Lecture 2- Microscopes Flashcards
1
Q
What did Antony Van Leeuwenhoek do?
A
Saw protozoa and bacteria for the first time
1838
2
Q
Who came up with cell theory?
A
Schleider and Schwann… in 1838
big gap from 1600s and 1800s bc textile revolution lead to dyes
3
Q
Reticular vs. Neuronal Theory
A
Reticular theory: neurons are not cells (like vascular network)
Neuronal theory: neurons are cells but atypical
4
Q
100 microns
A
can see with eye, plant cell
5
Q
10 microns
A
typical animal cell
can be seen with light microscope
6
Q
1 micron
A
mitochondria, bacteria
can be seen w light microscope
7
Q
100 nm
A
viruses and ribosomes
can be seen with electron microscope
8
Q
10 nm
A
proteins
can be seen with electron microscope
9
Q
1 nm
A
molecules
can be seen with electron microscope
10
Q
What are the two major choices when selecting a microscope?
A
- Which microscope to select (there are several, each with its own niche)
- How to process cells tissue (fixing/staining)
11
Q
What is a fixed cell?
A
cell that has been chemically treated to preserve its structure and essentially “kill” it, making it suitable for staining
12
Q
What is resolution?
A
the ability of a microscope to distinguish details of a specimen or sample. It’s defined as the minimum distance between two distinct points on a specimen that can still be seen as separate entities by the observer or microscope camera
ability to see two dots as distinct from each other
if resolution increases, a single dot will be seen as two smaller dots
13
Q
What did Abbe do?
A
One year after beginning the manufacture of the Carl Zeiss
compound microscope, in 1873, Ernst Abbe released a scientific paper describing the mathematics leading to the perfection of this wonderful invention. For the first time in optical design, aberration, diffraction and coma were described and understood. Abbe described the optical process so well that this paper has become the foundation upon which much of our understanding of optical science rests today. As a reward for his efforts Carl Zeiss made
Abbe a partner in his burgeoning business in 1876.
14
Q
What is Abbe’s equation
A
math describing theoretical resolution
15
Q
Theoretical vs practical limit of resolution
A
- Theoretical: Abbe’s equation, tell u the best resolution a microscope could acheive
- Practical: What can actually be acheived, as cells aren’t good canidates so you can’t see as much as theoretical
16
Q
How was Abbe’s rule overcome?
A
Eric Betzig, Stefan W. Hell and William E. Moerner are awarded the Nobel Prize in Chemistry 2014 for having
bypassed a presumed scientific limitation stipulating that an optical microscope can never yield a resolution better than 0.2 micrometres. Using the fluorescence of molecules, scientists can now monitor the interplay between individual molecules inside cells; they can observe disease-related proteins aggregate and they can track cell division at the nanolevel.
superresolution microscopy
17
Q
Why are cells bad candidates to be viewed under a microscope?
A
- Cells are mostly water, have little contrast
- cells are organic cells that absorb light, which leads to heat, which leads to movement
18
Q
Abbe’s equation (formula)
A
d=.61 wavelength/nsin(theta)
d=resolution
19
Q
relationship between d and λ
A
- D (resolution) is roughly half the wavelength of the imaging radiation
20
Q
Super resolution microscopy
A
- Super-resolution microscopy (SRM) is a technique that uses fluorescence microscopy to image cellular structures with more detail than conventional optical microscopy. SRM can achieve resolutions up to 20 times greater than conventional light microscopy, which has a resolution limit of about 200 nanometers
Is not limited by Abbe’s equation
21
Q
What ways is the problem of contrast in microscopy overcome?
A
- Dyes (colorimetric and fluorochromes)
- Light Manipulation (ex. phase and DIC)
- Computer enhancement
22
Q
What is hematoxylin?
A
primarily used in histology as a stain to color cell nuclei a deep blue-purple color
colorimetric dye
23
Q
What is eosin?
A
an acidic dye that stains the cytoplasm, muscle, and connective tissues in various shades of pink and orange
colorimetric dye
24
Q
Light microscope resolution limit
A
0.2 microns
25
Electron microscope resolution limit
2.4 angstroms
26
Who was Carl Zeiss
Businessman who worked with Abbe to refine microscopy (Zeiss optics)
27
Clarapath Introduces Robotic Tissue AI Processing/Staining System
* Clarapath said pathology labs currently rely on labor intensive, manual procedures to process tissue specimes for purposes of diagnosing cancer and other diseases.
* Faced with labor shortages, quality control challenges, and rapidly increasing sample volumes, pathology labs seek to improve the reliability and efficiency of creating glass slides for pathological review
| partners w mayo clinic
28
Bright-field microscopy physical properties
* light from a tungsten lamp is focused on the specimen by a condenser lense below the stage
* light travels yellow pathway
29
What is the oldest yet most often used type of microscopy?
bright field microscopy
30
Who uses bright field microscopy?
* pathologists
* cytochemists
* for dead tissue sections or dead cells
31
Steps to prepare cells for bright field microscopy
1. Fixation (killing cells to keep in lifelike state using chemicals like formaldehyde, which cross links proteins)
2. Dehydration (remove water but replace w ethanol)
3. Xylene replacement (makes tissue transparent and easier to read on slides)
4. Parrafin Infiltration (replace xylene with paraffin, results in wax block w tissue held in stasis, allowing sections to be cut)
5. Microtome cuts slices 10-15 microns
6. Put sections on slide
7. Remove wax, put cell back in water
32
Examples using bright field microscopy
* retina
* thyroid gland
* Covid lung fibrosis (can see lymphocytes and scar tissue)
* Basal cell carcinoma (skin cancer islands)
33
Cryostat use
* often used in operating rooms for quick analysis
* not typical for pathology
* ex. Moh's surgery (for melanoma, skin removed in consecutive rings until margin is found, rings are quickly analyzed)
| rather than paraffin, sections are quickly frozen
34
Who uses phase-contrast microscopy?
cell culture biologists
35
What is the main purpose of phase microscopy?
To look at living cells without fixation or dyes
## Footnote
good for single cells or thin cell layers, byt not thick tissues... particularly useful for examining the location and movement of larger organelles in live cells
36
How does phase microscopy work?
* Contrast created by light interference
* Recombines refracted and unrefracted light
* If light is in sync (in phase), it is made brighter
* If light is out of sync (out of phase), it is diminished
37
What did Robert Hooke do?
first to see cells- named them after monk's quarters
38
Differential Interference Contrast/Nomarski
* used for living cells
* different from phase bc u get a 3D image
39
Differential Interference contrast applications
* Single cell electrophysiology (use pipette and poke cell to look at change in membrane potential)
* Patch clamping (Monitors ion flow through single cell membrane channels inside out and outside-out)
40
Darkfield microscopy
* Used by microbiologists
* Dark field w bright image
* not increasing resolution, but increasing contrast w dark background
* can see bacteria very clearly, better than bright field/phase
41
How does polarizing light microscopy work
* Light passes through a polarizer (only light of one plane passes through)
* Polarized light hits specimen
* Highly ordered parallel specimen changes light
* changed light is observed to show specimen
42
What is polarizing light microscopy used for
* neurobiologists (microtubules)
* muscle cell biologists (actin/myosin)
* both are highly ordered structures
43
Why do microscopes have multiple capabilities in one machine
* allows for overloads of images
44
Why was confocal microscopy a revolution?
* greatly increased the theoretical limit of resolution
* governed by abbes equation still
45
Confocal microscope history
* 1957- original patent filed for theoretical design
* 1970s- idea starts maturing, tech still needed to be developed
* 1988- commercial launch of first confocal microscope
46
What are the components of confocal microscopy?
* monochromatic lasers (around 488 nm)
* confocal pinholes to narrow laser beam diameter (point by point scan of image is used to make the image more defined)
* Computer to display summed image
## Footnote
also needs fluorochromes
47
What were the advantages of confocal microscopy?
* More precise bc less stray imaging
* optical sectioning (allows for thin section images w/o mechanical slicing by removing out of focus light)... these can be z-stacked and added to make 3D cell
* Stereo/3D images
* Multiple labeling for differentiation of structures, allows to see more in living cells than before
48
What is the problem with confocal microscopy?
flurochromes can be photobleached by the laser (once a photon is emitted it can't be emitted again)
49
Spinning disk confocal microscope advantages
* can be better used for living cells bc much faster scanning, less lazer intensity/ heat (doesn't kill cell), and dynamic movement more visible (can reveal changes in endoplasmic reticulum)
* Reduced photobleaching
50
What is vivascope?
* handheld confocal imaging system used to view skin anomalies
* non-invasive
* can directly scan problem on skin and get results
51
"3D imaging Technique Visualizes Lung Tissue from COVID-19 Patients"
* new multi-scale phase contrast X-ray tomography is a new x-ray microscope technique
* scientists visualized large areas of lung tissue embedded in wax blocks like CT scans (which don't have required resolution)
rather than constructive and destructive light interference to generate contrast, they used different propagation velocities of C-rays to generate the intensity pattern
* technique can also map the 3D distribution and density of lymphocytes infiltrating the diseased tissue
52
What does deconvolution microscopy do
uses an algorithm to create a 3-D view of fluorescently stained cells
53
What is vital fluorescence microscopy?
* the use of fluorochromes to measure changes in cell behavior
* dyes are chemically modified w special group that allows permeability through cell membrane and detaches once inside the cell
54
What are vital dyes?
* flurescent dies that don't kill the cell
* diffuse easily
* are membrane insoluble once inside the cell
* include JC-1, Calcein-AM. and Fluo3-AM
55
What dye is used to visualize mitochondria activity?
* JC-1
* green=monomers=low proton motive force
* red= J-aggregates=high PMF
56
What dye is used for "live-dead" assays?
Calcein-AM (green)/ Propidium Iodide (red)(PI can get into the nucleus)
57
What dye is used to measure intracellular calcium concentrations in living cells?
Fluo3-AM (green)
58
What is microspectrofluorometry?
* qualitative assessment of fluorescent probes
* modifying images to allow for better understanding of results
* cells can be genotypically the same but w different phenotypes
59
What are plate reading spectroflurometers for?
* quantitative assessment of fluorescent probes
* sum the fluorescent signal from a large group of cells (u get control and compare experimental data)
60
What does FRAP stand for
Fluorescence recovery after photobleaching
61
What does FRAP do
* reveals exchange rates or membrane fluidity
* can show lateral fluidity of cell membrane proteins
* Works by illuminating laser on one spot (photobleached) and the spot will fill in
62
What is TIRF microscopy
* developed to see the edges of cells that you can't always see in the plate
* Total internal reflection fluorescence microscope
63
What is intracellular injection of lucifer yellow used for
* tracing neurons in vivo- you shoot in the dye when doing electrophysiology so you cal tell which exact cell you are working with
* allows you to tell the electrical connectivity between non neural cells (gap junctions, where ions can be passed)
* Requires: vital dye that is: fluorescnet, diffuses easily, and membrane insoluble
64
What is fluorescence immunocytochemistry
use of antibodies to identify proteins in cells
| can reveal cell polarity
65
# immunocytochemistry
What is an antigen
protein of interest (what we want to track inside the cell)
66
# immunocytochemistry
What is an epitope
part of the antigen that the antibody binds to
67
# immunocytochemistry
What is an antibody
a cell that responds to the antigen (bivalent- meaning 2 antigen binding sites)
68
# immunocytochemistry
specificity vs affinity
* specificity- the antibody you want to use should only bind to your POI and no others
* affinity- how well the antibody binds to the POI
69
# immunocytochemistry
Direct technique
* antibody enters cell with attached flurochrome
* binds to POI and fluoresces
70
# immunocytochemistry
Indirect technique
* primary antibody enters first
* secondary antibody enters and binds to primary antibody and fluroesces
* more complicated, but less likely to lose specificity and affinity of your primary antibody
71
Polyclonal antibodies
* high chance of cross reactivity
* supply ends w death of the rabbit
* less specific than monoclonal
* not used anymore in bio/medicine
| solution: monoclonal antibodies
72
Monoclonal antibodies
* high specificity and affinity
* can cryopreserve and thaw, so have better supply chain than polyclonal
| werent patented, now among top selling biologics in world
73
How are polyclonal antibodies made
* inject antigen into rabbit
* antigen activates B cells
* Plasma B cells produce polyclonal antibodies
* Obtain antiserum from rabbit containing polyclonal antibodies
74
"The process of generating monoclonal antibodies wasn't patented"
* In 1975 Kohler and Milstein gave a presentation of the process of making mAbs w a paper pending in Nature
* Vickers realized its importance and tried to patent the process before it was published in Nature but the UK patent process was too slow and the paper was published thuse preventing patent fliling
* Case was a big concern w Prime Minister Margaret Thatcher getting involved who was a chemist by training
75
Bispecific antibodies
* are trifunctional (bind tumor cells and immune cells and can link T cells to other cells)
* generated using quadroma cell lines
* have two different fab (antigen binding) ends
* used for treating cancer
76
What was the first FDA approved bispecific trifunctional antibody?
* catumxomab
77
"REGEN-COV Antibody cocktail is active against sars-Cov-2 variants- first identified in the uk and south africa"
* Bamlanivimab (Eli Lilly's mAb) recieved emergency use authorization from the FDA.
* 2 weeks later REGN-COV2 (Regeneron's mAb) granted EUA
* FDA says mAbs should be given asap after symptoms emerge and person tests positive. Bc of limited supply limited to high-risk patients
* mAbs in this regard work by binding to the spike protein, preventing the virus from binding to the ACE2 receptor
78
What mAbs in REGN-COV2
casirivimab and imdevimab
79
What is ELISA?
* enzyme linked immunosorbent assay
* used for quantifying a protein of interest
* can be colorimetric or fluorescent
80
What are the 2 types of cell death
necrosis and apoptosis
81
What does annexin V do
tags apoptotic cells by binding to phophatidylserine (a phospholipid)
82
What is necrosis
* pathological cell death
* cells explodes from something external like poison
83
What is apoptosis
* genetic cell death, internal
* ex. chemo, radiation, T cells
* cell membrane flips inside out (inner and outer leaflet)
84
What is GFP?
* Green Fluorescence Protein (from jellyfish)
* Can reveal protein expression in whold organisms as well as cells
* Used to see if a gene of interest is being expressed or not- "reporter molecule- tells you if something is happening"
* Chimeric gene including GOI + GFP is transfected into a cell (is never perfect/never 100% of cells are infected)
* other derivates of GFP now made in different colors so u can see 2 things at once
85
Regulated vs Constitutive reporter (GFP)
* constitutive reporter: is always on, leading to continuous expression of GFP (often used for labelin cells or protein localization)
* regulated reporter- expressed in response to specific stimuli/signals, gene is placed under control of specific promoter (hormones, drugs, etc)... good for studying gene regulation/signaling pathways (cellular responses to stimuli)
86
"Human-Pig Chimera Embryos Detailed"-GFP labeled human cells can be tracked
* Currently in sever organ shortage-one solution may be to generate human organs in other large mammals
* Researchers have successfully created chimeric embryos containing a combo of human and pig cells
* When transferred into surrogate pig mothers, the developing humanized kidneys had normal structure and tubule formation after 28 days.
* First time scientists have been able to grow a solid humanized organ inside another species
* Used DsRED (Red fluorescent protein) to track the human cells
87
What does FRET stand for
Forster Resonance Energy Transfer
88
What is FRET's purpose?
* designed to signal when two molecules are within close proximity
* molecules have to be within 1 and 10 nm of each other to work
* can also be used to look at enxyme activation as well (can tell when proteins are phosphorlyated- shape changes) by attaching cyanFP and yellowFP to different parts of the protein
89
How does FRET work?
* Proteins are each marked with different GFP-family proteins (cyan and yellow)
* when the proteins bind (1-10nm apart), only one GFP is visible (yellow)
* can also be used to see when proteins change shape
90
What are FRET biosensors and how do they work?
* biosensor: protein that reveals a change in cell behavior
* Calmodulin: changes shape when there is a large change in Ca2+ concentration within a cell
* FRET can be used to determine when this change occurs, serving as an indicator for calcium concentration
## Footnote
known as inomycin-indued activation of calmodulin (a calcium binding protein)
91
What is GFP/YFP/CFP and how does it work?
* Fluorescent Protein (green, yellow, cyan)
Fused to a protein or gene (chimeric gene) of interest and transfected into a cell. When expressed, the attached fluorescent protein emits a signal allowing researchers to track the target.
* Can be regulated reporter (sometimes on) or constituent reporter (always on)
* Fluorescnece can be quantified to measure gene expression levels
92
What research questions does GFP/CFP/YFP address/solve?
* can reveal protein expression in whole organisms as well as cells
* can measure gene expression levels or visualize cellular processes like protein trafficking, interaction, or localization
| ex. viewing human cells in humanized kidneys in pigs
93
What is audioradiography?
* trace molecule or process in cells over time using a radioactive protein
* ex. cell division: DNA syntthesis can be probed by 3H-Thymidine. Cells in culture- radioactve 3H-Thy will enter cells that undergo DNA synthesis. Then, film used to reveal radioactive decay partcles coming off of cell. Can be used to identify dividing cells in tumor.
* Other probes include amino acids (for protein synthesis): 3H-leucine, 35-Methoine
| around since 70s
94
What does FISH stand for?
* Fluorescence in situ hybridization
95
What does FISH do?
* Uses fluorochromes to identify mRNA of interest and DNA seq of interest... fluorochromes are put on RNA strand complimentary to mRNA OI
* Ex. detecting HPV, chromosome spread
96
What are the 4 types of single cell intracellular injection?
* single cell micropipette intracellular injection
* electroporation
* liposomes/nanoparticles
* viral transfection
97
# Intracellular Injections
Single cell micropipette intracellular injection
* living cell is punctured by the pipette and things are injected
* used for somatic cell nuclear transfer (transplanting nucleus of a body cell into an egg cell to create an embryo-used to clone): suction pipette holds cell, another pipette injects cell
* advantages: SCNT requires it
* disadvantage: can only be done one cell at a time
98
# Intracellular Injections
Electroporation
* uses controlled, millisecond electrical pulses (transient electrical charge) to induce temporary pores in the cell membrane allowing for diffusion into cell. cell membrane reseals and left unharmed
* can inject many cells at once but only some cells can survive (never 100% effective)
99
# Intracellular Injections
Liposomes and Nanoparticles
*
100
# intracellular injections
Viral tranfection
* can only bring genes into the cell
* not FDA approved
* virus carrying target DNA inserts itself into the genome of the target cell
101
What are the two types of electon microscopy?
* transmission electron microscope: "transmits" electrons through the specimen (most common)
* scanning electron microscope: "scans" the image
| resolution=2.4 Angstrom
102
How does transmission electron microscopy work?
* "Transmits" electrons through the specimen
* for visualizing internal structures
* techniques: plastic thin sectioning, freze fracture, ultrastructural immunocytochemistry, ultrastructural autoradiography
103
TEM vs light microscope properties
* Illumination: TEM= electrons; lm=light
* Tissue sections: 50-90nm (thin); lm=10-15um
* Lenses: TEM=electromagnetic; lm=glass
* Imaging: TEM=screen (easier to see); lm=eye
104
HVEM
* high voltage electron microscope
* theoretical limit of resolution is better because accelerating voltage is 1000kv, not 100kv as in typical TEMS
* but best used for imaging thick sections
105
# TEM
plastic thin sectioning process
1. Fixation (to keep in life-like state) using glutaraldehyde (crosslinks proteins) or OsO4 (cross links phospholipids)
2. dehydration
3. infiltration w epoxy plastic
4. cut ultra-thin sections using microtome (diamond knife, float sections) (50-90nm)
5. stain (sections on a copper grid, stain w heavy metals...lead for membrane, uranium radioactive counterstain for everything else)
106
# TEM
Freeze Fracture
* sees interior structure of membranes
* cells frozen and then fractured
* exposed surface sprayed with platinum and carbon, then cell is dissolved
* this creates a mold of what was in the cell
107
# TEM
Ultrastructural immunocytochemistry
* idenfity POI w/ mAbs
* fluorochromes swapped with gold particles
108
# TEM
negative vs positive staining
* positive staining: sample is stained, resulting in a dark image of the sample against a light background
* negative staining: background is stained, leaving sample untouched and visible against a dark background
109
Scanning electron microscopy
* scans the image using electrons
* can only see surface of specimen
110
How is resolution different for electron microscopes vs light microscopes?
* still limited by Abbe's equation but...
* wavelength of elecrons depends on their acceleration (voltage)
* 100,000 volts d=2.4 angstroms (lm=.2 um)
* shorter wavelength is better resolution (faster electrons/higher voltage)
111
Super Resolution microscopy
* includes SIM, STED, and PALM
* won nobel prize for breaking through Abbe's diffraction limit
* can Z stack like confocal for 3-D imaging
