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Basics of Image Acquisition

This presentation explores the fundamentals of image acquisition in cellular biology, focusing on the notable HeLa and 3T3 cell lines. It discusses the challenge of visualizing cells due to their high water content and introduces staining techniques that enhance visibility through specific dyes. Key methods such as Central Dogma-tagging using Green Fluorescent Protein (GFP) are covered. The presentation also compares different types of confocal microscopy, including Confocal Scanning Laser Microscopy (CSLM) and Spinning Disk Confocal Microscopy, highlighting their resolution, acquisition speed, and application in three-dimensional imaging.

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Basics of Image Acquisition

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  1. Basics of Image Acquisition Elvira Garcia Osuna Tad Merryman ITR Meeting 7 October 2004

  2. Cell Types • HeLa • Cells from Henrietta Lacks • Died in 1951 of cervical cancer • 3T3 • Cells from mouse embryo

  3. Cells are mostly water Since cells are about 70% water, there is nothing to impede light rays. For the most part cells are almost invisible in an ordinary light microscope. So what can we do to see them? Staining

  4. Staining • Dyes show a preference for particular parts of the cell • These dyes make the internal structures visible • For example, Hematoxylin has an affinity for negatively charged molecules (DNA, RNA, acidic proteins) • But dyes have relatively low specificity Central Dogma-tagging

  5. Central Dogma of Biology DNA exon intron exon intron transcription RNA translation PROTEIN

  6. Central-Dogma (CD) Tagging • Insertion of a CD-cassette into an intron • CD-cassette is a specially designed sequence DNA exon intron exon intron guest exon transcription RNA translation PROTEIN We use Green Fluorescent Protein (GFP)

  7. Types of Confocal Microscopy • Confocal Scanning Laser Microscopy (CSLM) • Spinning Disk Confocal Microscopy

  8. Confocal Scanning Laser Microscopy • Point by point acquisition • High resolution • Precise Control of capture locations • Slow acquisition

  9. Confocal Scanning Laser Microscopy

  10. Confocal Scanning Laser Microscopy

  11. Spinning Disk Confocal Microscopy • Fast acquisition • Lower resolution • Little control of acquisition locations

  12. Spinning Disk Confocal Microscopy

  13. Spinning Disk Confocal Microscopy

  14. Our Data Sets • 3D HeLa • Confocal Scanning Laser Microscope (100x) • DNA stain, all protein stain and fluorescent anti-body for a specific protein • 3D 3T3 • Spinning Disk Confocal Microscope (60x) • GFP for a specific protein

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