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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 on theme: "P. Moghe, 125:583 1 Microscopy Techniques for Biomaterials and Cell Based Interfaces Professor Prabhas V. Moghe October 26, 2006 125:583 Fall 2006."— Presentation transcript:

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

2 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)

3 P. Moghe, 125:583 3

4 4 Principle of Compound Light Microscopy

5 P. Moghe, 125:583 5 Modern Day Microscope

6 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

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

8 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.

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

10 P. Moghe, 125:583 10 Bright Field Microscopy

11 P. Moghe, 125:583 11 Dark Field Microscopy

12 P. Moghe, 125:583 12 Principle of Phase Contrast Microscopy Zernicke: Greatest advance in Microscopy (1953) Phase microscopy requires phase objectives and a phase condensor.

13 P. Moghe, 125:583 13 Phase Contrast Microscopy

14 P. Moghe, 125:583 14 Polarized Light Microscopy

15 P. Moghe, 125:583 15 Differential Interference Contrast 3-D like appearance DIC polarizer and prisms required; Individual prisms required for each objective. (Relatively expensive)

16 P. Moghe, 125:583 16 DIC Set-up

17 P. Moghe, 125:583 17 Differential Interference Contrast

18 P. Moghe, 125:583 18 Fluorescence Microscopy: Principle of Fluorescence

19 P. Moghe, 125:583 19 Fluorescence Microscopy

20 P. Moghe, 125:583 20 Fluorescence Microscope Mercury Light Source Exciter Filter Dichroic Mirror Barrier Filter Objective/ Condensor Specimen Exploded View of a Filter Cube

21 P. Moghe, 125:583 21 Immunofluorescence

22 P. Moghe, 125:583 22 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

23 P. Moghe, 125:583 23 Principle of Confocal Optical Microscopy illumination & detection apertures focus abovebelow lens

24 P. Moghe, 125:583 24 Schematic of Confocal Microscopy

25 P. Moghe, 125:583 25 Confocal Imaging for 3-D Projection of Cells in Scaffolds Moghe, Treiser, Kohn (unpublished) Copyright: P. Moghe et al.

26 P. Moghe, 125:583 26 Confocal Image of Cell- Assembled Matrix Proteins Moghe et al., Unpublished

27 P. Moghe, 125:583 27 Confocal Imaging of Cell Morphogenesis on Biomaterials: Case Study Chang, C.; Lieberman, S.; and Moghe, P.V. Biomaterials 20: 273-281 (1999)

28 P. Moghe, 125:583 28 Cell Morphogenesis via CLSM

29 P. Moghe, 125:583 29 Cell Morphometric Factors

30 P. Moghe, 125:583 30 Cell Morphology on Biomaterials Chang et al., Biomaterials, 20:273, 1999

31 P. Moghe, 125:583 31 Confocal Imaging of Cell 3D Morphology Chang et al., Biomaterials, 20:273, 1999

32 P. Moghe, 125:583 32 Integrated View of Cell Morphogenesis on Ligand/Biomaterials

33 P. Moghe, 125:583 33 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)

34 P. Moghe, 125:583 34 Semler et al., Biotechnol. Prog. 13: 630, 1997 3-D Biomaterial Topography Via Reflection CLSM Reflected light images At 4 um increments along Depth of PLAGA sponge Topographical reconstruction Of PLAGA surface profile

35 P. Moghe, 125:583 35 Quantitation of Texture Correlation

36 P. Moghe, 125:583 36 Single-photon vs Multiphoton Excitation Nature Biotech 21: 1370 (2003)

37 P. Moghe, 125:583 37 Schematic of MPM EOM: Electro-optical modulator (controls laser beam intensity) PMT: Photo-multiplier tube (detector)

38 P. Moghe, 125:583 38 Dye doped DTE - DTO Carbonate Blend: Multiphoton Image Courtesy: Drs. P. Johnson & P. Moghe

39 P. Moghe, 125:583 39 Dye doped DTE - DTO Carbonate Blend: 20X Multiphoton Image

40 P. Moghe, 125:583 40 Two-photon fluorescence and SHG

41 P. Moghe, 125:583 41 Second Harmonic Generation as a function of material polarizability (advanced material; not for review)

42 P. Moghe, 125:583 42 Extra slides

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