BioCity, 5th floor).
Moreover, FLIM is going to be available on some instruments,
like the STED microscope (BioCity, 5th floor).
Contact
Daniel Abankwa & Camilo Guzmn, FRET@bioimaging.fi
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Principles of FCS and RICS. Left: single point FCS where laser focus (red) is
static but recorded fluorescence fluctuates as fluorescent molecules (green dots)
transit through the focal point. Right: RICS, a scanning FCS technique. Laser
is not static, but is scanning a determined area. Periodicity of the scanning is
exploited to obtain additional temporal information from the scanned image. RICS has lower temporal resolution than FCS, while being able to average
fluctuation parameters such as diffusion over a larger area.
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3D image of an intact mouse lymph node demonstrating the possible imaging depth of two-photon microscopy. Red and green fluorescent lymphocytes
are visible as deep as 250 m below the capsule (blue) at the surface of the
lymph node.
unprocessed tissue pieces can be imaged. Although most fluorescent proteins are suitable for two-photon excitation, the technique can also be used with tissues not containing fluorescently
proteins, since specific fluorescence phenomena (so-called second- and third-harmonic generation) allow the visualization of
certain tissue structures (collagen fibers and muscle filaments)
without labeling.
Availability within Turku Bioimaging
Leica TCS SP5 MP
Multiphoton system with an upright DM6000 CFS microscope.
BioCity, floor -1, staircase A.
Contact
Tibor Veres, 2-photon@bioimaging.fi
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A live COS-7 cell transfected to express EGFP-tagged small GTPase Rab7. The
images show Rab7-positive vesicles in close proximity to the plasma membrane
(the TIRF channel) and throughout the cell body (the widefield channel).
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What is required?
Fluorescently labeled samples that would also be suitable for observation with a normal fluorescence microscope. For example
multi-well plates with transparent bottom, coverslips, etc.
How to get started?
Contact VTT Medical Biotechnology for guidance and assistance.
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Signifer microimager.
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The principle of time-resolved measurement showing the decay curve of a longlifetime label. The cycle commences with an excitation pulse and is followed,
after 2000 microseconds, by another pulse.
Contact
Tuomas Nreoja, TRFM@bioimaging.fi
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BioImageXD.
BioImageXD can create animations, for example by defining a flight path for
the camera, as shown on the left. Such animations can lead into a cell, as
displayed on the right, where a surface view of a nucleus inside a living cell
can be seen.
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Turku, which means that help and user support, as well as tailored analysis, are available. No other software solution offers
equal flexibility and support within Turku BioImaging. BioImageXD is especially suitable for processing of fluorescence-based
images, but many package tools are applicable to all kinds of images.
What is required?
BioImageXD will run on any normal personal computer, but
powerful workstations are recommended for more demanding
calculations or if large amounts of data are to be processed. The
software is available free of charge for PC, Mac, and Linux. A
basic understanding of digital images and the processing thereof
is required, and, in many instances, some user training is needed.
How to get started?
BioImageXD can be downloaded and installed on any computer
(see below) An overview of the user manual provided may be
helpful, as may attendance at a course or a training session organized every year in Finland.
Availability within Turku Bioimaging
The BioImageXD software is freely available for download at
www.bioimagexd.net, and user support is available through info@
bioimagexd.net. The email address below is for all other / general
issues related to image analysis.
Contact
Pasi Kankaanp, analysis@bioimaging.fi
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What is required?
The cells to be analyzed must be prepared as a single cell suspension. Many samples, including peripheral blood mononuclear
cells (PBMCs), confluent cell lines, or bacteria, can be injected
directly. Adherent cells must be detached. Tissue must be homogenized before analysis. Practically all commercial fluorophores can be measured with flow cytometry. However, if it is
desired to combine several different fluorophores in a single
sample, attention should be given to excitation and emission
wavelengths to prevent any possible overlap.
How to get started?
Experimenters with no previous experience of flow cytometry
should contact a responsible operator to request a training session. Training will cover all necessary steps, and will include tips
for sample preparation, hands-on training on the instrument,
how to set up the instrument for particular samples, and assistance in correct data analysis. The goal is to train all users to in48
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What is required?
Cell sorting is performed as a service. Thus, the details of the
process are not important. Simply contact the responsible operator and arrange a sorting time. It is always better to analyze cells
by regular flow cytometry before sorting, to estimate the proportion of target cells. This helps to determine the time needed for
cell sorting.
How to get started?
Contact the responsible operator.
Availability within Turku Bioimaging
BD FACSAria IIu cell sorter
The sorter has two lasers, 488 nm blue and 633 nm red.
BioCity, 5th floor, room 5126.
Contact
Perttu Terho, flow@bioimaging.fi
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used to measure the force between the tip and the sample, and
if, for example, the tip is coated with a ligand, the position of a
receptor molecule on the cell surface can be determined (recognition mapping). In addition, the tip can be replaced by an entire
cell attached to the cantilever, and the attachment of this cell to a
substrate such as collagen can be measured (cell adhesion measurements). The tip can also be used to physically manipulate a
sample, or to pull on a protein to study how the protein unfolds.
Confocal and atomic force microscopy images of the same cell and an example
of a force measurement curve recorded using an AFM.
the impact of the primary beam. The use of electrons allows detailed imaging of structure, and localization and identification
of molecules and elements to a resolution below 1 nanometer
(10-9 m). The Laboratory of Electron Microscopy at the University of Turku offers a full service, including consultation in
the planning of experiments, sample processing, microscopic
examination and recording of images, and reporting and interpretation of results. The goal of the research service is to provide
electron microscopic facilities for investigators within the medical faculty and in part also for scientists from all over Finland.
In addition to basic morphological transmission and scanning
electron microscopy, the facility offers immunocytochemistry,
cryo-ultramicrotomy, X-ray microanalysis, scanning tunneling
microscopy, atomic force microscopy, and morphometry.
When is this technique the right choice?
If detailed structural details at nanometer resolution are required, micro- and nano-structural imaging combined with molecular analysis is the method of choice, applicable to samples
of tissue, cells, bacteria, viruses, nanoparticles, macromolecules,
and other related materials. Samples must be fixed with an aldehyde, and dried either with ethanol or at the critical point of
carbon dioxide (for SEM), and embedded in epoxy or another
resin (for TEM) before being cut into thin sections.
The facility offers the following methods and instrumentation:
Cutting of ultrathin sections
Immunocytochemical light and electron microscopy
Recording and documentation of images taken using virtual,
light, and fluorescence microscopes, transmission electron
microscopes (TEMs) and scanning electron microscopes
(SEMs)
An atomic force microscope (AFM)
A scanning tunneling microscope (STM)
X-ray spectrometry for element analysis and localization
Crystal analysis using electron diffraction
Availability within Turku BioImaging
JEM 1200EX with EDS analyser (TEMSCAN)
120 kV scanning transmission electron microscope with the
NORAN SIX EDS-system. The X-ray detector is a NORWARwindow Si(Li) instrument with an energy resolution of 145 eV.
All elements heavier than boron can be analyzed.
Medisiina, 4th floor, room A408.
Philips EM410 (TEM)
Medisiina, 4th floor, room A425.
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Transmission electron micrograph of a thin section showing a portion of testicular Leydig cell cytoplasm with a sector of the nucleus in the bottom righ corner. The typical organelles that can be seen are smooth endoplasmic reticulum,
rough endoplasmic reticulum, polyribosomes, and mitochondria.
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Positron-emitting radionuclides are usually produced in cyclotrons. Short-lived (T= 2-110 min) radionuclides are then incorporated into molecules of interest using sophisticated radiochemical syntheses. Because of the short half-lives of positron
emitters, the syntheses must be very rapid. Tracers are delivered
to the subject, usually by injection, and a PET scan is performed.
Images are reconstructed as tomographic images by mathematical processing of data.
What can be studied with this technique?
Tissue function and metabolism.
The functions of neurotransmitters and receptors.
Drug pharmacokinetics and drug dynamics.
Gene expression.
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PET methodology.
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B-mode echocardiography at diastole and systole (cut from live loop) from
mouse with experimental apical infarct due to ligation of LAD coronary artery.
White area visible both in epicardium and endocardium at anterior wall is the
ligation stitch. Y-axis scale on millimeters.
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Contact
Jorma Mtt, CT@bioimaging.fi
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Imaging MS of rat brain. Mass peaks selected and visualized by color reveal the
regional distribution and abundance of peptides. Combining the three individual masses creates a composite image. In the overall mass spectrum plot, x-axis
corresponds to mass-to-charge ratio (m/z) and y-axis denotes relative intensity.
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Language requirements
A good knowledge of the English language is required. All applicants must prove that they have adequate English skills by appending the score from an international English-language test to
their applications.
Aims of the programme
Biomedical imaging has emerged as one of the most important
technologies in basic bioscience and biomedical research, as
well as in clinical medicine and translational studies. The current technologies enable detailed real-time non-invasive visualization of molecules, structures, and events in cells, tissues, and
whole organisms. Application areas include cellular and molecular biology, pharmacology, structural biology, nanotechnology
and biomaterials research, and patient care and diagnostics. The
Programme in Biomedical Imaging aims to give professionals a
thorough understanding of diverse imaging technologies along
with practical skills. The advanced portion of the curriculum is
also intended to offer training to graduate students and postdoctoral fellows.
Collaborators
The immediate collaborators are the national Turku PET Centre,
the Turku Centre for Biotechnology, the Turku Center for Disease Modeling (TCDM), and the Turku University Hospital. The
programme has collaborators in the Nordic countries, Europe,
the USA, and Asia, and also offers courses given by international
experts.
Career prospects
The interdisciplinary curriculum provides graduates with an excellent basis for careers in many different areas of life sciences,
both in academic research and industry. Importantly, the pro73
More information:
www.med.utu.fi/biomaging
www.abo.fi/bioimaging
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The Chair of Turku BioImaging, Professor John Eriksson (left), and researcher
Kimmo Isoniemi (right), explain new three-dimensional imaging technology to
the Rector of bo Akademi, Jorma Mattinen, and the Rector of the University
of Turku, Keijo Virtanen.
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Editors
John Eriksson
Pasi Kankaanp
Jari Korhonen
Maritta Lytmki
Eeva Rainio
Jouko Sandholm
Image editors
Pasi Kankaanp
Maritta Lytmki
Markku Saari
Design
Joacim Pivrinne
Printed by
Finepress Oy
October 2011
Authors
1. What is Turku BioImaging
Text: Maritta Lytmki & John Eriksson
Image: Sami Koho
2. A Brief history of Turku BioImaging
Text: Maritta Lytmki & John Eriksson
Images: Markku Saari / Turun yliopiston viestint
3. Turku BioImaging facilities
Text: Maritta Lytmki & John Eriksson
Images: Diana Toivola / Markku Saari / Heikki Minn / Mika
Okko / Lauri J. Pelliniemi / Pasi Kankaanp
4. Light microscopy
Text & image: Jouko Sandholm & Markku Saari
5. Confocal microscopy
Text & images: Pasi Kankaanp
6. High content analysis
Text: Kimmo Isoniemi & Jari Korhonen
Images: Kimmo Isoniemi / Vesa-Matti Vr
7. STED microscopy
Text & images: Sami Koho
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8. FRET microscopy
Text & image: Daniel Abankwa
9. FCS and RICS
Text & images: Daniel Abankwa & Camilo Guzmn
10. Two-photon microscopy
Text & images: Tibor Veres
11. Spinning disk confocal microscopy
Text: Antti Arjonen
Images: Yokogawa Electric Corporation / Antti Arjonen
12. TIRF microscopy
Text: Jorma Mtt
Image: Antti Arjonen
13. ScanR automated fluorescence microscopy
Text: Rami Mkel & Jeroen Powels
Images: Juha Rantala
14. Stereo microscopy with microinjection
Text: Jouko Sandholm
Image: Markku Saari
15. Time-resolved fluorescence microscopy
Text: Tuomas Nreoja & Roope Huttunen
Images: Pasi Kankaanp / Roope Huttunen / Tuomas Nreoja
16. Live cell imaging
Text: Jouko Sandholm & Markku Saari
Images: Pasi Kankaanp / Markku Saari
17. Image analysis
Text & images: Pasi Kankaanp
18. Flow cytometry
Text: Perttu Terho
Images: Perttu Terho / Perttu Terho / Markku Saari
19. Cell sorting
Text & images: Perttu Terho
20. Flow cytometry data analysis
Text & image: Perttu Terho
21. Atomic force microscopy
Text & images: Pasi Kankaanp
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Notes
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