Sima Salahshor, BSc, MSc, PMP, PhDDepartment of Laboratory of Medicine & Pathobiology,

Faculty of Medicine, University of Toronto & ScienceHA, Inc.

Lecture part of the “Cellular Imaging in Pathobiology” courseCourse ID #LMP1006H (http://www.lmp.utoronto.ca/course/lmp1006h)

Full body scan,

Whole animal scan

Organ, Tissue

imaging

Cellular imaging

Chromosome Genes

DNA

Molecular imagingSingle

molecule

Radiology, Sonography, Radioiodine whole body scanUltrasound, Spectroscopy MRI, CT scan, PET scan(General diagnostic imaging)

FISH, SKYC-banding, G-banding

Light-, Confocal-, Two-photon-, Fluorescence microscopy Electron microscopy (SEM, TEM)

FLIM, FRAP, TIR-FM , AFMFRET, X-ray crystallography,Super-resolution microscopy,Laser tweezers, Raman spectroscopy

Imaging Technologies

Whole body MRI scan Imaging of Multiple Myeloma and Related

Plasma Cell Dyscrasias

Full body scanner (detect object under clothing) Backscatter X-ray systems Millimeter wave technology

Employs non-ionizing submillimeter microwaveradiation (reflects extremely high frequency radiowaves off the body)

Reference: Ronald C. Walker et alJ Nucl Med July 1, 2012 vol. 53 no. 7 1091-1101http://jnm.snmjournals.org/content/53/7/1091.full.pdf+html

Health effect and safety of backscatter x-ray or millimeter wave scanners: Not fully investigated!Reference: http://www.iacrs-rp.org/

Small-animal imaging

Reference: Hoffman J M , Gambhir S S Radiology 2007;244:39-47

A. whole-body micro-PET imageB. micro-CT imageC. micro-SPECT imageD. Optical reflectance fluorescence imageE. T2-weighted micro–MR imageF. Optical bioluminescence image

MRI Magnetic Resonance Imaging uses a powerful magnetic field, radio frequency pulses and a computer to produce detailed

pictures of organs, soft tissues, bone and virtually all other internal body structures.

CT X-ray Computed Tomography combines special x-ray equipment with sophisticated computers to produce multiple images or

pictures of the inside of the body.

PET Positron Emission Tomography uses small amounts of radioactive material to diagnose or treat a variety of diseases, including

many types of cancers, heart disease and certain other abnormalities within the body.

SPECT Single-Photon Emission Computed Tomography a special type of computed tomography (CT) scan in which a small amount of a radioactive drug is

injected into a vein and a scanner is used to make detailed images of areas inside the body wherethe radioactive material is taken up by the cells. SPECT can give information about blood flow totissues and chemical reactions (metabolism) in the body.

Ultrasound exposing part of the body to high-frequency sound waves to produce pictures of the inside of the

body.

Optical Imaging the image formed by the light rays from a self-luminous or an illuminated object that traverse an

optical system.

The Compound Light MicroscopeThe Stereo Microscope (dissecting microscope) The Electron MicroscopeThe Scanning Probe Microscope (SPM)

An electron microscope is a type of microscope that produces an electronicallymagnified image of a specimen for detailed observation. The electron microscope (EM) uses a particle beam of electrons to illuminate thespecimen and create a magnified image of it.

TEM Transmission Electron Microscope

can achieve magnifications of up to 50,000,000x

SEM Scanning Electron Microscope

magnification up to 5,000,000x

Both SEM and TEM are useful in biology and geology, as well as in materials science.

Reference:Girard F, Batisson I, Frankel GM, Harel J and Fairbrother JM (2005). Interaction of enteropathogenic and Shiga-Toxin producing Escherichia coli with porcine intestinal mucosa: role of Intimin and Tir in adherence. Infection and Immunity 73: 6011

Floral primordia of Allium sativum (garlic) captured with the Epi-Illumination technique.

Credit: Dr. Somayeh NaghilooUniversity of Tabriz, Department of Plant Biology, Faculty of Natural Sciences http://www.nikonsmallworld.com/galleries/photo || Small World, 2012 Nikon competition winner

Mouse C2C12 cells in prophase imaged with 3Dstructured illumination microscopy (SIM).Condensed chromosomes are stained with DAPI(red), the nuclear lamina and microtubuli areimmunolabeled with an anti-lamin B (blue) and ananti-tubulin antibody (green), respectively.

Reference:Lothar Schermelleh, Ludwig-Maximilians-Universität München http://www.cell.com/cell_picture_show-superres

Imaging by WFM, LSCM and 3DSIM10 µm section of mouse small intestine, fixed in formaldehyde, cryosectioned, stained with DAPI (blue), anti-tubulin (red) and phalloidin (green) and mounted in glycerol stained with DAPI.A: WFM recorded on a Leica fluorescence microscope with a Hamamatsu Orca CCD camera. B: LSCM on a Zeiss 710 microscope. C: 3DSIM recorded on an OMX microscope.

Images courtesy of Paul Appleton and Emma King.

Reference:Innovation in biological microscopy: current status and future directions. Bioessays. 2012 May;34(5):333-40 by Swedlow JR.

WMF =A widefield Microscope (one type of Fluorescence Microscope)LSCM = Laser Scanning Confocal Microscopy3D-SIM = Structured Illumination Microscopy (super-resolution technique). SIM can image up to 10 microns past the coverslip into the sample.

A powerful instrument for microbiological Investigation.

AFM has the advantage ofimaging almost any type ofsurface, including polymers,ceramics, composites, glass,and biological samples. AFMprovides a 3D profile of thesurface on a nanoscale.

Reference: Atomic force microscopy: a nanoscopic view of microbial cell surfaces. Micron. 2012 Dec;43(12):1312-22 by Dorobantu LS, Goss GG, Burrell RE.

Karyotype Chromosome analysis gives you a full picture of the structure of each chromosome as

well as the number of chromosomes present in each cell.

FISH Fluorescent In Situ Hybridization FISH analysis doesn't give you a picture of each chromosome but it does tell you how

many copies of a certain chromosome are present in each cell.

SKY Spectral Karyotyping Permits the simultaneous visualization of each human or mouse chromosome in a

different color, facilitating the identification of chromosomal aberrations.

CGH Comparative Genomic Hybridization Utilizes the hybridization of differentially labeled tumor and reference

DNA to generate a map of DNA copy number changes in tumor genomes.

Reference: H.-U.G. WEIER, J. KWAN1, C.-M. LU, Y. ITO, M. WANG, A. BAUMGARTNER ,S.W. HAYWARD6 J.F. WEIER, H.F. ZITZELSBERGER

Credit: Cancer Genomics Program,Departments of Pathology and Oncology, University of Cambridge

Superresolution MicroscopyPhotoactivated localization microscopy (PALM)Stochastic optical reconstruction microscopy (STORM)Near-field scanning optical microscopy (NSOM)Stimulated emission depletion microscopy (STED)Saturated structured-illumination microscopy (SSIM)Ground state depletion (GSD) microscopytwo-photon laser-scanning microscopy (TPLSM).

The study of lymphocyte activation requires observation of samples that vary in size over six orders of magnitude. This figure shows the T cell receptor (TCR)-mediated signallingpathway and microscopy techniques used at three levels of sample size.

Reference:Imaging techniques for assaying lymphocyte activation in action. Lakshmi Balagopalan, EilonSherman, Valarie A. Barr & Lawrence E. Samelson

Applications of imaging techniques

Reference:Lakshmi Balagopalan, Eilon Sherman, Valarie A. Barr & Lawrence E. Samelson Imaging techniques for assaying lymphocyte activation in action

Reference: Cancer imaging by optical coherence tomography: preclinical progress and clinical potential. Vakoc BJ, Fukumura D, Jain RK, Bouma BE.

Overview of intravital imaging approaches in preclinical cancer research

Imaging Technique Spatial Resolution Key Use

Multi-photon Microscopy 15 – 1000 nm Visualization of cell structuresAtomic Force Microscopy 10 – 20 nm Mapping cell surfaceElectron Microscopy ~5 nm Discerning protein structureUltrasound 50 μm Vascular imagingCT/MicroCT 12 – 50 μm Lung and bone tumor imagingMRI/MicroMRI 4 – 100 μm Anatomical imagingfMRI ~1 mm Functional imaging of brain activityMRS ~2 mm Detection of metabolitesPET/MicroPET 1 – 2 mm Metabolic imaging

The various micro versions of the imaging modalities (MicroCT, MicroMRI, MicroPET) as well as themicroscopy techniques (Fluorescence, Multi-photon, Atomic, Electron) are primarily used in either cellular oranimal studies. The remaining modalities (Ultrasound, CT, MRI, MRS, PET) are more widely used clinically.

Reference: Kherlopian et al. BMC Systems Biology 2008, 2:74

Comparison of imaging technology for systems biology

No. 1: Magnetic resonance imaging (MRI) & Computed Tomography (CT) scanNo. 5: Functional Magnetic Resonance Imaging (fMRI)No. 8: Molecular Breast Imaging (MBI)

Medical imaging technology has revolutionized health care.

MRI CT scan fMRI MBI

Reference: Ann Tracy Mueller, 2013, http://bit.ly/1QOkYgr

Email: s.salahshor@utoronto.ca || URL: http://bit.ly/1hTp48aTwitter: @SSalahshor

For more information about LMP1006H course visit: http://www.lmp.utoronto.ca/course/lmp1006h