Molecular Cell Biology

Light Microscopy in Cell BiologyCooper

Modified from a 2010 lecture byRichard McIntosh, University of Colorado

Images from a light microscope can be strikingly informative about cells

How are these images made? What questions can they answer?What are their limitations? Can you make and use them?

Scales of absolute size: powers of 10

Wavelength sets limitson what one can see

Light behaves as a Wave

Resolution = 0.61 x wavelength of light NA (numerical aperture)

The effect of NAon the image ofa point.

The need forseparation to allow resolution

θθ

θ

Lower limits on spatial resolution aredefined by the Rayleigh Criterion

NA = nsinθn = refractive index of the mediumθ = semi-angle of an objective lens

Contrast in the Image is Necessary:Types of Optical Microscopy Generate Contrast

in Different Ways

•Bright field - a conventional light microscope•DIC (Differential Interference Contrast -

Nomarski)•Phase contrast•Fluorescence•Polarization•Dark field

Bright-field Optics: Light Passing Straight Through the Sample

•Most living cells are optically clear, so stains are essential to get bright field contrast

•Preserving cell structure during staining and subsequent observation is essential, so cells must be treated with “fixatives” that make them stable

•Fixing and staining is an art

Classic drawings and modern images made from Giemsa-stained blood smears

Plasmodium falciparum Histidine-rich Protein-2

Generating Contrast

• Staining• Coefficients of absorption among different materials

differ by >10,000, so contrast can be big• Without staining

• Everything is bright• Most biological macromolecules do not absorb visible

light• Contrast depends on small differences between big

numbers• Need an optical trick

Mammalian Cell: Bright-field and Phase-contrast Optics

Principles of bright fieldand phase contrast optics

Differential Interference Contrast (DIC)

•Optical trick to visualize the interference between two parts of a light beam that pass through adjacent regions of the specimen

•Small amounts of contrast can be expanded electronically

•Lots of light: Video camera with low brightness & high gain

Brightfield vs DIC

DIC has shallow depth-of-field:Image a single plane in a large object

QuickTime™ and aCinepak decompressor

are needed to see this picture.

Worm embryo

DIC: Good contrast. Detection vs Resolution.Microtubules: 25 nm diameter (1/10 res.lim.) but visible in DIC

Fluorescent staining:High signal-to-noise ratio (white on black)

Principle of Fluorescence

• Absorption of high-energy (low wavelength) photon

• Loss of electronic energy (vibration)

• Emission of lower-energy (higher wavelength) photon

Design of a Fluorescence Microscope

QuickTime™ and aCinepak decompressor

are needed to see this picture.

Fluorescent tubulin injected into aDrosophila embryo, plus a DNA stain

Green Fluorescent Protein - Considerations

• Color - Not just green• Brightness • Time for folding• Time to bleaching

QuickTime™ and aCinepak decompressor

are needed to see this picture.

Live-cell Imaging of Microtubule Ends:EB1-GFP chimera

QuickTime™ and aCinepak decompressor

are needed to see this picture.

GFP-Cadherin in cultured epithelial cells

Immunofluorescence

•Primary Abs recognize the antigen (Ag)•Secondary Abs recognize the primary Ab•Secondary Abs are labeled

Immunofluorescence Example

•Ab to tubulin•Ab to kinetochore

proteins•DNA stain (DAPI)

Biological microscopy problem: Cells are 3D objects, and pictures are 2D images.

•Single cells are thicker than the wavelength of visible light, so they must be visualized with many “optical sections”

•In an image of one section, one must remove light from other sections

•Achieving a narrow “depth-of-field”•A “confocal light microscope”

Laser-Scanning Confocal Light

Microscopy

• Laser thru pinhole• Illuminates sample with

tiny spot of light• Scan the spot over the

sample• Pinhole in front of

detector: Receive only light emitted from the spot

Light from points that are in focus versus out of focus

Spinning-disk confocal microscopy:Higher speed and sensitivity

Example: Confocal imaging lessensblur from out-of-focus light

Optically Sectioning a Thick Sample: Pollen Grain

Multiple optical sections assembled to form a 3D image

QuickTime™ and aCinepak decompressor

are needed to see this picture.

3D Image Reconstructed From Serial Optical Sections Obtained with a Confocal Microscope

QuickTime™ and aCinepak decompressor

are needed to see this picture.

Fluorescence can Measure Concentration of Ca2+ Ions in Cells:

Sea Urchin egg fertilization

Phase Contrast Fluorescence

Summary

•Light microscopy provides sufficient resolution to observe events that occur inside cells

•Since light passes though water, it can be used to look at live as well as fixed material

•Phase contrast and DIC optics: Good contrast•Fluorescence optics: Defined molecules can be

localized within cells•“Vital” fluorescent stains: Watch particular

molecular species in live cells

End