BLG 311 Section 2 - Visualizing Cells - CHAPTER 9 -...

This preview shows page 1 - 14 out of 94 pages.

Visualizing cells and beyond
VISUALISINGCELLSCHAPTER 9:579-598; 604-6112
Section 2: VISUALIZING CELLS1.Imaging tools needed to observe cells2.Light microscopy- Resolution, magnification, detection, numericalaperture- Typesof light microscopy (Bright Field, DarkField, Phase-contrast, DIC)- Fixation, embedding, sectioning- Staining with Dyes- Digital Image Processing3. Fluorescence Microscopy-Deconvolution and confocal microscopy- Immunocytochemistry- Fluorescent Proteins: GFP and others and relatedtech. (dynamics, photoactivation, FRAP)- Fluorescent Probes/Indicators4. Electron Microscopy- Transmission EM (incl. sample processing, immuno EM)- Scanning EM- Metal showdowing- Deep etching- Negative staining- Cryoelectron microscopy (incl. computer reconstruction)3
ells are too small for our eyes to resolvA typical animal cell is10-100m in diameterHuman eye can onlyresolve >100mparticlesThus the need forinstruments thatmagnify and resolve:microscopes4
5Light Microscopy and Resolution
Figure 9-3aMolecular Biology of the Cell(© Garland Science 2008)The Compound Light Microscope6Visualizing CellsWhite light illuminates specimenCondenserfocus light onto specimenCompound objectives(objective lens & ocularlens)magnify the image of the specimen bybending lightMagnified specimen image is focused onto thehuman eyeLight rays travel as a waveLight rays are bent by entering and leavingdifferent media: water, glass, airFirst lensssecond len
7Magnification, Detection andResolutionMagnify 15x: increase in size withincreased resolutionMagnify 15x:increaseinsize butpoor resolutionDetect:BUT poorresolution orseparation of detailBlow up image verygrainyNice pic
8Magnification, Detection andResolutionGood detectionPoor detection
Light microscopes have aresolution limitLight microscopesmagnify and increase the resolutionof imagesBUT, only particles > 0.2m apart are resolved as individual particlesParticles that are < 0.2m apart cannot be “separated” orresolved– they willappear as one particle.This is theresolution limit.9200 nm
10The Light MicroscopeDetection vs. Magnification vs.ResolutionResolution: “visual” separation of the individualcomponents of an object, which previously appeared asone; quality of the image.Detection: able to visualize light signals from objectsmuch smaller than 0.2m, including a single molecule.However, objects appear like they are 0.2m in size (dueto diffraction).Magnification: apparent “visual” increase in the size ofan object. There is no limit to magnification.Resolution limit: is reached when additionalmagnification does not separate further detail. The resultis simply an enlarged blurry image.
The Light Microscope: basic setup
Figure 9-4Molecular Biology of the Cell(© Garland Science 2008)12Why is there a resolution limit?
Diffraction of light: sometimes hard to understand,but there’s a silver lining (literally)See:Diffraction of light:Water dropletsin a cloud

Upload your study docs or become a

Course Hero member to access this document

Upload your study docs or become a

Course Hero member to access this document

End of preview. Want to read all 94 pages?

Upload your study docs or become a

Course Hero member to access this document

Term
Winter
Professor
N/A
Tags
Biology, cell biology, molecular biology, Notes, Chapter 9, Cell, Filled Powerpoint With Notes, BLG 311, Garland Science, Molecular Biology of the Cell

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture

  • Left Quote Icon

    Student Picture