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Z BIO103- Microbiology > Lecture 3A: Microscopy and Staining > Flashcards

Lecture 3A: Microscopy and Staining Flashcards

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Biology 101 flashcards
1
Q

basic tool for viewing cells

A

Microscope

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2
Q
  • specialized optical instrument designed to generate
  • enlarged, visible ______________ of specimens
  • key features : ______________ and _______________
A
  • images
  • Magnifying and Resolving power (which depends on the lens system)
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3
Q

Micrometer Symbol

A

µm

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4
Q

Unaided human eye

A
  • 200 µm-
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5
Q

Compound light microscope (LM)

A
  • 200nm-10nm
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6
Q

Scanning electron Microscope (SEM)

A
  • 1nm to 1mm
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7
Q

Transmission Electron Microscope (TEM)

A
  • 10nm -100 µm
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8
Q

Atomic force Microscope (AFM)

A
  • 1nm-10nm
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9
Q

Scanning tunneling microscope (STM)

A
  • 0.5 nm-10nm
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10
Q

Atom Size

A
  • 0.1 nm
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11
Q

Why are cells so small?

A
  • Cells are designed to be small for efficiency.
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12
Q

Why are cells so small?;
- As surface area to the volume ratio gets ______________ as the cell gets _______________.

A
  • Smaller
  • Bigger
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13
Q

if the cell grows beyond a certain limit, not enough material will be able to cross the _____________ fast enough to accommodate the increased cellular volume

A
  • Membrane
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14
Q

(4) General Principles of Microscopy

A
  • Wavelength of Radiation
  • Magnification
  • Resolution
  • Contrast
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15
Q

Wavelength Visible for Human

A
  • Visible light 400nm-700nm
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16
Q

Wavelengths from Weaker/low to strongest/biggest

A
  • Radio waves and television, microwave, infrared, Ultraviolet light, X-rays, Gamma rays
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17
Q

Weakest Wavelength

A
  • Radio waves and television
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18
Q

Strongest Wavelength

A
  • Gamma rays
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19
Q

Simple magnifier lenses are _____

A
  • Bi-convex
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20
Q

Means they are thicker at the center that the periphery.

A
  • bi-convex
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21
Q

occurs when the image continues to be enlarged, but no additional detail is resolved.

A
  • Empty Magnification
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22
Q

Magnification must be accompanied by _________

A
  • Improved resolution
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23
Q

The _______ of a microscope is its capacity for discerning detail.

A
  • Resolution
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24
Q

Microscope resolution Formula

A
  • D= 0.61λ / n sin v
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25
Q

Symbol of λ in microscope resolution formula

A
  • The wavelength of the light source
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26
Q

What does n in the formula?

A
  • The refractive index of air or liquid between the objective lens and the specimen
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27
Q

What does ‘v’ mean in the formula?

A
  • The aperture angle (a measure of the light-gathering ability of the lens)
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28
Q

Expression n sin v

A

Called numerical aperture.

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29
Q

What does the answer from formula D= 0.61λ / n sin v mean in practical terms?

A
  • Limit of resolution, D- is the smallest dimension that we can see clearly/distinctly.
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30
Q

Types of microscopy

A
  • Bright-field microscopes
  • Dark-field microscopes
  • Phase microscopes
  • Fluorescent microscopes
  • Immunoinfluorescent microscopes
  • Electron Microscopes
  • Probe Microscopes
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31
Q

differences in intensity between two objects, or between an object and background.

A
  • Contrast
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32
Q

In __-_____ _______ , ______ results when cells absorb or scatter light differently from their surroundings.

A
  • Bright-field microscopy
  • Contrast
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33
Q

What is the theoretical limit for light microscope?

A
  • 0.2 µm
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34
Q

Types of Light Microscope

A
  • Bright-Field Microscope
  • Dark-field Microscope
  • Phase Microscope
  • fluorescent microscope
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35
Q

Contain a single magnifying lens, -similar to magnifying glass, -Leuwenhoek used simple microscope to observe microorganisms.

A
  • Bright-field microscope, simple
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36
Q

Contain a ____ magnifying lens, -similar to magnifying glass.
-_______ used simple microscope to observe microorganisms

A
  • Single
  • Leuwenhoek
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37
Q

2 lens systems
– Light rays pass through specimen and into objective lens – Specimens illuminated directly from above or below – Oil immersion lens increases ___________
– Total magnification = magnification of objective lens X magnification of ocular lens
– Most have _________________ to direct light through specimen

A
  • Resolution (because light does not refract)
  • Condense Lens
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38
Q

2 lens system
– Light rays pass through specimen and into objective lens – Specimens illuminated directly from above or below – Oil immersion lens increases Resolution (because light does not refract)
– Total magnification = magnification of objective lens X magnification of ocular lens
– Most have Condense Lens to direct light through specimen

A
  • Bright-filed microscopes, compound
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39
Q

Advantage of Bright-filed microscopes, compound

A
  • Convenient, relatively inexpensive, available
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40
Q

Disadvantage of Bright-filed microscopes, compound

A
  • R.P 0.2 µm at best; can recognize cell but not fine details
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41
Q

Needs contrast of Bright-filed microscopes, compound

A
  • Easiest way to view cells is to fix and stain.
42
Q

The ____ _____ and _____ ___ combine to produce a magnified image of the specimen.

A
  • Objective lens and eyepiece lens
43
Q

Light rays from the specimen AB pass through the objective lens to give____________________________________________.

A
  • A magnified inverted and real primary image.
44
Q

The eyepiece lens magnifies this further to produce a _______ of the specimen

A
  • Virtual Image
45
Q

A typical light microscope ‘s light path consists of:

A
  • A transillumination light source
  • A condenser lens
  • An objective lens
  • Oculars and or camera
46
Q

A typical light microscope ‘s light path consists of:
-a transillumination light source, commonly a ____________in the microscope stand;

A
  • Halogen lamp
47
Q

A typical light microscope ‘s light path consists of:

-a _______________, which focuses light from the light source onto the sample;

A
  • A condenser lens
48
Q

A typical light microscope ‘s light path consists of:

-an _____________, which collects light from the sample and magnifies the image;

A
  • Objective lens
49
Q

A typical light microscope ‘s light path consists of:

___________ and/or a _____________ to view the sample image.

A
  • Oculars, a camera
50
Q

The total magnification is obtained by multiplying this by the eyepiece value (usually __)

A
  • 10x
51
Q

Kinds of objective (lens)

A
  • 4x Scanning
  • 10x Low-dry
  • 40x High-dry
  • 100x oil immersion
52
Q

4X scanning

A
  • Find the object
53
Q

10X low-dry

A
  • Course focusing
54
Q

40X high-dry

A
  • Fine focusing
55
Q

100X oil immersion

A
  • Fine focusing and improved resolution.
56
Q

-Best for observing pale objectives.
–Only light rays scattered by specimen enter the objective lens
– Specimen appears light against dark background
– Increases contrast and enables observation of more details

A
  • Dark-field microscopes.
57
Q

Best for observing __________________
–Only light rays scattered by specimen ____________ the objective lens
– Specimen appears __________ against _________ background
– Increases ___________ and enables observation of more details.

A
  • Pale objectives
  • Enter
  • Light
  • Dark
  • Contrast
58
Q

– occludes direct light, passes wide angle light
– angle too wide to enter objective

A

Special condenser diaphragm

59
Q

Used to examine living organisms or specimen that would be damaged or altered by attaching them to slides or staining them –
-best for highly transparent specimen
– Treat one set of light rays differently from another set
– Light rays in phase produce brighter image, while light rays out of phase produce darker image
– Contrast is created because light waves are ½ wavelength out of phase
-– Uses two specific microscope components, the condenser annulus and the objective phase plate, to create a phase/shift light that results in an image with greater contrast perceived by the observer

A
  • Phase microscopes
60
Q

Used to examine _________________________ that would be damaged or altered by attaching them to slides or staining them –
-best for __________________
– Treat one set of light rays differently from another set
– Light rays ______________ produce brighter image, while light rays ______________ produce darker image
– Contrast is created because light waves are ______________ out of phase
-– Uses two specific microscope components, the _________________ and the _______________________ , to create a ____________________ that results in an image with greater contrast perceived by the observer

A
  • living organisms or specimens
  • highly transparent specimen
  • in phase; out of phase
  • ½ wavelength out of space
  • condenser annulus and objective phase plate
  • to create phase/shift light
61
Q

Two types of phase microscopes

A

-Phase-contrast microscopes
-Differential interference contrast microscopes

62
Q

light rays through objects of different n → change in phase, not intensity

A
  • Phase contrast microscopy
63
Q

This component is a specialized condenser meant specifically for phase contrast microscopy.

A
  • Condenser annulus
64
Q

These specialized objectives are built with a phase plate that works in conjunction with the condenser annulus to achieve the phase shift required for phase contrast microscopy.

A
  • Phase contrast objective(s)
65
Q

– Direct UV light source at specimen; causes the specimen to radiate energy back as a longer, visible wavelength
– UV light increases resolution and contrast
– Some cells and molecules are naturally fluorescent, while others must be stained
– Used in immunofluorescence to identify pathogens and to locate and make visible a variety of proteins

A
  • Fluorescence Microscopes
66
Q

– Direct _____________ source at specimen; causes the specimen to radiate energy back as a longer, visible wavelength
– UV light increases __________ and ____________
– Some cells and molecules are naturally fluorescent, while others must be stained
– Used in immunofluorescence to identify pathogens and to locate and make visible a variety of proteins UV light

A
  • UV light
  • Resolution and contrast
67
Q

an assay which is used primarily on biological samples and is classically defined as a procedure to detect antigens in cellular contexts using antibodies.

A
  • Immunofluorescence
68
Q

The property of certain dyes absorbing light rays at one particular wavelength (ultraviolet light) and emitting them at a different wavelength (visible light) is known as _______

A
  • Fluorescence
69
Q

The dye which gives yellow-green fluorescence

A
  • Fluorescein isothiocynate
70
Q

Immunofluorescence tests are also termed as

A
  • Fluorescent anti body test (FAT)
71
Q

Fluorescent dyes that can be tagged with antibody molecules

A
  • fluorescein isothiocyanate and lissamine rhodamine
72
Q

– Light microscopes cannot resolve structures smaller than 200 nm
because the shortest wavelength of visible light is 400 nm
– Electrons produce wavelengths of 0.01 nm to 0.001 nm, so electron
microscopes have greater revolving power and greater magnification
– Magnifies the image (not the object) 10,000X to 100,000X
– Gives detailed views of bacteria, viruses, internal cellular structures,
molecules, and large atoms

A

Electron Microscopes

73
Q

– Light microscopes cannot resolve structures smaller than ________ nm
because the shortest wavelength of visible light is ________ nm

A

200nm; 400nm

74
Q

– Electrons produce wavelengths of 0.01 nm to 0.001 nm, so electron
microscopes have greater _____________ and ____________________

A

revolving power and greater magnification

75
Q

– Magnifies the images ____________ to _______________

A

10,000X to 100,000X

76
Q

‘Magnificies objects’ Is this statement correct??

A

no, because the microscopes magnifies the IMAGE, and not the object

77
Q

Two Types of Electron Microscopes

A
  • Transmission electon microscopes (TEM) and;
  • Scanning Electron microscopes (SEM)
78
Q

TEM stands for

A

Transmission electron microscopes

79
Q

SEM stands for

A

Scanning electron microscopes

80
Q
  • form images using electrons that are transmitted (pass) through a specimen
  • Like 2D or planar
A

TEM (Transmission electron microscopes)

81
Q
  • Utilize electrons that has bounced off the surface of the specimen
  • some kind of 3D image
A

SEM (Scanning electron microscopes)

82
Q

Points the electron beam

A
  • Electron gun
83
Q

defines the size of the electron beam

A
  • Condenser Lens (magnet)
84
Q

to focus and initially magnify the image (electron microscope)

A
  • Objective lens (magnet)
85
Q

Similarities of Electron Microscopes and Light Microscopes

A
  • Form Larger (magnified) and more detailed images of small objects or small areas of larger objects e.g. a
    leaf, part of a bone, etc. than can be formed by the human eye.
    *Used in study and research in biology and medical sciences,
    material sciences e.g. metallurgy and other aspects of science.
    *Specimens must be carefully prepared using techniques appopriate for both the equipment and the sample
    e.g. slicing, staining, mounting, etc
86
Q

Differences of EM and LM Size;

A

Light microscopes are smaller and lighter, so are easier to move and set up.

87
Q

Differences of EM and LM cost and availability;

A

Light microscopes are less expensive than EM

88
Q

Differences of EM and LM radiation type;

A

LM uses UV (Visible light) approx. 400-700nm, while Electron Microscopes use electrons approx equivalength wavelength to 1 nm

89
Q

Differences of EM and LM Control image;

A

LM via glass lenses, beams electrons can be focused using electromagnets due to negative charge on electrons

90
Q

Differences of EM and LM Resolution;

A

EM have much higher resolution than LM

91
Q

Differences of EM and LM Magnification;

A

EM have higher magnifications than LM

92
Q

Differences of EM and LM Colour Images;

A

Light microscopes form images including the range of wavelengths
(colors) provided by the light source

93
Q

Differences of EM and LM Preparation of specimens;

A

Generally involves harsher processes, e.g. using
corrosive chemicals, for viewing via electron microscope than preparation of slides for
viewing using a light microscope. Therefore more skill required

94
Q

Differences of EM and LM Image Formation;

A

Light microscope images can be viewed directly. Electron
microscopes require use of a fluorescent screen, photographic plate or electronic
display because electrons cannot be observed directly by the human eye.

95
Q

Differences of EM and LM Usage Limitations;

A

Living specimens cannot be viewed using electron microscopes
because electron microscopes require there to be a vacuum in the tub

96
Q

Use minuscule, pointed, _________________ to magnify more
than _____________________ times

A
  • electronic probes; 100,000,000 times
97
Q

Use minuscule, pointed, electronic probes to magnify more than 100,000,000 times

A

Probe Microscopes

98
Q

Two Types of Probe microscopes

A

-Scanning tunneling microscopes (STM)
- Atomic force Microscopes (AFM)

99
Q

Detects the surface structure of the objects based on the tunnel effect of the quantum mechanics.

A

STM (Scanning Tunneling Microscopes)

100
Q

Comparison between AFM and Electronic Microscopes

A
  • Optical and electron microscopes can easily generate 2D images of sample surface. 1000X for optical microscope and a few hundred thousands for an EM
  • These microscopes cannot measure the Vertical dimension (z-direction) of the sample, the height/depth of the surface features.
  • AFM uses a sharp tip to probe the surface features by RASTER scanning
  • Measurement of AFM is made in Three dimensions, the Horizontal (X-Y plane) and the vertical Z dimention

Z BIO103- Microbiology (8 decks)

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