Microscopy as an analytical tool Dr Imran. Wavelength and resolution Resolution (r) = λ / (2NA) R= resolution λ = wavelength of light used for imaging.

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Presentation transcript:

Microscopy as an analytical tool Dr Imran

Wavelength and resolution Resolution (r) = λ / (2NA) R= resolution λ = wavelength of light used for imaging NA = numerical aperture of the lense

Manipulate Refractive index to increase contrast Specimen and medium………By using stains At higher magnification… Use of oil keep the magnification and contrast

Uses of light microscope Effects of nitrate, phosphate and salinity stress on cell division, chloroplast morphology and cell wall architecture in a filamentous green alga Spirogyra punctulata Jao Gram + and Gram -

When you can not stain your sample BrightDarkPhase contrast

Fluorescense microscopy Absorbs shorter WL and emits at longer WL Natural fluorescence…..image as such Fluorochromes….Auramine O Yellow fluorescence..TB fluorescein isothiocyanate….green Immunofluorescence

GABAAγ2 molgra Anti-GABAAγ2 20x Confocal image

What are Z axis in confocal? What is optical sectioning? Why blurred image is produced in florescent microscope and how confocal help to get a better resolution?

Two Photon microscopy Staining same as for confocal….but uses infrared beam instead of blue…so two photons needed to exit the fluorochrome hence TFM Live sample can be seen 1mm depth can be imaged Sample damage is minimized

Energy, wavelength, frequency

EM With a best light microscope…maximum magnification…….2000X µm or below can not be r e solved with light microscope. Why? How we see SEM (Scanning Electron Microscope) TEM (Transmission Electron Microscope)

The Cu grid, and EM micrograph

Resolution and magnification 2.5 nm 10,000 to 100,000X Contrast is created with…..using stains which absorbs electron and appear darker Stains….Uranil acetate, Osmimum, tungsten, lead, molybdate etc. Staining is of two types…positive and negative stain

Limitations Less penetration…..100 nm Many samples can not be studied in 3D Fixed, dehydrated, high vacuum, kills samples… Artifacts…..

TEM use to analyze protein structure