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TRACKING
TECHNIQUES
M.Vinoth
MVM09003
ADVISORY COMMITTEE
Chairman:
Dr.A.PALANISAMY,Ph.D
Professor,
Department of Animal Biotechnology,
Members:
Dr.K.KUMANAN, Ph.D
Professor and Head ,
Department of Animal Biotechnology,
Dr.S.BALASUBRAMANIAN,Ph.D
Associate Professor,Department of Animal
Reproduction, Gynaecology &obstetrics
Stem cell
A cell that has the ability to continuously divide
and differentiate (develop) into various other
kind(s) of cells/tissues
Stem cell tracking techniques / in vivo stem cell
imaging
Techniques which has ability to non invasively
monitor stem cell trafficking in vivo
and engraftment
Expansion
and viability
Differentiation
CHARACTERISTICS OF AN IDEAL
IMAGING TECHNOLOGY
Biocompatible, safe, and nontoxic
No genetic modification or perturbation to the stem
cell
Single-cell detection at any anatomic location
Quantification of cell number
Minimal or no dilution of contrast agent with cell
division
Minimal or no transfer of contrast agent to non
stem cells
Non invasive imaging in the living subject over
months to years
No requirement for injectable contrast agent
(frangioni et al.,2004)
Fluorescence
Radionuclear Imaging
Positron
Emission Tomography(PET)
Single Photon Emission Computed Tomography
(SPECT)
(zhao et al.,2010)
Bioluminescence
radiotracer
fluorescent agent
Fluorescent tag
DIRECT LABELING
Advantages
simple
Can use clinically approved agents
Disadvantages
Signal
(zhao et al.,2010),
Very
promoter
reporter
transcription
mRNA
translation
protein
Fluorescent protein (OPT)
Receptor (NUC)
Enzyme (OPT, NUC, MRI)
[18F]-FHBG
Advantages
Disadvantages
Kraitchman et al.,2009
T1-agents
gadolinium-based
Paramagnetic
loaded via pino/endocytosis into stem cells
permit tracking for up to 6 weeks
T2 agents
(Nohroudi et al.,2010)
MR IMAGING OF MIGRATION
OF STEM CELLS IN RAT BRAIN
From: Modo M, Hoehn M, Bulte JWM: Cellular MR Imaging. Molecular Imaging 4: 143-164, 2005.
ADVANTAGE (MRI)
MRI scan is harmless to the patient. It uses
strong magnetic fields and non-ionizing
radiation, unlike CT scans and traditional X-rays
use ionizing radiation
High spatial resolution
Outstanding anatomic imaging
MRI meets the requirements of penetration
depth
clinical availability
sensitivity 10-3-10-5 M
DISADVANTAGE (MRI)
Dilution of contrast with cell division
Difficulty in quantification because of
susceptibility artefact
The potential transfer of contrast to non stem
cells, such as macrophages, after stem cell death.
A significant clinical problem common to all MRI
methods is that certain implantable devices, such
as pacemakers and defibrillators,
Long scan times for large volumes/high
resolution
NUCLEAR IMAGING
Positron Emission Tomography (PET)
Single Photon Emission Computed Tomography (SPECT)
gambhir et al .,2000
ADVANTAGE (PET)
The cross-sectional information and threedimensional (3D) reconstruction capability offer
more informative than the optical imaging
techniques
Sensitivity 10-11-10-12 M
Quantification possible
DISADVANTAGE (PET)
Resolution in PET is less than that which can be
achieved by MRI.
Ionizing radiation
Requires genetic modification of stem cell
Intravenous injection of contrast agent
It is not readily available
ADVANTAGE (SPECT)
Sensitivity 10-10-10-12 M
3D full-body scanning,
No dilution of effect size with cell division
(transgenic approaches)
Blackwood et al 2009,
DISADVANTAGE (SPECT)
Requires genetic modification of stem cell
Intravenous injection of contrast agent
Ionizing radiation
Quantification can be difficult
OPTICAL IMAGING
BIOLUMINESCENCE IMAGING
(BLI)
Bioluminescence is the process of light emission
in living organisms.
The DNA encoding the luminescent protein
(luciferase )is incorporated into stem cell via a
viral vector
Bioluminescence utilizes light generated by the
enzyme luciferase to detect cells in vivo.
The reporter probes for these proteins are
substrates that are oxidized and generate light.
Ultra-sensitive CCD camera can image
bioluminescence
CCD
image luminescence at
surface of animal
Xenogen Corp.
ADVANTAGE (BLI)
High sensitivity 10-15-10-17 M
No ionizing radiation
DISADVANTAGE (BLI)
Requires genetic modification of stem cell
Intravenous injection of contrast agent,
luciferase genes and substrates described to date
generate only visible (400 to 700 nm) light, which
has very high absorption and scatter in living
tissue.
Limited to small animal use
Even in mice false-negative scanning can occur,
dependent on cell depth
FLUORESCENCE IMAGING
Fluorescence imaging utilizes organic (eg, green
fluorescent protein) as exogenous contrast agents
for in vivo imaging.
Because of high photon absorption and scatter at
visible wavelengths are recorded
(Frangioni et al.,2009)
ADVANTAGE
High sensitivity 10-9-10-12 M
No ionizing radiation,
Fast
Signals from relatively superficial sites, such as
skin and subcutaneous tissues, or from deep sites
after removal of overlying tissues, can offer highresolution images.
DISADVANTAGE
Limited to small animal or intraoperative use
The major problem with NIR fluorescence is that
even with tomographic imaging methods,
detection is limited to only 4 to 10 cm of tissue
X-RAY COMPUTED
MICROTOMOGRAPHY (MICROCT)
X-RAY COMPUTED
MICROTOMOGRAPHY (MICROCT)
Microtomography uses x-rays to create crosssections of a 3D-object
The term micro is used to indicate that the pixel
sizes of the cross-sections are in the micrometer
range
Advanced microCT is capable of achieving a
spatial resolution up to 0.3 m
Cancedda et al 2007
ADVANTAGE (MICROCT)
high definition and resolution human cells after
transplantation
quantification of the number of cells
Readily available,
3D, full-body scanning
DISADVANTAGE (MICROCT)
Requires high molar concentrations of contrast
agent,
Artifacts from bone and cardiac devices,
Ionizing Radiation
SUMMARY
Zhao et al.,2010
CONTINUE
Frangioni et al 2010