Section 2: Microscopy and Cell Manipulation Flashcards
(92 cards)
1
Q
Human eye can only resolve particles above
A
100 micrometers
2
Q
Compound light microscope steps
A
- White light illuminates specimen.
- Condensers focus light onto specimen.
- Compound objectives and eyepiece magnify image of specimen by bending light.
- Magnified specimen image is focused onto human eye.
3
Q
Bad detection
A
Bad detection comes from not enough light rays entering the camera
4
Q
Resolution limit of light microscopes happens at
A
Particles above 0.2 micrometers apart are resolved as individual particles. Limit hits at wavelengths smaller than that of light.
5
Q
Magnification
A
apparent visual increase in size of an object. No limit to magnification.
6
Q
Resolution
A
Separation of the individual components of an object, which previously appeared as one; quality of the image. Ability to tell two objects apart.
7
Q
Resolution limit
A
Reached when additional magnification does not separate further detail. Result is simply an enlarged blurry image.
8
Q
Detection
A
Ability to observe signal aka light rays entering.
9
Q
Light Microscope basic setup
A
- Light travels from source through condenser lens onto specimen.
- Specimen interferes with passage of light & the objective collects the light.
10
Q
How does optical diffraction lead to resolution limit?
A
As light interacts with an object and diffracts, the phase relationships of light waves changes in complex waves.
* Two light waves in phase – increased brightness
* Two light waves out of phase – decreased brightness
11
Q
How close do objects have to be to give diffraction?
A
If closer than a wavelength, then the wavelength of light will look blurry and give diffraction.
As the size of the object gets smaller, the ratio of the “blurring” to the size of the object is larger
12
Q
Objects smaller than ___ will always look ___ in size because of diffraction in light microscopy.
A
200 nm
13
Q
R in diffraction light microscopy
A
Radius of how big a small point appears because of diffraction.
14
Q
D in diffraction light microscopy
A
Distance between two objects
15
Q
if D»_space; R then
A
We see both distinct objects.
16
Q
if D < R then
A
We can’t tell them apart because diffraction patterns overlap.
17
Q
How does resolution depend on wavelength
A
The smaller the wavelength, the smaller the resolution limit.
18
Q
Resolution limit formula
A
0.61λ/nsinθ
19
Q
What is n in resolution limit
A
refractive index of medium between objective and sample.
20
Q
What is θ in resolution limit
A
half angular width of cone of light collected by objective lens.
21
Q
Numerical aperture (NA)
A
A measure of the number of highly diffracted light rays collected by an objective.
22
Q
Numerical Aperture formula
A
NA=nsinθ
23
Q
Relationship between resolution limit and numerical aperture
A
Resolution limit/quality is proportionally impacted by wavelength & inversely impacted by numerical aperture.
24
Q
Relationship between numerical aperture and sample closeness to objective
A
The higher the numerical aperture, the closer the sample has to be to objective.
25
Phase-contrast Microscopy
* Light is in phase before entering sample (no constructive or destructive interference).
* As light passes through sample, some of the light interacts with the material, and thus falls out of phase.
* The out of phase light produces constructive and destructive interference and thus produces contrast.
26
Dark-field microscopy
Light is emitted at 90 to the lens and scattered by cell parts (scattered light is what is detected, over a dark background).
27
How do we label a sample?
Organic dye binds to molecule to indicate presence of that molecule in numbers.
28
When are tissue samples processed in microscopy?
Before dye staining
29
Fixation
Covalent cross-linking locks proteins into place.
30
Embedding
Wax permeates (spreads through) and then solidifies to harden and stabilize tissue.
31
Sectioning
Tissue is thinly sliced with a microtome for observation under a light microscope.
32
What do fluorescents emit?
Fluorescents emit light with longer wavelength (lower energy photons) that decay and release energy in the form of light after excitation with shorter wavelength, higher energy photons.
33
Fluorescent dyes have:
Each dye has a specific excitation wavelength that it prefers and a corresponding and higher emission wavelength and spectrum.
34
First barrier filter
AKA Excitation filter. Removes light that is above and below excitation light, so that only excitation light is allowed to pass.
35
Objective lens
Focuses excitation light onto the sample and also collects emission (fluorescent) from sample.
36
Beam-splitting mirror
Reflects light of a set of wavelengths (lower), transmits light of different set of higher wavelengths (dichroic mirror)
37
Second barrier filter
AKA Emission filter. Removes light that is above and below emitted light so only emitted (fluorescent) light is allowed to pass.
38
Detector
Detects fluorescent light (your eye or camera).
39
Out-of-Focus light
When every dye in the sample is fluorescent and illuminates.
40
How to remove out of focus light
1. Image processing or deconvolution
2. Confocal microscope
3. Spinning disc confocal microscope*
41
What does Confocal Microscope use?
Uses a single-wavelength laser for excitation
42
Confocal Microscope
Laser is focused on a single point but still “shines” as a line through the specimen.
Pinhole permits focused light to pass while out-of-focus light is blocked.
The result is the detection of a single focused point of the specimen (pixel).
To obtain a full image, laser is scanned across specimen.
Image is a composite of many in-focus dots.
43
Optical sections
Taking a series of confocal microscopy images at different depths.
44
Methods of fluorescence labeling specific molecules
1) Use of antibodies for immunocytochemistry
2) Fluorescent proteins
3) Fluorescent dyes
45
B-cells
Cells of the immune system that make antibodies (professionals at making proteins that recognize other specific molecules).
46
Antibodies
Protein molecules that the immune system uses to recognize foreign (i.e. pathogenic) molecules. They're generated by b-cells.
47
VDJ recombination
Each b-cell goes through this during development. It makes each b-cell make only one type of antibody that recognizes a specific protein.
48
What happens when a b-cell detects a molecule that their antibody binds to?
The B-cell divides to make lots of that specific antibody.
49
How do we make antibodies in lab that recognize specific proteins?
Get B-cell out of an animal by:
1. injecting animal with purified protein we wanna study the antibody of.
2. Animal will have B-cells that make an antibody that recognizes the protein.
3. Draw animal blood and find this type of B-cell.
4. fuse with tumour cell
50
How do we keep B-cells alive in the lab?
Fuse with a tumour cell.
51
Hybridomas
Fused cells of B-cell and a tumour cell which generate specific type of antibody needed indefinitely.
52
Primary antibody
Recognizes and binds to the protein that you want to study.
53
Secondary antibody
additional (different) antibody that recognizes and binds to primary antibody
* is covalently attached to a specific fluorescent molecule.
54
Green Fluorescent Protein (GFP)
Isolated from jellyfish.
Forms a fluorochrome that emits green light after excitation with blue light.
GFP gene can be recombined with any other gene to generate a fusion protein of both.
55
Two functions of GFP
1. GFP fusion proteins localize proteins in cells.
2. GFP as a tool to study gene expression.
56
How does GFP fusion proteins localize proteins in cells?
1. GFP gene fused to protein-coding gene.
2. Resulting protein is chimera (fusion) of normal protein & GFP.
3. The fluorescent protein fused to the protein of interest allows study of the location of the protein of interest
57
Chimera
Resulting protein that is a fusion of normal protein and GFP.
58
How can GFP be used as a tool to study gene expression
We can know in which cell a protein is expressed.
1. GFP gene can be fused to any promoter sequence.
2. Promoters drive gene expression.
3. Indicates where, when and how much of a protein is made.
59
GFP has been genetically modified to change:
* Maturation (photoactivation)
* Stability
* Color (Blue, Yellow, Cyan)
* pH sensitivity
60
FM4-64
Becomes fluorescent only when it reaches hydrophobic part of lipid bilayer.
It intercalates into membranes allowing detection of where lipid bilayers are in a cell.
61
Fura2
measures changes in Ca2+ concentration in cells. Intensity increases with increasing calcium [Ca2+].
* In most cells at rest, intracellular [Ca2+] is very low.
* intracellular [Ca2+] increases dramatically as part of a cell response.
62
Fluorescein
Intensity decreases with lower pH.
63
use of antibodies and immunofluorescence labeling requires cell to be alive or dead
Dead
64
What permits live-cell imaging?
GFP and fluorescent protein variants permit live-cell imaging.
65
How can movement/changes in living cells be detected?
Using fluorescently-tagged proteins.
66
Fluorescence photobleaching
The permanent change to a fluorescent dye.
67
Fluorescence photobleaching
Photobleaching is the permanent change to a fluorescent dye.
* Caused by nonradiative decay of excited fluorophores, which leads to damage of fluorophores.
* They absorb light then don't emit the light back out, so the energy changes the chemical composition of that dye.
68
Fluorescence recovery after photobleaching (FRAP)
* Photobleach a patch of the cell with a strong laser
* Observe fluorescence recovery
* Indicates rate of diffusion and movement of molecules
69
Three ways to do super-resolution microscopy
* Structured Illumination microscopy (SIM)
* Simulated EMission Depletion Microscopy (STED)
* Single Molecule Localization Microscopy (SMLM)
70
Single-Molecule Microscopy
Illuminating only one or a few fluorophores at a time.
Do that 10,000 times & it gives better image after reconstruction.
70
Single-Molecule Microscopy
Illuminating only one or a few fluorophores at a time.
Do that 10,000 times & it gives better image after reconstruction.
71
Scanning Electron Microscopy Steps
1. Samples fixed, dried & coated with thin layer of heavy metal
2.Electron beam goes through deflector and bombards/scans the sample.
3.Detector detects shape and angle that light comes off surface.
4. Gives picture of surface of something.
72
Scanning in SEM
Electron beam shoots at sample and gets reflected.
73
Transmission Electron Microscopy (TEM) steps
* Before TEM, cells are cut into thin sections
* Electron beam passes through specimen.
* Collected by objective lens.
* Forms image on a screen on the other side.
74
Dark regions in TEM indicate:
Electrons blocked by dense material
75
Bright regions in TEM indicate:
Electrons passed because of less density.
76
In TEM we ___ live cells
Can't use
77
Preparing sample for TEM (4 steps)
Fixation, treatment, dehydration, slicing
78
Sample Fixation in TEM
Fixation with glutaraldehyde to cross-link proteins in place
79
Sample Treatment in TEM
Treatment with osmium tetroxide to stain lipid membanes with a heavy metal.
Heavy-atom salts can be used (lead and uranium) to increase electron contrast
80
Sample Dehydration in TEM
Dehydration and embedding in resin
81
Sample Slicing in TEM
Sliced into 50-100 nm thick slices.
82
Immunogold Electron Microscopy
(Form of TEM)
Identifies where a specific protein is using antibodies again.
Using gold because it’s e- dense and scatters e- so we see it.
83
What if we don't wanna slice our sample but still wanna take an image of what’s inside cell using TEM?
Metal Shadowing and Deep-etching Technique
84
Deep Etching (In TEM)
(like cracking open an egg)
*Samples are flash-frozen (quickly so they don't spill everywhere)
*Fractured for access to internal surfaces
*Ice is evaporated in vacuum by freeze-drying
85
Metal Shadowing (In TEM)
*Deep-etched or normal samples are sprayed with a thing coat of a heavy metal at an angle (important)
*This creates a 3D-like dimension and shadows corresponding to different parts of the cell.
86
What if we want a higher resolution image of a macromolecule?
You can if you give up looking at the rest of the cell and work with purified proteins (or other macromolecules or particles).
87
Negative Stain Electron Microscopy Steps
1. Macromolecules on a thin film of carbon
2. Coated with heavy metal salt (uranium acetate) and dried
3. Salt covers the carbon film except where macromolecules are present
4. Thus, it is a negative image (dark is absence of molecule).
88
Negative stain electron microscopy is used for ___
Macromolecules not the whole cell
89
Cryoelectron Microscopy (CryoEM)
Resolving structure of single molecules (purified samples).
Doesn’t use metal labeling of sample.
90
Cryoelectron Microscopy (CryoEM) Steps
1. Take suspension of purified sample (viruses, ribosomes, etc)
2. Flash-freeze on the microscope grid as a thin film (100 nm)
3. Freezes so quickly with no ice crystals forming
4. Imaging done in a vacuum, at -160°
Electrons pass through frozen water easily; Electrons are deflected by the object, so image shows loss of deflected electrons.
91
Problem and solution of CryoEM
PROBLEM: Weak electron beam is used to avoid breakdown of organic sample results in high-noise background.
SOLUTION: acquire thousands of images and have a computer construct an average image.
