Lecture 8 - Microscopy Flashcards
(55 cards)
1
Q
What can you see with naked eye?
A
can see from 100um to 1m+
2
Q
what size can you see through light microscope (includes fluorescence microscopy)?
A
- thickness of hair, cell, bacterium
can see from 100nm to 1mm
3
Q
What size can you see through electron microscopy?
A
- virus, macromolecules, small molecule, atom
can see from 10^-10m to 10um
4
Q
What are the 3 necessary elements for imaging?
A
- source of illumination (light or electrons)
- a specimen to be examined
- a system of lenses to focus the illumination on the sample and form an image
5
Q
What was Robert Hooke’s compound microscope?
A
- microscope with 2 lenses
- light microscope that uses visible light and glass lens to form image that can be detected by eye or camera
6
Q
explain the components of the light microscope
A
- Light source - illuminate sample
- condenser lens - in front of light source to focus light at desired point on the specimen
- specimens
- Objective lens - forms primary image of specimen (lens closest to object of interest
- intermediate lenses
- Ocular lens - magnifies the primary image produced by objective lens
- Magnification - refers to the size of image
7
Q
What is magnification a product of?
A
- product of the magnification of the individual lens
ex. objective lens is 100x and ocular lens is 10x = 1000x magnification
8
Q
What is resolution
A
the minimum distance 2 points can be apart and remain apart
9
Q
Magnification vs. Resolution
A
- without resolution, details won’t be visible (empty magnification)
- resolving power (resolution) allows objects to be distinguished
- limit of resolution (how far apart objects must be to appear as distinct)
- the smaller the limit of resolution, the greater its resolving power
10
Q
Why can we see smaller things with an electron microscope?
A
- longer wavelength = larger objects observed (small samples not seen as it has no affect to wavelength)
- shorter wavelength = smaller objects observed (large samples not seen)
- Light microscopy has longer wavelength and Electron microscopy has shorter wavelengths
this is why we can see smaller things with an electron microscope
11
Q
what does resolution consider?
A
- wavelength of illumination
- refraction index
- angular aperture
12
Q
explain angular aperture
A
- half angle of the cone of light entering the objective lens
- measure of how much illumination that leaves the specimen passes through the lens
13
Q
explain focal length
A
light (parallel rays) go through midline of lens, then these rays come together after a certain length (the focal length!) till it comes together to a point (called focal point!)
14
Q
what is the refractive index?
A
- measure of the change in the velocity of light as it passes from one medium to another
- relative velocity of light in the medium compared with the velocity in air
15
Q
What is the Abbe Equation?
A
r = (0.61λ) / (n sin α)
n sin α = NA
r = (0.61λ) / NA
r = resolution
λ = wavelength of illumination
n = refractive index
α = angular aperture
- the higher the NA, the better
16
Q
What is the 0.61 in the Abbe equation?
A
- constant that represents the degree to which image points can overlap and still be recognized as separate
17
Q
explain visibility in light microscope
A
- lighting: in the macroscopic world, we see how light reflects off an object. In the microscopic world, we view the light that is transmitted through
- constrast: difference in appearance between an object and its background
18
Q
explain the 6 different types of light microscopy (briefly)
A
- unstained
- stained
- phase contrast
- differential interference contrast
- fluorescence
- confocal
19
Q
Explain bright-field microscopy (unstained)
A
passes light directly through specimen, unless cell is naturally pigmented or artificially stained, image has little contrast
- visualizing white light passed through
- living samples can be examined
- live cell and tissues lack compounds that absorb light and are nearly invisible
20
Q
explain bright-field microscopy (stained)
A
staining with various dyes enhances contrast but need to be fixed cells (dead)
- can make thin, translucent specimens visible
- stain with dye that absorb a specific wavelength
- different dyes bind to different biomolecules
ex. Feulgen stain specific for DNA
21
Q
Explain fixation
A
preserves cells; prevents degradation
- using formalin and formaldehyde
22
Q
explain section specimen
A
fixed and embedded tissue that is cute into thin pieces and placed on a slide
23
Q
explain whole mount specimen
A
an intact object placed on a slide
24
Q
What are Haematoxylin and Eosin (H&E)?
A
- a common routine stain in histology that stains nuclei blue (due to Haematoxylin) and cytoplasm pink (due to eosin)
- Haematoxylin stain is basic dye and it stains acidic
- Eosin is acidic and dyes proteins
- easy to carry out on paraffin or frozen sections
FUN FACT! in histopathology, a high proportion of diseases can be diagnosed by using an H&E stain alone
25
explain phase-contrast microscopy
enhances contrast in unstained cells by amplifying variations in refractive index within specimen; especially useful for examining living, unpigmented cells
- takes advantage of differences in refractive index and thickness to image living cells without the need to section and stain
- converts phase differences into alterations in brightness
- most useful for examining dynamic events (movement of organelles within cell)
26
explain differential interferences contrast
- uses optical modifications to exaggerate differences in refractive index (enhanced contrast)
- has a shadow-casting effect that makes cells appear dark on one side and light on the other as results of diff. in the optical path due to phase
- 3D effect
27
explain fluorescence microscopy
- detects fluorescent proteins or dyes to show locations of substances in the cell
- energy from external source of light (photons) is absorbed and immediately re-emitted
- fluorophores absorb light
28
explain confocal scanning microscopy
- uses a laser beam to illuminate a single plane of a fluorescently labeled specimen
29
explain excitation filters
transmits only light of a particular wavelength
30
explain dichroic mirror
reflect light below a certain wavelength and transmits light above a certain wavelength
31
explain emission filter
prevents light that does not match the emission wavelength from exiting the microscope
32
explain the epifluorescence light path
1. light source from mercury lamp
2. goes through excitation filter that transmits only desired wavelength
3. through objective lens to specimen
4. light bounces back up to objective lens and to dichroic mirror which transmits light above 510nm (what we see)
5. this transmitted light goes to emission filter that blocks unwanted signals and comes to us!
33
what are the approaches to fluorescent microscopy?
1. Fluorescent Protein technology: green fluorescent protein is fused to "your favourite protein" and visualized in the cell
2. Immunohistochemistry: a method of detecting proteins with a primary antibody and fluorescent secondary antibody (indirect) - detects when it binds
3. Fluorescent Stains: there are fluorescent dyes that associate with specific cellular structures
34
what is GFP and why is it revolutionizing cell biology?
- Green Fluorescent Protein
- to visualize patterns of gene expression and subcellular localization of proteins in living cells and organisms
- Shimormura originally isolated the GFP protein from jellyfish
- got 2008 noble prize in chem with Chalfie and Tsien
35
What did Chalfie demonstrate?
- showed that GFP in its fluorescent form can be expressed in both E.coli and C. elegans without addition of auxiliary heterologous factors
- showed that the feasibility of GFP as a universal genetic marker
36
Explain Transcriptional/Promoter Fusion and Translational fusion
Transcriptional
- promoter+GFP
- promoter drives GFP expression in 6 neurons
- promoter determines where it gets expressed
- GFP expressed to mRNA then to a GFP protein 27kDa (green cell)
Translational
- Promoter+goi+GFP
- becomes mRNA+goi+GFP then a fusion protein!
- goi cannot have a stop codon so that the GFP can also be transcribed
37
what happened to Doug Prasher?
cloned GFP, struggled with grant funding and quit academic science
so sad
38
What can you do with proteins and GFP?
- proteins can be tagged with GFP and used to look at the subcellular localization of the fusion protein
- gene X + GFP
- becomes one long transcript (translational fusion)
39
Explain DAPI and Phalloidin
- DAPI stains nuclei blue
- Phalloidin stains cytoskeleton green
40
How did Tsien create the rainbow palette? <3
- mutated GFP and isolated variants that shone in diff colours
- added red fluorescent protein from Discosoma (REDDD) then took variants of this too for diff. shades
- using this, you can use these variants to image more than 1 protein in the same image (diff colours)
41
What are the advantages and disadvantages of GFP?
Advantage
- cellular events can be observed in living cells
- can follow dynamic events over time
Disadvantage
- GFP is a 27kDa protein that might negatively affect your "fav protein" (could kill it)
- introducing the GFP-tagged protein/gene into cells
42
what does immunostaining use?
antibodies!
43
What are antibodies
- y-shaped proteins produced by cells of immune system
- bind with very high specificity to antigens
- immune system uses this to bind to and neutralize bacteria and viruses
- can raise antibodies against a protein of interest by injecting the protein into an animal (usually mouse, rat or rabbit), generate immuno response, then purify the antibody from sera
44
what is direct immunofluorescence microscopy?
- dye is coupled directly to primary antibody
- this antibody is then applied to cell, tissue sample, embryo, etc where they bind to target
- usually fixed cells
- fixation conditions aim to preserve cellular morphology and the target antigen (epitope)
45
What is indirect immunofluorescence microscopy?
- more common
- dye is conjugated to secondary antibody
1. primary are added to sample where they recognize and bind (no dye)
2. secondary with dye are added, binding to primary
46
What are the advantages of indirect over direct immunofluorescence?
- can commercially buy secondary antibodies coupled to a whole range of fluorescence dyes
- amplifies signal (multiple secondary recognizing primary - direct only has one)
47
What are the advantages and disadvantages of immunofluorescence microscopy?
Advantages
- high specificity
- strong signal (from indirect)
- identifies endogenous proteins in native environment
- can stain more than one protein at a time (distinguish using diff. secondary antibodies)
Disadvantages
- done on dead cells
- limited availability of primary antibodies
48
Explain the process where confocal microscope produces optical sections
- ability to produce in-focus images of thick specimens (optical sectioning)
- excludes out-of-focus light
- fluorescent specimen is illuminated with a focused point of light (specific) from a pinhole
- uses a laser with specific nm
- emitted fluorescent from in-focus point is focused at pinhole and reaches detector (where we see)
- emitted light from out of focus point is not at pinhole and blocked (will not see)
- image acquired point by point then reconstructed with computer
49
briefly explain FRAP
- "bleach" area and see how long it takes for unbleached mol. to return
- measure how fast a molecule moves
50
what is FRET?
- fluorescence resonance energy transfer
- living cells
- when dyed mol are close together, the emission of one fluorophore can be used to excite the second
- measures if two mol. are touching
ex. emission spectra of Cyan Fluorescent Protein will excited Yellow FP
51
What is electron microscopy?
- a beam of electrons and electromagnets, rather than light and glass lenses
uses SEM and TEM
- theoretical limit of resolution of transmission electron microscope is around 0.003 nm
- the practical limit is higher, because the NA is very small
- gives a better resolution
52
What is SEM?
- scanning electron microscopy
- surface of a specimen is scanned by detecting electrons deflected from the outer surface
- provides 3D image of surface of an unsectioned specimen
53
What is TEM?
- transmission electron microscopy
- electrons transmitted through the specimen
54
How do prepare samples for EM?
- very thin fixed sections, 50-100 nm thick, using common fixatives (glutaraldehyde)
- samples embedded in resin that dehydrates to form solid block (then cut to thing sections)
- usually stained with heavy metals (electron dense) which will scatter electrons
- can not study live cells
- challenge to determine how the fixed sample resembles the original
55
What happens in immuno EM?
- antibodies are conjugated/linked to substances that are electron dense
- ex. AB coupled to gold
