P. Moghe, 125:583 1 Microscopy Techniques for Biomaterials and Cell Based Interfaces Professor Prabhas V. Moghe October 26, 2006 125:583 Fall 2006.

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Presentation transcript:

P. Moghe, 125:583 1 Microscopy Techniques for Biomaterials and Cell Based Interfaces Professor Prabhas V. Moghe October 26, :583 Fall 2006

P. Moghe, 125:583 2 Outline Physics of Compound Light Microscopy Light Microscopy Modes Bright Field & Dark Field Phase Contrast Differential Interference Contrast Fluorescence Confocal Laser Scanning Mode Multiphoton Microscopy Applications Cell-Scaffold Imaging; Biomaterial Topography Cell Morphogenesis Analysis (Case Study)

P. Moghe, 125:583 3

4 Principle of Compound Light Microscopy

P. Moghe, 125:583 5 Modern Day Microscope

P. Moghe, 125:583 6 Physics of Optical Microscopy The ability of a microscope objective to "grasp" the various rays coming from each illuminated part of the specimen is related to the angular aperture of the objective. N.A. = n. sin (u); n= refractive index; u=1/2 subtended angle - Max theoretical N.A. of a dry objective is 1 - Max theoretical N.A. of oil immersion objectives is 1.5

P. Moghe, 125:583 7 Compound Microscopy: Optical Issues

P. Moghe, 125:583 8 Optical Microscopy Issues: Resolution Resolution is defined as the ability of an objective to separate clearly two points or details lying close together in the specimen. where R=resolution distance; , the wavelength of light used; N.A. = the numerical aperture. - As N.A. increases, resolution gets better (R smaller). - Longer wave lengths yield poorer resolution.

P. Moghe, 125:583 9 Bright and Dark Field Contrast

P. Moghe, 125: Bright Field Microscopy

P. Moghe, 125: Dark Field Microscopy

P. Moghe, 125: Principle of Phase Contrast Microscopy Zernicke: Greatest advance in Microscopy (1953) Phase microscopy requires phase objectives and a phase condensor.

P. Moghe, 125: Phase Contrast Microscopy

P. Moghe, 125: Polarized Light Microscopy

P. Moghe, 125: Differential Interference Contrast 3-D like appearance DIC polarizer and prisms required; Individual prisms required for each objective. (Relatively expensive)

P. Moghe, 125: DIC Set-up

P. Moghe, 125: Differential Interference Contrast

P. Moghe, 125: Fluorescence Microscopy: Principle of Fluorescence

P. Moghe, 125: Fluorescence Microscopy

P. Moghe, 125: Fluorescence Microscope Mercury Light Source Exciter Filter Dichroic Mirror Barrier Filter Objective/ Condensor Specimen Exploded View of a Filter Cube

P. Moghe, 125: Immunofluorescence

P. Moghe, 125: Confocal Fluorescence Microscopy Confocal Microscopy –Pin hole eliminates out of focus light Multiphoton Microscopy –Only focus plane is fluorescently excited –Increased penetration depth –Second Harmonic Generation Reflectance Allow for simultaneous scaffold/biomaterial and cell imaging

P. Moghe, 125: Principle of Confocal Optical Microscopy illumination & detection apertures focus abovebelow lens

P. Moghe, 125: Schematic of Confocal Microscopy

P. Moghe, 125: Confocal Imaging for 3-D Projection of Cells in Scaffolds Moghe, Treiser, Kohn (unpublished) Copyright: P. Moghe et al.

P. Moghe, 125: Confocal Image of Cell- Assembled Matrix Proteins Moghe et al., Unpublished

P. Moghe, 125: Confocal Imaging of Cell Morphogenesis on Biomaterials: Case Study Chang, C.; Lieberman, S.; and Moghe, P.V. Biomaterials 20: (1999)

P. Moghe, 125: Cell Morphogenesis via CLSM

P. Moghe, 125: Cell Morphometric Factors

P. Moghe, 125: Cell Morphology on Biomaterials Chang et al., Biomaterials, 20:273, 1999

P. Moghe, 125: Confocal Imaging of Cell 3D Morphology Chang et al., Biomaterials, 20:273, 1999

P. Moghe, 125: Integrated View of Cell Morphogenesis on Ligand/Biomaterials

P. Moghe, 125: Confocal Imaging Topographical Imaging of Biomaterials Via Reflection CLSM 3 um 10 um25 um Laser-scanning confocal microscopy using reflected light. Biodegradable materials fabricated to different pore sizes using freeze-drying of solvent dissolved polymer (PLAGA)

P. Moghe, 125: Semler et al., Biotechnol. Prog. 13: 630, D Biomaterial Topography Via Reflection CLSM Reflected light images At 4 um increments along Depth of PLAGA sponge Topographical reconstruction Of PLAGA surface profile

P. Moghe, 125: Quantitation of Texture Correlation

P. Moghe, 125: Single-photon vs Multiphoton Excitation Nature Biotech 21: 1370 (2003)

P. Moghe, 125: Schematic of MPM EOM: Electro-optical modulator (controls laser beam intensity) PMT: Photo-multiplier tube (detector)

P. Moghe, 125: Dye doped DTE - DTO Carbonate Blend: Multiphoton Image Courtesy: Drs. P. Johnson & P. Moghe

P. Moghe, 125: Dye doped DTE - DTO Carbonate Blend: 20X Multiphoton Image

P. Moghe, 125: Two-photon fluorescence and SHG

P. Moghe, 125: Second Harmonic Generation as a function of material polarizability (advanced material; not for review)

P. Moghe, 125: Extra slides