Programs

Facilities

Undertaking the sophisticated research in the Molecular Sciences Program requires state-of-the art facilities and instrumentation. Here, we highlight a few of the key research tools and instruments available to our graduate students. Not only can you read about the instrument but also see the type of information that one can extract.

Advanced Microscopy and Imaging Instrumentation:

1. Zeiss Confocal Imaging System inverted scope for live cell imaging
2. Two-photon Zeiss Confocal Microscope (two-phton with MuhammadAli)
3. Leica Epifluorescence Microscope (epifluorescence scope with Rojia)
4. Digital Instruments Atomic Force Microscope (afm – with Amra)
5. Acoustic-enabled Microscope (Acoustic Micrsocpe with Eric Strohm)
6. Gel Documentation Systems (Gel doc. With Zafar)
7. Scanning Electron Microscope (with Eric da Silva)

Analytical Instrumentation:

1. Glove box for anaerobic conditions   (Gove box with Salma)
2. High performance liquid chromatography systems with UV/VIS, fluorescence and refractive index detection (HPLC)
3. Bruker 400 MHz nuclear magnetic resonance spectrometer (NMR with Salma)
4. SciEx Q-star MALDI-Esi-Qq-TOF mass spectrometer 

Environmental and Growth Facilities:

1. Biosafety Cabinets and CO2-jacketed incubators for cell culture (Cell Culture with David)
2. Mesocosms (environmental setups)
   
   

Zeiss Confocal LSM Microscope: A state of the art Zeiss Laser Scanning Microscope permits Veronica to acquire images of cells undergoing phagocytosis without out-of-focus light. This microscope is equipped with three laser lines and can be used for live-cell imaging.

Data: Macrophage expressing GFP-Akt-PH in the process of engulfing sheep red blood cells in a process called phagocytosis. The GFP probe shows that the signaling lipid, phosphatdiylinositol-3,4,5-trisphosphate, is produced in response to receptor engagement when external particles touch the surface of the macrophage. The green channel is overlayed over the Differential Interference Contrast channel (grayscale) showing other macrophages and the smaller red blood cells. Image provided by Dr. R. Botelho.

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Zeiss Two-photon Scanning Laser Microscope: The Two-photon Confocal uses two long-wavelength laser beams that combine at a single point in space to form shorter-wave, higher-energy photons that excite target fluorochromes only at that single point of space. This reduces excitation of out-of-focus fluorochromes and permits Muhammad to image thicker samples such as food emulsions.
	Data: COS cells expressing GFP-fusion of a lipid kinase (green). These cells were stained with antibodies against a marker of late endosomes called lyso-bisphosphatidic acid (LBPA). Courtesy of Dr. R. Botelho.

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Epifluorescence Microscope: Rojia is using a Leica DM 5000B Advanced Research Microscope with bright field, fluorescence, and Differential Interference Contrast optics with Leica DC 300F cooled color digital camera. This microscope permits imaging of cells and transparent organisms such as the C. elegans worm.
	Data: The image shown is of some of the DA and DB classes of motor neurons in an unc-5(e53) mutant animal in the microscopic nematode C. elegans. The motor neuron axon was visualized due to expression of green fluorescent protein and was detected by epi-fluorescence. Image courtesy of Dr. M. Killeen and S. Sybingco.

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Atomic Force Microscope: The atomic force microscope (AFM) is used to measure the height profile, or topography, of a surface with nanometer resolution. A very sharp probe tip attached to a flexible cantilever interacts with the sample surface. As the probe is rastered across the sample, it moves up and down with the features on the surface. The changes in the height of the probe, which can be as small as a few nanometres, are detected by measuring the deflection of a laser beam from the back of the cantilever. Amra can operate the AFM in 'contact mode' for hard surfaces such as semiconductors or 'tapping mode' for soft samples such as food or biological samples. Our AFM is also capable of "lateral force" measurements, which is a way of measuring the local coefficient of friction over microscopic distances.

Data: With the Atomic Force Microscope, one can detect volcano-like cones on the surface of chocolate. These cones develop with time and is an interesting feature of fat bloom formation, a complex phenomenon whose origins remain somewhat murky. Image courtesy of Dr. D. Rousseau.

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Acoustic Microscope: A student uses the acoustic microscope as a tool for studying the internal structure of cells using high frequency ultrasound (100-1000 MHz). The biomechanical properties (speed of sound, density, attenuation, bulk modulus) of cells can be determined from the ultrasound echoes from within the cell, and these properties can be measured as the cells undergo specific cellular functions such as apoptosis (programmed cell death).

Data: Shown is a combined optical image acquired with white light (left) and an acoustic image (right) of two MCF-7 breast cancer cells. The acoustic image was measured using a 400 MHz transducer, and shows ultrasound echoes back-scattered from regions within the cell. The scale shown is the same for both images. Image courtesy of Drs. M. Kolios and E. Strohm.

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Gel Documentation System: There are several gel documentation systems at Ryerson. Here, Zafar is using a Kodak Gel Logic 1500 Imaging System equipped with the Kodak Molecular Imaging software. Among other things, it permits visualization and acquisition of digital images of ethidium bromide-labeled DNA molecules separated by electrophoresis using agarose gels.
	Data: This ethidium bromide-stained gel shows the separation of amplified DNA molecules of several types of small Ras GTPases produced by reverse-transcription PCR. Image courtesy of Dr. R. Botelho.

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Scanning Electron Microscope: This JEOL Scanning Electron Microscope (SEM) permits scanning of surfaces with electron beams to generate very high magnification and resolution of various types of Materials. Eric uses the SEM to obtain high-resolution images of silver sulfide crystals.
	Data: This is a SEM image of a daguerrotype photograph display a silver sulfide crystal. A daguerrotype are the very first photographs taken. The crystals form from the reaction of the silver metal with sulphur from the air and crystallize over time. Image courtesy of Mike Robinson.

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Analytical Instrumentation

High Performance Liquid Chromatography System: Sossina prepares to use the HPLC. Our system is equipped with an autosampler, computerized 4-solvent delivery system, and column heater, with detector systems based on UV-Visible absorption, refractive index or conductivity. HPLC separates compounds according to properties such as size and charge with very high resolutions.
	Data Chromatogram: The HPLC performs analysis by separating components in a mixture. The area under the peaks in the chromatogram is proportional to the amounts of each component. This chromatogram shows that the mixture being analyzed contains two stereoisomers in a 79:21 ratio (i.e. a 58% excess of one isomer over the other). Courtesy of Dr. R Viire.

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An Inert Atmosphere for Synthetic Chemistry: Gloveboxes are an important tool for the synthetic inorganic, organic, organometallic, polymer and materials chemistry laboratories here at Ryerson. They provide an inert nitrogen atmosphere, which allows for the preparation, recrystallization, handling and storage of air and moisture sensitive materials including pyrophoric solids. Salma has access to a low temperature fridge, balances, and an advanced solvent removal system all located within the Glovebox.

Data: The synthetic inorganic and coordination chemistry laboratories use modern instrumentation to characterise newly synthesized compounds for catalysis, chemotherapy and materials science. This tri-nuclear Nickel complex is being studied for its unusal magnetic and thermodynamic properties. ORTEP image courtesy of R. A. Gossage.

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400 MHz Nuclear Magnetic Resonance Spectrometer: Salma operates the NMR spectrometer to analyze a new compound she has synthesized in the lab. The NMR places a sample inside a very strong magnetic field and then very sensitively detects the subtle effects of this field on the nucleus of nearly every atom in a molecule, allowing the user to very accurately determine the structure of the molecules in their sample. Our spectrometer has a multinuclear broadband probe with digital automated tuning and a variable temperature unit for acquisition of data at any temperature between -150 �C and +180 �C.

Data: Pictured at right is a 2-dimensional NMR spectrum called a COSY (short for Correlation Spectroscopy). In this spectrum, the 1-D spectrum of all hydrogen atoms in the molecule being analyzed is laid across both axes. Any 'correlations' (spots on the graph) that do not lie on the diagonal line indicate that the hydrogen atom giving rise to a signal on one axis is 'coupled' (attached through two or three bonds) to the hydrogen atom causing the corresponding signal on the other axis. Using COSY, and a suite of other 1- and 2-dimensional techniques, we routinely determine and verify the structures of new molecules made in the synthetic labs. Image provided by Dr. R. Viirre.

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Mass Spectrometers: Molecular Science have access to several high-end mass spectrometers including this new ESI-MS/MS spectrometer that is in the process of being set up. With it, Amra can identify proteins based on fragmentation and ionization of peptides by measuring their exact mass/ion ratio and matching those values to expected amino acid composition and sequence.
	Data: The raw MS data can be analyzed with a breadth of bioinformatics tool to identify unknown proteins in a biological sample. Above is the identification of Angiotensinogen found in human serum using a LC-ESI ion trap MS/MS spectra. Image is courtesy of Monika Tucholska and Dr. J. Marshall.

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Environmental and Growth Set-ups

Cell Culture and CO2 incubators: David uses the biosafety cabinet to culture mammalian macrophage cell lines. The Biosafety cabinet protects the user and minimizes contamination of cells during handling. Our Cell Culture Facility is also equipped with several CO2-jacketed incubators for cell culture and is licensed to handle Level 2 Biohazards.

Data: The Cell Culture Facility allows molecular and cell biology researchers to manipulate cells. This includes introduction into cells of DNA vectors (Transfection) expressing proteins such as this green fluorescent protein-chimera called GFP-Akt-PH, a protein probe that specifically detects a major signaling lipid called phosphatdiylinositol-3,4,5-trisphosphate. Image provided by Dr. R. Botelho.

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Mesocosm: Joseph measures soil temperature in a plant growth experiment. Our facility includes freshwater and terrestrial mesocosms. Our mesocosms allow us to mimick many of the aspects of natural systems, while giving us greater experimental control than is possible in field experiments. Mesocosms are currently used to study contaminant impacts on plants and animals, run-off impacts on aquatic communities, and ecosystem function.

Data: Terrestrial mesocosms are being used in plant growth experiments to study potential impacts of soil contaminants. The availability of mesocosms allows us to run replicated experiments comparing reference soil conditions against soil amended with a study contaminant. Here, Phaseolus vulgaris, the common bean, is being grown under reference conditions. Image provided by Dr. R. Botelho.

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