Microscopy Flashcards
What is resolution?
The ability to distinguish to objects from each other
What is resolution limit (D)?
The smallest distance between two points on a specimen that can be distinguished as two separate entities.
How is resolution increased?
To increase the refractive index
Decreasing the wavelength
Increasing the angular aperture
What is the practical limit of resolution of human eye?
0.2mm
What is the practical limit of resolution of light microscope and fluorescence microscope?
Light microscope - 200nm
Fluorescence microscope - 100nm
What is the greatest magnification light microscope can provide?
1000x
With immersion oil 1400x
Give example of immersion oil
Paraffin oil and cedarwood oil
Compound light microscope works on what principle?
Amplitude contrast
Dark field microscopy uses a central circular disk for what?
To remove the unscathered light
What kind of specimen is observed in a dark field microscope?
Transparent, unstained
What kind of cells are observed in dark field microscope?
Sperms and flagella
Is dark field microscope useful for low resolution?
Yes
What kind of image do you get in phase contrast microscope?
Black and white
How does phase contrast microscope work?
Deduce the difference in refractive index and thickness of the sample
What kind of samples are observed in phase contrast microscope?
Live unstained cell
What is the better version of phase contrast microscope and how is it better?
Differential interference contrast microscope is better because it shows live 3D cells
What is the light source for fluorescence microscope?
The specimen
What is the use of excitation filter in fluorescence microscope?
Helps in providing excitation light to the sample
How is fluorescence microscope helpful?
Provides high contrast, specificity, live cell imaging and quantitative analysis
Name the dyes used in fluorescence microscope
EtBr
DAPI
Phalloidin peptide
Fluorescein
Rhodamine
Why is phalloidin peptide used?
To stain actin filament
What kind of light do confocal microscope blocks?
Out of focus light
What is the use of confocal microscope?
It provides a 3D image by collecting serial section from thick specimen
How does electron microscope has high resolution?
Because the wavelength of electrons are very short and uses electromagnetic lenses.
Name the two types of electron microscope?
Scanning electron microscope
Transmission electron microscope
Which electron microscope forms a 3D image?
Scanning electron microscope
Why does scanning electron microscope uses coating of metal?
To increase the solution by increase in the conductivity
How is transmission electron microscope helpful?
It forms a 2D image of internal structure of fixed cell.
Which kind of microscopy helps in observation of plasma membrane?
Freeze fracturing microscopy
Maximum value of angular aperture.
70°
Hence sin a is 0.94
In freeze fracture electron microscopy, what is used to freeze cells?
Liquid nitrogen
What is a cryoprotectant and what is its use?
It helps to prevent distortion from ice crystal formation
Give example of cryoprotectant
Glycerol
