Lecture 2: Microscopy Flashcards
General size the human eye can detect
0.1mm/100 micrometers
Range of resolution for the human eye
100-200 micrometers
Range of resolution for light microscopy (Conventional Light Microscopy)
200 nm
Range of resolution for light microscopy (Super-Resolution)
50 nm
Atomic Force Microscopy
Enables visualization of untreated cells by detecting Van der Waals forces. SCANS THE SURFACE
Fluorescence microscopy overlap zone
Region where absorption and fluorescence overlap preventing a clear image
Ways to improve contrast in light microscopy
Staining, fluorescence, dark-field, and phase-contrast
Sample Requirement for Scanning Electron Microscopy
Sample must be dried and cells need to be fixed
Resolution
The smallest distance between 2 objects that allows separation (How clearly we can see an image)
Magnification
The increasing of an objects apparent dimensions
Atomic Force Microscopy Range of Resolution
1-2 Angstroms
Scanning Electron Microscopy Range of Resolution
2 nm
X-Ray Crystallography Range of Resolution
1-2 Angstroms
Transmission Electron Microscopy Range of Resolution
2 nm
Cons of light microscopy
Poor contrast due to transparency of bacterial cells
Cryo-electron microscopy resolution
1-2 Angstroms
Consequence of higher frequency light in microscopy
Higher energy causing higher resolution
What bright field microscopy image quality depends on
Wavelength, magnification power, and focus
Numerical Aperture
Value directly linked to better resolution (High NA = Low R= High Resolution) (High NA = Low R)
Numerical Aperture Formula
NA = n*sinθ
n=refractive index (medium/solution holding the sample)
Consequence of a large n value on resolution
Large n values increase NA values causing a smaller R value. Causes an overall better resolution.
Gram positive bacteria
Single membrane & thick cell wall containing peptidoglycan
Gram negative bacteria
Inner and outer membranes with thin cell wall
Gram staining result in gram-negative cells
Safranin counterstains and binds to nucleic acid causing pink appearance in cells.
Gram staining result in gram-positive cells
Absorbs safranin but remains dark purple
Acid fast stain
Carbolfuchsin used to stain mycobacterium
Endospore staining
Malachite green specifically binds to an endospores coat
Process of fluorescence microscopy
-Light excites electrons
-Electrons move to more stable high energy state
-Electron begins to drop to a lower orbital to release energy
-As electron drops fluorescence is released
Absorption and fluorescence terms
-Absorption/Excitation
-Fluorescence/Emission
Benefits of fluorescence microscopy
-Can study bacterial cells inside a complex such as biofilms
-Can be used to study bacterial motion
Considerations for choosing fluorescence microscopy
-Excitation and emission
-Orthogonality between fluorescent proteins (ability for proteins to be used simultaneously in the same system without interference)
-Monomeric structure (Easier to experiment with, higher resolution, monomeric proteins are less likely to disrupt the cellular function of what is being observed)
-Maturation time (Events or functions that occur fast enough may not be observable since fluorescence takes 10-15 mins)
-Brightness (Visibility)
-Autofluorescence (Some cells have weak fluorescent ranges)
Single-Molecule localization in light microscopy
Approximation of location in order to calculate the best probability of fluoroform
Single-molecule tracking in light microscopy
Tracks consecutive images of the same molecules in order to gain unique frames to reconstruct a path
Dark field microscopy
Central aperture above the light source allowing visibility of cellular structures
Cryo-electron microscopy
Rapid freezing of samples to preserve native structure
Sample requirement for transmission electron microscopy
An electron dense negative stain from salt or heavy metal
Benefit of scanning electron microscopy
Effective for visualizing cells in complex communities such as biofilms
Benefit of cryo-electron microscopy
Maintains native structure of protein
