Microscopy

Microscopy

Magnification, Contrast, Resolution

Light Microscopy (200 nm)Brightfield (To visualize structures → stain with dyes, can only be used on fixed (dead) cells)Fluorescent (Fluorescent dyes “glow” against dark background, Cells may be living or dead)Advanced:

• Phase contrast (Permits observation of transparent living cells) (standard phase contrast and differential interference contrast)

• Confocal (gives 3D image)

Electron Microscopy (1 nm)Transmission (TEM)Scanning (SEM)

The optical microscope

Specimen preparation for brightfield microscopy

Most cells are colorless or transparent. Tissues are sectioned.

Therefore fixing and staining of cells is often nessecary for brightfield imaging

Fluorescent microscopy

Permits localization of specific cellular moleculesFluorescent dyes “glow” against dark backgroundDye may be indirectly or directly associated with the cellular moleculeMultiple fluorescent dyes may be used simultaneouslyCells may be fixed or living

Immunofluorescense microscopy3T

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DAPI Anti-ArxesAnti-Calnexin(ER) Merge

DAPI Anti-ArxesAnti-Pparγ Merge

bar: 10µm

Advanced light microscopy –phase contrast

Permits observation of transparent living cellsLight phase shifts induced by specimen are used to generate contrast

Phase contrast (refracted and unrefracted light)Differential interference contrast (two light beams)

Brightfield, DIC, and PC image of the same cells

Advanced light microscopy – confocal imaging

Confocal Scanning MicroscopyGenerates focused images of living cells(one optical section per image)

Can look inside thick specimens (eggs, embryos, tissues)

Advanced light microscopy – confocal imaging

Several (confocal) optical sections can be deconvoluted into one sharp 3D image

Electron microscopy

-Transmission electron microscopy (TEM)

-Scanning electron microscopy (SEM)

Operates in vacuumSpecimen usually fixed, embedded, sectioned, and stained with an electron-dense material

Special techniques:Metal shadowing: visualize surface structures, cell componentsCryoelectron: visualize unfixed, unstained samples

Scanning coils move the beam across the sample

Electron microscopyCan visualize surfaces of tissues, cells, isolated cell parts Specimen is fixed and can additionally be coated with thin layer of heavy metal (metal shadowing)Images secondary electrons, resolution = 10 nm

TEM generates 2D image of electron dense structures

SEMgenerates 3Dimage