STRUCTURED ILLUMINATION MICROSCOPY (SIM) Karen Porter … · 2018-04-19 · Structured Illumination Microscopy (SIM) casts a structured light pattern onto the sample. The illumination

Super-resolution Imaging of Frustrated PhagocytosisSTRUCTURED ILLUMINATION

MICROSCOPY (SIM)Karen Porter-Davis

STEP-UP Program 2014

PHAGOCYTOSIS

Fig. 1: Phagocytosis video from http://academic.brooklyn.cuny.edu/biology/bio4fv/page/phago.htm

Fig. 2: Diagram of the IC binding with the Fc-Receptors on the Phagocyte. Image courtesy of the Curtis Group.

Immune Cell

ACTIN

• A protein found in many forms and has many functions within animal cells.

• F-actin = Filament actin (forms microfilaments)

• Actin that makes up the cytoskeleton:

1) stabilizes cell shape

2) assist in cell movement (whole cell or parts of the cell)

FRUSTRATED PHAGOCYTOSIS

Fig. 3: Macrophage undergoing frustrated phagocytosis; labeled actin cytoskeleton with phalloidin (FITC), cell membrane with WGA633 and the nucleus with DAPI.Imaged with anOlympus Confocal Microscope –Georgia Institute of Technology 2014

RESOLUTION• The goal of any

microscope is to improve resolution

• Resolution: ability of an optical instrument to resolve detail in the object that is being imaged. It depends on wavelength and aperture width. Fig. 4 : Demonstrates how aperture size and wavelength

affect the diffraction of light. http://www.imagen-estilo.com/Articles/Photography-basics/lens-diffraction-

limit.html

DIFFRACTION LIMIT• The resolution of

an optical instrument can be limited by factors such as flaws in the lenses or misalignment.

• There is a max to the resolution of any optical instrument due to diffraction (~ 200 nm).

Fig. 5: Diagram of Diffraction Limit. Image from http://www.xenophilia.com/zb0012a.htm

Fig. 6: image from http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html

Fig. 7: Comparison of actin imaging from conventional microscopy and SIMImage from UC Davis: http://cbst.ucdavis.edu/research/summaries/optical-microscope-experimental-omx 6/6/2014

Structured Illumination Microscopy (SIM) casts a structured light pattern onto the sample.

The illumination pattern interacts with the fluorescent labeled structures in the sample which create interference patterns know as moiré fringes.

By changing the illumination pattern, collecting and reconstructing the subsequent images, super-resolution images lateral (x, y) resolution ~100 nm (compared to ~200 nm) and axial (z) resolution ~ 300 nm.

Fig. 8 : Depiction of the rotational illumination pattern of SIM; Image from “Super-resolution Technology.”

[6]

Fig. 10: RAW264.7 Phagocytosing IgG-opsonized red blood cell (green). Actin (red) concentrated in region of contraction. [Swanson et al. J. Cell Sci. 112, 307-316 (1999)]Fig. 11: Drawing of the point of constriction with a dense clustering of actin during phagocytosis. Drawing courtesy of Dan Kovari of the Curtis Group, Georgia Institute of Technology.

Proposed Contractile Forces Created During Phagocytosis

Fig. 13: Macrophage undergoing frustrated phagocytosis; labeled actin cytoskeleton with phalloidin (FITC) and the nucleus with DAPI. Pre-parallel bundling of the actin fibers around perimeter. Images created by K. Porter-Davis at Georgia Tech Microscopy Lab Summer 2014.

Fig. 14: Macrophage undergoing frustrated phagocytosis; labeled actin cytoskeleton with phalloidin (FITC) and the nucleus with DAPI. Parallel banding around the perimeter and tethering can be seen. Images created by K. Porter-Davis at Georgia Tech Microscopy Lab Summer 2014.

Fig. 15: Macrophage undergoing frustrated phagocytosis; labeled actin cytoskeleton with phalloidin (FITC) and the nucleus with DAPI. Parallel banding around the perimeter and tethering can be seen. Images created by K. Porter-Davis at Georgia Tech Microscopy Lab Summer 2014.

RESULTS AND FUTURE WORK

• The actin banding seen in our images lends support to the proposed mechanism of the constriction around the target particle.

• Effectively label myosin II and the cell membrane to further understand the mechanics behind phagocytosis.

UP-DATED LESSON PLAN• Expanded Interference and Diffraction of

Light Lesson Plan from last summer

• Added in:

• Diffraction and Optical Instruments PowerPoint

• Microscope Internet Scavenger Hunt

• Morie’ Pattern Demo

• Interference S.T.E.A.M project

• Microscope Background Information

ACKNOWLEDGEMENTS

• Thank you for the guidance and support:

• Dr. Leyla Conrad and the STEP-UP Program participants

• Dr. Jennifer Curtis

• Dan Kovari

• Andrew Shaw

• Curtis and Payne Group members

REFERENCES• Cooper, G.M. The Cell: A Molecular Approach. Sunderland, MA: Sinauer Associations, 2000. Web. 22 July 2014.• “DAPI Nuclear Counterstain.” Pierce Protein Biological Product. Thermo Scientific. 2014. Web. 23 July 2014.• “History of the Microscope”. Vision Engineering. n.p.n.d. pg. 1-7. Web. 16 July 2014. <

http://www.visioneng.com/resources/history-of-the-microscope#>• “Introduction to Super resolution Microscopy”. Education in Microscopy and Digital Imaging. Carl Zeiss and

Molecular Expressions. Web. 06 June 2014 http://zeiss-campus.magnet.fsu.edu/articles/superresolution/introduction.html

• Lodish, Harvey, et al. Molecular Cell Biology. New York: W.H. Freeman and Company, 2008. Print.• “Mechanics and Signaling in Phagocytosis.” The Curtis Group: Cell Physics Laboratory. Georgia Institute of

Technology. n.d. Web. 22 July 2014.• Purves, William K., David Sadava, Gordan H. Orians, and H. Craig Heller. Life: The Science of Biology.

Sunderland, MA: Sinauer Associations, 2004. Print.• Silfies, Joel S.; Schwartz, Stanley A.; Davidson, Michael W. “The Diffraction Barrier in Optical Microscopy”,

Microscopy U: Nikon. Copyright 2000-2013. Web. 16 July 2014. http://www.microscopyu.com/articles/superresolution/diffractionbarrier.html

• Silfies, Joel S.; Schwartz, Stanley A.; Martini, Sunita; Price, Stephen P., Coker, Alex B.; Davidson, Michael W. “Fundamental Concepts in Super-resolution Microscopy”, Microscopy U: Nikon. Copyright 2000-2013. Web. 16 July 2014. http://www.microscopyu.com/articles/superresolution/index.html

• Spring, Kenneth R.; Davidson, Michael W. “Introduction Fluorescence Microscopy”, Microscopy U: Nikon. Copyright 2000-2013. Web. 16 July 2014. http://www.microscopyu.com/articles/fluorescence/fluorescenceintro.html

• “Super-resolution Technology.” Applied Precision. GE Healthcare Company. 2014. Web. 05 June 2014. http://api.gehealthcare.com/api/super-res-tech.asp

• Swanson, Joel A., Hoppe, Adam D. “The Coordination of Signaling during Fc Receptor-Mediated Phagocytosis.” Journal of Leukocyte Biology Volume 76, December 2004: 1093. Web. 22 July 2014.