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BSCI443: Microbial Physiology > Lecture 2: Microscopy > Flashcards

Lecture 2: Microscopy Flashcards

1
Q

General size the human eye can detect

A

0.1mm/100 micrometers

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2
Q

Range of resolution for the human eye

A

100-200 micrometers

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3
Q

Range of resolution for light microscopy (Conventional Light Microscopy)

A

200 nm

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4
Q

Range of resolution for light microscopy (Super-Resolution)

A

50 nm

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5
Q

Atomic Force Microscopy

A

Enables visualization of untreated cells by detecting Van der Waals forces. SCANS THE SURFACE

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5
Q

Fluorescence microscopy overlap zone

A

Region where absorption and fluorescence overlap preventing a clear image

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6
Q

Ways to improve contrast in light microscopy

A

Staining, fluorescence, dark-field, and phase-contrast

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7
Q

Sample Requirement for Scanning Electron Microscopy

A

Sample must be dried and cells need to be fixed

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8
Q

Resolution

A

The smallest distance between 2 objects that allows separation (How clearly we can see an image)

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8
Q

Magnification

A

The increasing of an objects apparent dimensions

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9
Q

Atomic Force Microscopy Range of Resolution

A

1-2 Angstroms

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9
Q

Scanning Electron Microscopy Range of Resolution

A

2 nm

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10
Q

X-Ray Crystallography Range of Resolution

A

1-2 Angstroms

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10
Q

Transmission Electron Microscopy Range of Resolution

A

2 nm

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11
Q

Cons of light microscopy

A

Poor contrast due to transparency of bacterial cells

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12
Q

Cryo-electron microscopy resolution

A

1-2 Angstroms

13
Q

Consequence of higher frequency light in microscopy

A

Higher energy causing higher resolution

14
Q

What bright field microscopy image quality depends on

A

Wavelength, magnification power, and focus

14
Q

Numerical Aperture

A

Value directly linked to better resolution (High NA = Low R= High Resolution) (High NA = Low R)

15
Q

Numerical Aperture Formula

A

NA = n*sinθ
n=refractive index (medium/solution holding the sample)

16
Q

Consequence of a large n value on resolution

A

Large n values increase NA values causing a smaller R value. Causes an overall better resolution.

17
Q

Gram positive bacteria

A

Single membrane & thick cell wall containing peptidoglycan

18
Q

Gram negative bacteria

A

Inner and outer membranes with thin cell wall

19
Q

Gram staining result in gram-negative cells

A

Safranin counterstains and binds to nucleic acid causing pink appearance in cells.

20
Q

Gram staining result in gram-positive cells

A

Absorbs safranin but remains dark purple

21
Q

Acid fast stain

A

Carbolfuchsin used to stain mycobacterium

22
Q

Endospore staining

A

Malachite green specifically binds to an endospores coat

23
Q

Process of fluorescence microscopy

A

-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

24
Q

Absorption and fluorescence terms

A

-Absorption/Excitation
-Fluorescence/Emission

25
Q

Benefits of fluorescence microscopy

A

-Can study bacterial cells inside a complex such as biofilms
-Can be used to study bacterial motion

26
Q

Considerations for choosing fluorescence microscopy

A

-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)

27
Q

Single-Molecule localization in light microscopy

A

Approximation of location in order to calculate the best probability of fluoroform

28
Q

Single-molecule tracking in light microscopy

A

Tracks consecutive images of the same molecules in order to gain unique frames to reconstruct a path

29
Q

Dark field microscopy

A

Central aperture above the light source allowing visibility of cellular structures

30
Q

Cryo-electron microscopy

A

Rapid freezing of samples to preserve native structure

31
Q

Sample requirement for transmission electron microscopy

A

An electron dense negative stain from salt or heavy metal

32
Q

Benefit of scanning electron microscopy

A

Effective for visualizing cells in complex communities such as biofilms

33
Q

Benefit of cryo-electron microscopy

A

Maintains native structure of protein

BSCI443: Microbial Physiology (10 decks)

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