Lecture 3A: Microscopy and Staining Flashcards
1
Q
basic tool for viewing cells
A
Microscope
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)
3
Q
Micrometer Symbol
A
µm
4
Q
Unaided human eye
A
- 200 µm-
5
Q
Compound light microscope (LM)
A
- 200nm-10nm
6
Q
Scanning electron Microscope (SEM)
A
- 1nm to 1mm
7
Q
Transmission Electron Microscope (TEM)
A
- 10nm -100 µm
8
Q
Atomic force Microscope (AFM)
A
- 1nm-10nm
9
Q
Scanning tunneling microscope (STM)
A
- 0.5 nm-10nm
10
Q
Atom Size
A
- 0.1 nm
11
Q
Why are cells so small?
A
- Cells are designed to be small for efficiency.
12
Q
Why are cells so small?;
- As surface area to the volume ratio gets ______________ as the cell gets _______________.
A
- Smaller
- Bigger
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
14
Q
(4) General Principles of Microscopy
A
- Wavelength of Radiation
- Magnification
- Resolution
- Contrast
15
Q
Wavelength Visible for Human
A
- Visible light 400nm-700nm
16
Q
Wavelengths from Weaker/low to strongest/biggest
A
- Radio waves and television, microwave, infrared, Ultraviolet light, X-rays, Gamma rays
17
Q
Weakest Wavelength
A
- Radio waves and television
18
Q
Strongest Wavelength
A
- Gamma rays
19
Q
Simple magnifier lenses are _____
A
- Bi-convex
20
Q
Means they are thicker at the center that the periphery.
A
- bi-convex
21
Q
occurs when the image continues to be enlarged, but no additional detail is resolved.
A
- Empty Magnification
22
Q
Magnification must be accompanied by _________
A
- Improved resolution
23
Q
The _______ of a microscope is its capacity for discerning detail.
A
- Resolution
24
Q
Microscope resolution Formula
A
- D= 0.61λ / n sin v
25
Symbol of λ in microscope resolution formula
- The wavelength of the light source
26
What does n in the formula?
- The refractive index of air or liquid between the objective lens and the specimen
27
What does ‘v’ mean in the formula?
- The aperture angle (a measure of the light-gathering ability of the lens)
28
Expression n sin v
Called numerical aperture.
29
What does the answer from formula D= 0.61λ / n sin v mean in practical terms?
- Limit of resolution, D- is the smallest dimension that we can see clearly/distinctly.
30
Types of microscopy
- Bright-field microscopes
- Dark-field microscopes
- Phase microscopes
- Fluorescent microscopes
- Immunoinfluorescent microscopes
- Electron Microscopes
- Probe Microscopes
31
differences in intensity between two objects, or between an object and background.
- Contrast
32
In __-_____ _______ , ______ results when cells absorb or scatter light differently from their surroundings.
- Bright-field microscopy
- Contrast
33
What is the theoretical limit for light microscope?
- 0.2 µm
34
Types of Light Microscope
- Bright-Field Microscope
- Dark-field Microscope
- Phase Microscope
- fluorescent microscope
35
Contain a single magnifying lens, -similar to magnifying glass, -Leuwenhoek used simple microscope to observe microorganisms.
- Bright-field microscope, simple
36
Contain a ____ magnifying lens, -similar to magnifying glass.
-_______ used simple microscope to observe microorganisms
- Single
- Leuwenhoek
37
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
- Resolution (because light does not refract)
- Condense Lens
38
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
- Bright-filed microscopes, compound
39
Advantage of Bright-filed microscopes, compound
- Convenient, relatively inexpensive, available
40
Disadvantage of Bright-filed microscopes, compound
- R.P 0.2 µm at best; can recognize cell but not fine details
41
Needs contrast of Bright-filed microscopes, compound
- Easiest way to view cells is to fix and stain.
42
The ____ _____ and _____ ___ combine to produce a magnified image of the specimen.
- Objective lens and eyepiece lens
43
Light rays from the specimen AB pass through the objective lens to give____________________________________________.
- A magnified inverted and real primary image.
44
The eyepiece lens magnifies this further to produce a _______ of the specimen
- Virtual Image
45
A typical light microscope ‘s light path consists of:
- A transillumination light source
- A condenser lens
- An objective lens
- Oculars and or camera
46
A typical light microscope ‘s light path consists of:
-a transillumination light source, commonly a ____________in the microscope stand;
- Halogen lamp
47
A typical light microscope ‘s light path consists of:
-a _______________, which focuses light from the light source onto the sample;
- A condenser lens
48
A typical light microscope ‘s light path consists of:
-an _____________, which collects light from the sample and magnifies the image;
- Objective lens
49
A typical light microscope ‘s light path consists of:
___________ and/or a _____________ to view the sample image.
- Oculars, a camera
50
The total magnification is obtained by multiplying this by the eyepiece value (usually __)
- 10x
51
Kinds of objective (lens)
- 4x Scanning
- 10x Low-dry
- 40x High-dry
- 100x oil immersion
52
4X scanning
- Find the object
53
10X low-dry
- Course focusing
54
40X high-dry
- Fine focusing
55
100X oil immersion
- Fine focusing and improved resolution.
56
-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
- Dark-field microscopes.
57
Best for observing __________________
–Only light rays scattered by specimen ____________ the objective lens
– Specimen appears __________ against _________ background
– Increases ___________ and enables observation of more details.
- Pale objectives
- Enter
- Light
- Dark
- Contrast
58
– occludes direct light, passes wide angle light
– angle too wide to enter objective
Special condenser diaphragm
59
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
- Phase microscopes
60
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
- 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
Two types of phase microscopes
-Phase-contrast microscopes
-Differential interference contrast microscopes
62
light rays through objects of different n → change in phase, not intensity
- Phase contrast microscopy
63
This component is a specialized condenser meant specifically for phase contrast microscopy.
- Condenser annulus
64
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.
- Phase contrast objective(s)
65
– 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
- Fluorescence Microscopes
66
– 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
- UV light
- Resolution and contrast
67
an assay which is used primarily on biological samples and is classically defined as a procedure to detect antigens in cellular contexts using antibodies.
- Immunofluorescence
68
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 _______
- Fluorescence
69
The dye which gives yellow-green fluorescence
- Fluorescein isothiocynate
70
Immunofluorescence tests are also termed as
- Fluorescent anti body test (FAT)
71
Fluorescent dyes that can be tagged with antibody molecules
- fluorescein isothiocyanate and lissamine rhodamine
72
– 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
Electron Microscopes
73
– Light microscopes cannot resolve structures smaller than ________ nm
because the shortest wavelength of visible light is ________ nm
200nm; 400nm
74
– Electrons produce wavelengths of 0.01 nm to 0.001 nm, so electron
microscopes have greater _____________ and ____________________
revolving power and greater magnification
75
– Magnifies the images ____________ to _______________
10,000X to 100,000X
76
'Magnificies objects' Is this statement correct??
no, because the microscopes magnifies the IMAGE, and not the object
77
Two Types of Electron Microscopes
- Transmission electon microscopes (TEM) and;
- Scanning Electron microscopes (SEM)
78
TEM stands for
Transmission electron microscopes
79
SEM stands for
Scanning electron microscopes
80
- form images using electrons that are transmitted (pass) through a specimen
- Like 2D or planar
TEM (Transmission electron microscopes)
81
- Utilize electrons that has bounced off the surface of the specimen
- some kind of 3D image
SEM (Scanning electron microscopes)
82
Points the electron beam
- Electron gun
83
defines the size of the electron beam
- Condenser Lens (magnet)
84
to focus and initially magnify the image (electron microscope)
- Objective lens (magnet)
85
Similarities of Electron Microscopes and Light Microscopes
- 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
Differences of EM and LM Size;
Light microscopes are smaller and lighter, so are easier to move and set up.
87
Differences of EM and LM cost and availability;
Light microscopes are less expensive than EM
88
Differences of EM and LM radiation type;
LM uses UV (Visible light) approx. 400-700nm, while Electron Microscopes use electrons approx equivalength wavelength to 1 nm
89
Differences of EM and LM Control image;
LM via glass lenses, beams electrons can be focused using electromagnets due to negative charge on electrons
90
Differences of EM and LM Resolution;
EM have much higher resolution than LM
91
Differences of EM and LM Magnification;
EM have higher magnifications than LM
92
Differences of EM and LM Colour Images;
Light microscopes form images including the range of wavelengths
(colors) provided by the light source
93
Differences of EM and LM Preparation of specimens;
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
Differences of EM and LM Image Formation;
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
Differences of EM and LM Usage Limitations;
Living specimens cannot be viewed using electron microscopes
because electron microscopes require there to be a vacuum in the tub
96
Use minuscule, pointed, _________________ to magnify more
than _____________________ times
- electronic probes; 100,000,000 times
97
Use minuscule, pointed, electronic probes to magnify more than 100,000,000 times
Probe Microscopes
98
Two Types of Probe microscopes
-Scanning tunneling microscopes (STM)
- Atomic force Microscopes (AFM)
99
Detects the surface structure of the objects based on the tunnel effect of the quantum mechanics.
STM (Scanning Tunneling Microscopes)
100
Comparison between AFM and Electronic Microscopes
- 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
