Lecture 3A Microscopy and Staining Flashcards
(79 cards)
1
Q
representative microbiological organism of 1 meter
A
length of pork tapeworm, Taenia solium (1.8-8.0m)
2
Q
metric units of length: decimeter
meaning of prefix: __
metric equivalent: __
A
- 1/10
- 0.1 m = 10^-1 m
3
Q
metric units of length: centimeter
meaning of prefix: __
metric equivalent: __
representative microbiological application: __
A
- 1/100
- 0.01 m or 10^-2 m
- diameter of a mushroom cap (12cm)
4
Q
metric units of length: millimeter
meaning of prefix: __
metric equivalent: __
representative microbiological application: __
A
- 1/1000
- 0.001 m or 10^-3 m
- diameter of a bacterial colony (2.3mm); length of a tick (5.7mm)
5
Q
metric units of length: micrometer
meaning of prefix: __
metric equivalent: __
representative microbiological application: __
A
- 1/1,000,000
- 0.000001 m or 10^-6
- diameter of white blood cells (5um-25um)
6
Q
metric units of length: nanometer
meaning of prefix: __
metric equivalent: __
representative microbiological application: __
A
- 1/1,000,000,000
- 0.000000001 or 10^-9
- diameter of poliovirus (25nm)
7
Q
What can a scanning electron microscope (SEM) see?
A
10nm-1mm
- large protozoa (Euglena)
- human red blood cell
- chloroplast
- mitochondrion
- typical bacteria or archaea
- viruses
- ribosomes
8
Q
What can a transmission electron microscope (TEM) see?
A
10nm-100um
- human red blood cell
- chloroplast
- mitochondrion
- typical bacteria or archaea
- viruses
- ribosomes
9
Q
What can an atomic force microscope (AFM) see?
A
1nm-10nm
- proteins
- diameter of DNA
- amino acids
10
Q
What can an unaided human eye see?
A
200um-above
11
Q
What can a compound light microscope (LM) see?
A
200um-10mm
12
Q
What can a scanning tunneling microscope (STM) see?
A
0.5nm-10nm
- proteins
- diameter of DNA
- amino acids
13
Q
As the surface area to the volume ratio gets __ as the cell gets __
A
- smaller
- larger
14
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.
When this happens, the cell must divide into __ with a favorable surface area/volume ratios, or __.
A
- membrane
- smaller cells
- cease to function
15
Q
General Principles of Microscopy
A
- wavelength of radiation
- magnification
- resolution
- contrast
16
Q
the lower the nanometer, the __ the resolution
A
higher
17
Q
the bigger the wavelength, the __ powerful it is
A
less
18
Q
the weaker the radiation, the __ the resolution
A
lower
19
Q
Electromagnetic spectrum
A
- radio waves and television
- microwave
- infrared
- visible light
- UV rays
- X rays
- Gamma rays
20
Q
Electromagnetic spectrum: high-penetrability (also WC)
A
- X-rays
- Gamma rays
21
Q
What have mutagenic-affects on DNA
A
UV light
22
Q
Wavelength of radiowaves
A
10^3 m
23
Q
Wavelength of microwave
A
10^-2 m
24
Q
Wavelength of infrared
A
10^-4 m
25
Wavelength of visible light
5 x 10^-6 m
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Wavelength of UV light
10^-6 m
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Wavelength of X-ray
10^-8 m
28
Wavelength of Gamma-ray
10^-12 m
29
produces an image of the object upon which the microscope or magnifying glass is focused.
A simple microscope or magnifying glass (lens)
30
__ are bi-convex, meaning they are thicker at the center than at the periphery
Simple magnifier lenses
31
A __ allows us to see a considerably magnified image of our specimen. To continue magnifying an image beyond a certain point, however, serves little purpose, if it is not accompanied by an __.- Hoggs, 2005
- combination of two lens systems
- increase in detail
32
Magnification must be accompanied by __
improved resolution
33
increase in magnification but no further information
empty magnification
34
The __ (__) of a microscope is its capacity for discerning detail. More specifically, it is the ability to distinguish between two points a short distance apart, and is determined by the equation:
___
where λ is the __, n is the __ between the objective lens and the specimen and θ is the __ (a measure of the light-gathering ability of the lens).
- resolution (resolving power, or limit of resolution, d)
- d = 0.61λ / n sin θ
- wavelength of the light source
- refractive index of the air or liquid
- aperture angle
35
The expression n sinθ is called the __ and for high-quality lenses has a value of around __. The lowest wavelength of light visible to the human eye is approximately __, so the maximum resolving power for a light microscope is approximately
- numerical aperture
- 1.4
- 400 nm
- d = 0.61 × 400 / 1.4
= 0.17μm
36
differences in intensity between two objects, or between an object and background.
intensity
37
Most cells are colorless and therefore __ is often utilized to increase __
- staining
- contrast
38
In __, contrast results when cells absorb or scatter light differently from their surroundings.
bright-field microscopy
39
theoretical limit for light microscope is __
0.2um
40
the smallest distance by which two objects can be separated and still be distinguishable as two separate objects.
limit of resolution
41
types of light microscopes
I. Bright-field microscope
a. simple
b. compound
II. Dark-field microscope
III. Phase microscope
IV. Fluorescent microscopes
42
- contain a single magnifying lens
- similar to a magnifying glass
- Leeuwenhoek used a simple microscope to observe microorganisms
Bright-field microscope (simple)
43
- 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
Bright-field microscopes: Compound microscope
*resolution because light does not refract
*condenser
44
Bright-field microscopes: Compound microscope: Advantages and Disadvantages
Advantages:
- convenient
- relatively inexpensive
- available
Disadvantages:
- resolving power (RP) 0.2um at best can recognize cells but not fine details
- needs contrast: easiest way to view cells is to fix and stain
45
How an image is generated in a compound microscope
The objective lens and eyepiece lens combine to produce a magnified image of the specimen.
46
How an image is generated in a compound microscope
(a) Light rays from the specimen AB pass through the objective lens to give __, __, and __
(b) The eyepiece lens magnifies this further to produce a __ of the specimen
* a magnified, inverted, and real primary image
*virtual image
47
A typical light microscope‘s light path consists of:
* a __ light source, commonly a __ in the microscope stand;
* a __, which focuses light from the light source onto the sample;
* an __, which collects light from the sample and magnifies the image;
* __ and/or a __ to view the sample image.
* transillumination; halogen lamp
* condenser
* objective
* occulars; camera
48
* 4X __
* 10X __
* 40X __
* 100X __
* Scanning
* Low-Dry
* High-Dry
* Oil immersion
49
lights ability to bend
refraction
50
– Best for observing pale objects
– Only light rays scattered by the specimen enter the objective lens
– Specimen appears __ against __ background
– Increases __ and enables observation of more details
Dark-field microscopes
* light; dark
* contrast
51
– occludes direct light, passes wide-angle light
– angle too wide to enter the objective
special condenser diaphragm
(dark-field microscopy)
52
– Used to examine living organisms that would be damaged or altered by attaching them to slides or staining
them
– -best for highly transparent specimens that do not absorb much light
– Treat one set of light rays differently from another set
– Light rays __ produce brighter images, 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.
Phase microscopes
* in phase; out of phase
* 1/2 wavelength
* condenser
* objective phase plate
* phase shift of light
53
Phase Contrast Microscopy
* light rays through objects of different n ->
→ change in phase, not intensity
54
Phase Contrast Microscopy
- Human eyes are not able to pick up details with the use of diffracted light that is out of step by about __ of a wavelength with the un-diffracted light. Our eyes are not able to pick up details with this organization of light waves because of the __.
- the phase contrast technique slows the un-diffracted light by a __ making the waves further out of phase by a net difference of __.
* the wavelength difference the human eye is able to see much more __ on the image produced by the microscope.
* ¼
* lack of contrast
* ¼ of a wavelength
* ½ a wavelength
* contrast
55
Phase Contrast Microscopy special parts
* special ring-shaped condenser diaphragm
* special glass disc in objective
56
Phase Contrast Microscopy
- The first component is the __ with __. This component is a __ meant specifically for phase contrast microscopy. The condenser contains a __ which has a flat black light-absorbing plate with transparent annular rings
* phase contrast condenser
* condenser annulus
* specialized condenser
* phase annulus
57
Phase Contrast Microscopy
- The next component is a __. These __ are built with a phase plate that works in conjunction with the __ to achieve the phase shift required for phase contrast microscopy. These types of objectives have a phase plate built into the rear of the objective.
* phase contrast objective(s)
* specialized objectives
* condenser annulus
58
- Light coming through the annular aperture of the condenser passes through the object. Those rays, which are not deviated by the object, pass through the __ of the __ and acquire __.
- Those rays, which are deviated by the object structures, due to different refractive indices, pass through the __ not covered by the phase-altering pattern. Thus, their wavelength __. The difference in phase (wavelength) gives the __ for clear object visibility.
- phase-altering pattern
- phase plate
- longer wavelength
- phase-plate
- remains unchanged
- contrast
59
– Direct UV light 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 __
– Used in __ to identify pathogens and to
locate and make visible a variety of proteins
Fluorescent microscopes
- resolution; contrast
- stained
- immunofluorescence
60
an assay that is used primarily on biological samples and is classically defined as a procedure to detect antigens in cellular contexts using antibodies.
Immunofluorescence
- immunofluorescence
61
The specificity of antibodies to their antigen is the base for __. The property of certain dyes absorbing light rays at one particular wavelength (__) and emitting them at a different wavelength (__) is known as __.
- immunofluorescence
- ultraviolet light
- visible light
- fluorescence
62
In an immunofluorescence test, a fluorescent dye that illuminates in UV light is used to detect/show the specific combination of an __ and __. The dye usually used is fluoresce in __, which gives yellow-green fluorescence. Immunofluorescence tests are also termed as __. Fluorescent dyes, such as __ and __, can be tagged with antibody molecules. They emit blue-green and orange-red fluorescence, respectively under ultraviolet (UV) rays in the fluorescence microscope. This forms the basis of the immunological test.
- antigen; antibody
- isothiocyanate
- fluorescent antibody test (FAT)
- fluorescein isothiocyanate
- lissamine rhodamine
63
Passes light directly through the specimen; unless the cell is naturally pigmented or artificially stained, the image has little contrast.
Brightfield (unstained specimen)
64
Staining with various dyes enhances contrast, but most staining procedures require that cells be fixed (preserved).
Brightfield (stained specimen)
65
Enhances contrast in unstained cells by amplifying variations in density within specimens; especially useful for examining living, unpigmented cells.
Phase-contrast
66
Like phase-contrast microscopy, it uses optical modifications to exaggerate differences in density/refractive index
Differential-interference-contrast (Nomarski)
67
Uses lasers and special optics for "optical sectioning." Only those regions within a narrow depth of focus are imaged. Regions above and below the selected plane of view appear black rather than blurry. This microscope is typically used with fluorescently stained specimens, as in the example here.
Confocal
68
– Light microscopes cannot resolve structures smaller than __ nm because the shortest wavelength of visible light is __nm
– Electrons produce wavelengths of 0.01 nm to 0.001 nm, so electron microscopes have greater __ and __
– Magnifies objects __ to __
– Gives detailed views of bacteria, viruses, internal cellular structures, molecules, and large atoms
ELECTRON MICROSCOPES
- 200
- 400
- resolving power
- greater magnification
- 10,000x; 100,000x
69
parts inside a compound microscope (upside down)
- Lamp
- condenser lens
- specimen
- objective lens
- eyepiece
- final image seen by eye
70
parts inside an electron microscope
- electron gun (cathode (source of electrons))
- condenser lens (magnet)
- specimen
- objective lens (magnet)
- projector lens (magnet)
- final image in fluorescent screen
71
Similarities of light microscope and electron microscope
* Form larger (magnified) and more detailed (highly resolved) images of small objects or small areas of larger objects e.g. a leaf, part of a bone, etc. that 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 appropriate for both the equipment and the sample e.g. slicing, staining, mounting, etc.
72
Differences of Light microscope and electron microscope (size, cost/availability, radiation type, control of image formation, resolution, magnification)
* Size: LM smaller and lighter
* Cost / Availability: LM less expensive than EM
*Radiation Type:
LM - light (~WV 400-700nm)
EM - beam of electron (~WV 1nm)
* Control of image formation:
LM - glass lenses
EM - beams of electrons focused using electromagnets -- due to charge on electrons
* Resolution: EM higher than LM
* Magnification: EM higher than LM
73
Differences of Light microscope and electron microscope (Colour Images, Preparation of specimens, Image formation, Usage Limitations)
- Light microscopes form images including the range of wavelengths (colors) provided by the light source
- 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
- 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.
- Living specimens cannot be viewed using electron microscopes because electron microscopes require there to be a vacuum in the tube
74
Use minuscule, pointed, electron probes to magnify more than __ times
PROBE MICROSCOPES
- 100,000,000
75
Two types of probe microscope
- Scanning tunneling microscopes (STM)
- Atomic force microscopes (AFM)
76
detects the surface structure of the objects on the runner effect of the quantum mechanics
Scanning tunneling microscopes (STM)
77
not only gives images in 3D but also provides various types of surface measurements to the needs of scientists and engineers
Atomic force microscopes (AFM)
78
- Optical and electron microscopes can easily generate __ images of a sample surface, with a magnification as large as __ for an optical microscope, and a few hundred thousands __ for an electron microscope.
- However, these microscopes cannot measure the __ dimension (z-direction) of the sample, the __ (e.g. particles), or __(e.g. holes, pits) of the surface features.
- two dimensional
- 1000x
- ~100,000x
- vertical
- height
- depth
79
- AFM, which uses a sharp tip to probe the surface features by __, can image the __ with extremely high magnifications, up to 1,000,000x, comparable to or even better than electronic microscopes.
- The measurement of an AFM is made in __, the horizontal X-Y plane and the vertical z dimension. Resolution (magnification) at the Z-direction is normally __ than X-Y plane.
- raster scanning
- surface topography
- three dimensions
- higher
