Microscopy as an analytical tool

Dr Imran

Wavelength and resolution

Resolution (r) =  λ/(2NA)

R= resolutionλ = wavelength of light used for imagingNA = numerical aperture of the lense

Manipulate Refractive index to increase contrast

Specimen and medium………By using stainsAt higher magnification…Use of oil keep the magnification and contrast

Uses of light microscopeEffects 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

Bright Dark Phase 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 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

• Anything @0.2 µm or below can not be resolved 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