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
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
Confocal image
GABAAγ2
GABAAγ2
Anti-GABAAγ2
gra
20x
mol
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
e
• Anything @0.2 µm or below can not be r 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