Study Guide Terms Exam 1- Microscopy

Question 1

What is Cyvenio and how does it work?

Answer 1

• liquid biopsy device for the immunomagnetic and microfluidic capture of CTCs
• Uses antibodies that bind to proteins on CTCs surface. The antibodies are attached to magnetic particles, so once the antibodies bind to the CTCs the particles are separatd from the rest of the blood using a magnetic field
• Is more specific than vortex but may miss CTCs that do not have the marker

Question 2

What research question does Cyvenio address?

Answer 2

• cancer progression and metastasis
• treatment efficacy
• cancer detection and monitoring

amount of CTCs correlated to cancer progression/if cancer has spread

Question 3

What is Vortex and how does it work?

Answer 3

• device for microfluidic extraction of CTCs from liquid biopsies
• relies on physical characteristics of CTCs to capture them in a microfluidic vortex
• WBCs and RBCs move down the river faster leaving CTCs behind. They are collected and counted- no antibodies used to count

Question 4

What research question does Vortex address?

Answer 4

• CTC collection (same as Cyvenio)
• But does not use antibodies and can capture a broader range of CTCs

Question 5

What is CellInsight CX5 instrument and how does it work?

Answer 5

• automated imaging system designed for high-throughput analysis of cells using fluorescent and brightfield microscopy to capture high-resolution images of cells in multi-well plates
• can scan 1000s of samples automatically
• extracts quantatative data on cell morphology, intracellular markers, protein expression, and other metrics

Question 6

What research questions does CellInsight Cx5 attempt to address?

Answer 6

• drug discovery: researchers can study how potential drug candidates affect cellular phenotypes such as cell cycle progression/mitosis
• Toxicology studies: can assess how chemicals or drugs impact cell viability/structure/function
• Cell morhphology and phenotype changes

Class example: used to see if GVA halts cells in mitosis

Question 7

What is brightfield/compound light microscopy and how does it work?

Answer 7

• oldest and most often used microscope, used to look at dead cells and tissue sections (often used by pathologists and cytochemists)
• resolving power of .2 microns
• Process:
• 1. fixation using chemicals like formaldehyde (cross-links proteins)
• 2. dehydration, replace H2O w ethanol
• 3. xylene replacement
• 4. infiltration w paraffin- results in wax block w tissue held in statis
• 5. microtome to cut 10-15 micron sections
• 6. place sliced sample onto slide
• 7. remove wax and return sliced sample to being filled with water

Question 8

What research questions does bright field microscopy attempt to address?

Answer 8

• Often used in pathology and cytochemistry- used for dead tissue sections/cells
• Best for specimens that naturally contrast with their background or can be easily stained (hematoxylin for nucleus, teosin for cytoplasm)
• Lasks intracellular detail

Question 9

What is a cryostat and how does it work?

Answer 9

• easier to get tissue samples cut than paraffin
• sections are quickly frozen

Question 10

What research questions does a cryostat attempt to address?

Answer 10

• often used in operating rooms for quick analysis- not typically used for pathology reports
• Ex. Mohs surgery- skin removed in consecutive rings until margin is found and rings are quicklly analyzed

Question 11

What is a phase microscope and how does it work?

Answer 11

• light microscope, contrast created from light interference- adds wavelengths in sync to enhance or out of sync to diminish
• used by cell culture biologists, designed to look at living cells without fication or dyes

Question 12

What research questions does phase microscopy attempt to address/solve?

Answer 12

• used in living cells w/o fixation or dyes (non-invasive, preserves natural cellular behavior)
• allows researchers to observe finer details of live cells that would otherwise be difficult to see in brightfield microscopy (enhanced contrast, idealfor trasnparent spcimens that lack contrast under brightfield microscopy)
• Real-time dynamics

Question 13

What is Differential Interference Contrast/Nomarski microscopy and how does it work?

Answer 13

• light microscope that enhances contrast of transparent unstained specimens
• converts phase shifts into variations in light intensity
• provides 3-D image

Question 14

What research questions does DIC/Nomarski optics attempt to address/solve?

Answer 14

• Provides pseudo-3D images that highlight surface details and edges
• Applications include single cell electrophysiology (slender pipette and poke cell to look at change in membrane potential) and patch clamping (monitors ion flow through single cell membrane channels)

Question 15

What is patch clamping and how does it work?

Answer 15

• Glass micropipette with a very fine tip used to form a tight seal with the cell membrane, suction is applied
• membrane can be pulled to study of intracellular side of ion channels (inside-out) or extracellular side (outside-out)
• amplifier connected to pipette controls voltage accross the membrane or can measure the current that flows through ion channels

Question 16

What research questions does patch clamping attempt to address/solve?

Answer 16

• ion channel function
• action potential generation/signal propagation
• NT and drug effects on ion channel activity
• synaptic transmission

Question 17

What is darkfield microscopy and how does it work?

Answer 17

• illuminates specimen with scattered light from the specimen
• creates bright images of the sample against a dark field
• useful for small/thin structures that would otherwise be difficult to see using brightfield microscopy

Question 18

What research questions does darkfield microscopy attempt to address/solve?

Answer 18

• often used by microbiologists because it can detect very small particles/microorganisms that scatter light
• can see bacteria very clearly, much better than bright field/phase

Question 19

What is point scanning confocal microscopy and how does it work?

Answer 19

• confocal microscope (type of microscopy governed by Abbe’s equation with much better theoretical limit of resolution than light microscopes
• Uses confocal pinholes to narrow a monochromatic laser beam and point-by-point scans, then uses a computer to display summed images

significantly improved visualization of cells bc removed out of focus light

Question 20

What research questions does Point Scanning Confocal microscopy attempt to address/solve?

Answer 20

• More precise imaging
• Can do optical sectioning: can Z stack data and generate 3-D image
• Can use multiple labeling for structure differentiation- can see a lot more in living cells than before (including 3D cellular architecture)
• Especially useful for thick/complex specimens

but fluorochromes photobleach when radiated by laser

ex. allowed for invention of VivaScope for skin issues (handheld)

Question 21

What is spinning disk confocal microscopy and how does it work?

Answer 21

• differs from point scanning by using large number of pinholes arranged in spiral pttern and spun rapidly
• works much faster

Question 22

What research questions does spinning disk confocal microscopy attempt to address/solve?

Answer 22

• High speed imaging-fast dynamic processes in live cells (such as endoplasmic reticulum changes)
• reduced phototoxicity due to collecting light more efficiently
• larger scale 3d imaging

Question 23

What is polarizing light microscopy and how does it work?

Answer 23

• Uses interaction of polarized light with structures that have optical anisotrpy (different optical properties in different directions)

Question 24

What research questions does polarizing light microscopy attempt to address/solve?

Answer 24

• looking at highly ordered structures organization/orientation
• often used by neurobiologists (microtubles) and muscle cell biologists (actin/myosin)

Question 25

What is fluorescence microscopy and how does it work?

Answer 25

* Use of flurochromes to measure changes in cell behavior * Dyes are chemically modified with a special group that allows permeability through cell membrane and detaches once inside the cell * A light source emits light that is absorbed by the flchrome, exciting the electrons... the flchrome emits light as the electrons return to their ground state. Emitted light is longer wavelength (lower energy) than the excitation light

Question 26

What research questions does fluorescent microscopy address/solve?

Answer 26

* protein localization * cell structures * gene expression

Question 27

What is FRAP and how does it work?

Answer 27

* Fluorescence recovery after photobleaching * Steps: molecules of interest labeled w fluorochrome. Specific region of sample photobleached to damage flurophores attached to the molecules in that region. OVer time, unbleached molecules from the surrounding areas diffuse into the bleached region. * The recovery is tracked by images taken at regular intervals

Question 28

What research questions does FRAP address/solve?

Answer 28

* lateral fluidity of cell membrane proteins * diffusion rates of molecules within membranes or cytoplasm * how fluid/dynamic a cell membrane is * how proteins move within/netween organelles

Question 29

What are vital fluorescent dyes and how do they work?

Answer 29

* dyes used to stain live cells/tissues without killing them- do not require fixation so maintain the functional and physiological integrity of the cells * are fluorescent, diffuse easily, and are membrane insoluble once inside the cell * JC1:mitochondria (green=monomers=low PMF aka dysfunctional mitochondria, red=J-aggregates=high PMF aka healthy mitochondria) * Calcein (not membrane soluble, green)-AM/Propidium-Iodide (red): live-dead assay * Flu3-AM (green): measures intracellular calcium concentrations in living cells

Question 30

What research questions do vital fluorescent dyes address/solve?

Answer 30

* cell viability (calcein and propidium iodide) * Membrane integrity * Mitochondria function (JC-1, based on PMF) * dynamics of calcium signaling in live cells (Flu-3)

Question 31

What is TIRF and how does it work?

Answer 31

* microcope that enables visualization of molecules near a surface with high spatial resolution * works by selectively exciting fluorophores within a thin region of the sample using total internal reflection * developed to see the edges of cells that you can't always see in the plate

Question 32

What research questions does TIRF attempt to address/solve?

Answer 32

* receptor and signaling molecules behavior at cell membrane (dynamic events at cellular interfaces)

Question 33

What is fluorescence immunocytochemistry and how does it work?

Answer 33

* use of antibodies to idenfity proteins in cells * Direct method: primary antibody enters cell w attached flurochrome, binds to POI and fluoresces * Indirect method: after primary antibody enters, secondary antibody enters and binds to primary antibody, and fluoresces. Much more specificity and affinity (and signal amplification bc multiple secondary antibodies can bind to each primary)

Question 34

What research questions/techniques does fluorescence ICC attempt to address/solve?

Answer 34

* protein localization: where a protein is within a cell * cellular pathways/processes: how does a protein change location during cell signaling/cell cycle

Question 35

What is ELISA and how does it work?

Answer 35

* enzyme linked immunobsorbent assay- measures low concentrations of biomolecules w precision * plate coated w antigen or antibody that binds to target of interest. sampe is added and binds to coating in well. detection antibody that recognizes and binds to target molecule added- often linked to an enzyme to catalyze colorimetric reaction. substrate for enzyme added, causing color change proportional to the amount of target molecule bound in the well * spectrophotometer used find absorbance to quantify the concentration | can be colorimetric or fluorescent

Question 36

What research questions does ELISA attempt to address/solve?

Answer 36

* how much of a specific protein or hormone is present in a sample (protein quantification)

Question 37

What are polyclonal/monoclonal antibodies and how do they work?

Answer 37

* Polyclonal: mix of antibodies produced by different B-cell clones. generated by immunizing an animal w an antigen an collecting their antibodies. Recognize multiple epitopes on a single antigen so they bind to various parts of the antigen simultanesouly. Cross-reactive and supply ends w animal death. * Monoclonal: Identical antibodies by single B-cell clone that recognize and bind to one specific epitote (highly precise/specific). Created by fusing B-cell that produces desired antibody w immortalized myeloma cell to form hybridoma that can be cultured to produce large quantities. Easier reproducibility and high specificity and affinity

Question 38

What research questions do polyclonal/monyclonal antibodies attempt to address/solve?

Answer 38

* used in fluorescence immunocytochemistry and ELISA * used for potein detection and quantification * pathogen detection * therapeutic antibodies *

Question 39

What is microspectrofluorometry and plate reading spectrofluorometers, and how do they work?

Answer 39

* Techniques used to measure fluorescence properties of molecules for detection and quantification of fluorescent signals in samples * Micro spectrofluorometry- qualitative assessment using fluorescence emission spectrum, involves modifying images to allow for better understanding of results * Plate reading spectrofluorometers- quantitative assessment of fluorescent probes. Sums the fluorescent signal from a large group of cells. You get a control that you compare experimental data to

Question 40

What research questions do microspectrofluorometry and plate reading spectrofluorometers attempt to address/solve?

Answer 40

* Quantification of fluorescent models * Qualification of fluorescent models (cells can be genotypically the same but with different phenotypes)

Question 41

What is annexin V and propidium iodide and how does it work? (in relation to cell death)

Answer 41

* Annexin V binds to phosphatidylserine (PS- phospholipid normally on inner cell membrane in healthy cells). PS translocated outside during early apoptosis- where it becomes accessible to Annexin V. So, Annexin V binding serves as an indicator of cells in early apoptosis. * Steps for Assay: * 1. Live Cells: intact membrane and PS inside membrane, so no Annexin V nor PI binds * 2. Early Apoptotic Cells: PS translocated so Annexin V binds, but PI cannot enter because membrane is still intact * 3. Late apoptotic/necrotic cells: membrane integrity is compromised so PI enters and stains DNA. Annexin is also binding to exposed PS

Question 42

What research questions are adressed by Annexin V/PI assay?

Answer 42

* Detection of apoptosis: what proportion of cells are undergiong apoptosis? * Distinguishing necrosis from apoptosis: necrosis (PI+?Annexin V+) or apoptosis (Annexin V+, PI:-)

Question 43

What is FRET and how does it work?

Answer 43

* FRET=Fluorescence Resonance Energy Transfer * Relies on energy transfer between two fluorophores (YFP and CFP) * When donor molecule excited by light, it can transfer energy to the acceptor if they ae in close proximity (1-10nm), which decreases fluorescence from donor and increases fluorescence from the acceptor

Question 44

What research questions does FRET attempt to address/solve?

Answer 44

* protein-protein interactions * structural changes within a single protein (enzyme activation/phosphorylation) ## Footnote solves problem of detecting real-time, close-range molecular interactions non-invasively and in live cells, providing insight into dynamic processes at the molecular level

Question 45

What are FRET biosensors and how do they work?

Answer 45

* Biosensors: proteins that reveal a change in cell behavior (ex. Calmodulin, changes shape when there is a large change in Ca2+ concentration within a cell) * FRET biosensor consists of 2 fluorophores connected by molecular domain (protein, peptide, etc that undergoes conformational changes) sensitive to a specific biochemical signal * If the biosensor detects a biochemical change, the molecular conformation changes, altering the distance/orientation between the donor and acceptor fuorophores

Question 46

What research questions do FRET biosensors address/solve?

Answer 46

* cell signaling pathways: real-time monitoring of signaling molecules (ex. calcium) * protein conformational changes * enzymative reactions

Question 47

What is GFP/YFP/CFP and how does it work?

Answer 47

* 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

Question 48

What research questions does GFP/CFP/YFP address/solve?

Answer 48

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

Question 49

What research questions does autoradiography attempt to address/solve?

Answer 49

* Distribution of radiolabeled nucleotides: allows track of DNA synthesis during cell division * Cell division and proliferation rates: good for tumors/cancer research

Question 50

What is autoradiography and how does it work?

Answer 50

* technique used to detect radioactive molecules in biological samples * Radioactive isotopes incorporated into biological molecule (ex. DNA) and then introduced into the cells where they participate in natural processes * Then the cells are placed in close contact with a film and the radiation emitted by the radioactive molecules interact with the film- causing dark sports to appear w intensity proportional to concentration of radioactive material * exposed film developed revealing a radiographic image showing the distribution of the radioactively labeled molecules ## Footnote Probes: 3H-thymiding, 3H-leucin, 3-H methlone (used for protein synthesis)

Question 51

What is FISH and how does it work?

Answer 51

* Fluorescence In Situ Hybridization, used to detect and localize specific DNA or RNA sequences within cells/tissue sections * Uses fluorescently labeled DNA or RNA probes that are complementary to the specific sequences of interest * Fluorescently labeled probes indicate the location of the target sequences, revealing patterns of gene localization or expression

Question 52

What research questions does FISH attempt to address?

Answer 52

* gene mapping (physical location of genes on chromosomes) and chromosomal abnormalites * detecting mutations * microbial identification- can identify and localize specific microorganisms within environmental samples/human tissues; particularly useful in infectious disease diagnostics (ex. HPV)

Question 53

# Intracellular Injections What is single cell micropipette and how does it work?

Answer 53

* living cell is punctured by micropipette and things (DNA, RNA, proteins) are injeted into cell. * injects only one cell at a time

Question 54

# Intracellular injections What research questions does single cell micropipette attempt to address?

Answer 54

* used for somatic cell nuclear transfer for cloning * gene editing * developmental biology

Question 55

# Intracellular injections What is electroporation and how does it work?

Answer 55

* Cells are placed in a solution containing the molecule of interest (drugs, DNA, RNA) and transient electrical charge applied that temporarily creates pores in membrane that reseal when pulse is turned off * injects many cells at once but not 100% effective

Question 56

# Intracellular injections What research questons does electroporation attempt to address?

Answer 56

* gene transfer/genome editing * drug delivery * cancer research (delivering treatments to tumor cells)

Question 57

# Intracellular Injections What is liposome/nanoparticle injection and how does it work?

Answer 57

* Liposomes: small, spherical vesicles made of lipid bilayers. Package inside lipids binds to cell membrane, sometimes will be taken into the cell and released * Nanoparticles: tiny particles taken up cells through endocytosis (bind to cell membrane) * works on several cels at once but never 100% effective

Question 58

# Intracellular injections What research questions do liposomes/nanoparticles try to address/solve?

Answer 58

* used in Moderna/Pfizer mRNA delivery and siRNA/shRNA delivery (vaccine development and drug delivery)

Question 59

# Intracellular injections What is viral transfection and how does it work?

Answer 59

* Gentically engineered viruses used to deliver genetic material into cells, inserts itself into the genome of the target cell | not FDA approved

Question 60

# Intracellular injections What research q's does viral transfection address/solve?

Answer 60

* how can we achieve long-term expression of therapeutic genes in cells/tssues * what are the best viral vectors for gene therapy, and how can they treat genetic disorders or cancer

Question 61

What is transmission electron microscopy and how does it work?

Answer 61

* uses a beam of electrons instead of light ("transmits" electrons through the specimen). electrons have much shorter wavelengths, allowing much greater resolution than light microscopes * used to look at internal structures * samples must be extremely thin so the electron beacm can pass through them

Question 62

What research questions does TEM address?

Answer 62

* extensively in cell bio for internal structures of cells: organells, cytoskeleton, membranes, and viruses * bacteria, virus, and other microorganism visualization * nanoparticles

Question 63

What is high voltage electron microscopy and how does it work?

Answer 63

* specialized form of TEM w better theoretical limit of resolution because accelerating voltage is 1000kv (typical TEM is 100kv) * increased voltage allows electron beam to penetrate thicker samples, enabling imaging of larger specimens w better contrast

Question 64

What research questions does HVEM address?

Answer 64

* biological ultrastructure in thick specimens (ex. brain slices and whole cells) that cannot be adequately imaged using conventional TEM

Question 65

What is scanning electron microsopy and how does it work?

Answer 65

* "scans" the image for visualization of specimen's surface * provided detailed 3D images of a sample's surface, making it ideal for studying topography

Question 66

What research questions does scanning electron microscopy address?

Answer 66

* surface topgraphy and morphology

Question 67

What is plastic thin sectioning and how does it work?

Answer 67

* common method used to prepare specimens for TEM 1. Fixation (tissue is as life-like as possible): using glutaraldehyde (X link proteins) or OsO4 (X link phospholipids) 2. dehydration through ethanol washes 3. infiltration with epoxy plastic and hardened 4. Sectioning w ultramicrotome (50-90nm) 5. Staining w lead (membrane) and uranium (counterstain, radioactive)

Question 68

What research questions does plastic thin sectioning attempt to address?

Answer 68

* ultrastructure of cells and tissues (organelles, membranes, cytoskeletal structures at high resolution)

Question 69

What is freeze fracture and how does it work?

Answer 69

* used to visualize internal membrane structures * cells frozen and then fractured in line of least resistance * exposed surface sprayed w platinum and carbon, then cell is dissolved to create a mold of what was in the cell

Question 70

What research questions does freeze fracture attempt to address?

Answer 70

* internal mebrane structure (lipid bilayers and protein distribution in membranes) * cellular junctions * viral insertion into cellular membranes

Question 71

What is ultrastructural immunocytochemistry and how does it work?

Answer 71

* identifies POI in a cell using antibodies, but fluorochromes swapped for gold particles * antibodies conjugated to gold nanoparticles of varying sizes applied to sections * under TEM, gold particles appear as dark spots- shows location of target proteins

Question 72

What research questions does ultrastructural immunocytochemistry address?

Answer 72

* protein localization within ultrastructure of cells

Question 73

What is ultrastructural autoradiography and how does it work?

Answer 73

* combines radioactive labeling with TEM to localize molecules and track distribution of specific molecues at the ultrastructural level * Cells are incubated w a radioactive compound and placed on a photographic emulsion sensitive to radiation. Radiation from labeled molecules exposes emulsion forming silver grains. Emulsion is developed lie photographic film, leaving black spots where radioactive decay has occured. Silver grains represent location of radiolabeled molecules and are visualized using TEM

Question 74

What research questions does ultrastructural autoradiography attempt to address?

Answer 74

* DNA and RNA synthesis: to locate newly synthesized DNA or RNA at cellular or sub-cellular level * where radiolabeled drugs are distributed in tissues

Question 75

What is electron tomography and how does it work?

Answer 75

* used in electron microscopy to reconstruce 3D structures at high resolution * sample is placed under TEM and series of 2D images taken at various angles (tilt series) * Tilt series images aligned and 2D images combined to generate 3D model of the sample

Question 76

What research questions does electron tomograpy address?

Answer 76

* 3D visualization of cellular structures like organelles, can reveal complex arrangements of protein complexes (such as ribosomes on ER) * studying macromolecular complexes in stiu (more natural environment), can see structures within their cellular context

Question 77

What is super resolution microscopy and how does it work?

Answer 77

* breaks through Abbe's diffraction limit * can Z-stack images * can be used for live cells * includes STED, PALM, and SIM | acheives resolution of 10-50 nm

Question 78

What research questions does super resolution microscopy attempt to address?

Answer 78

* subcellular structure and organization (cytoskeletal filaments, nuclear pores, synapses, vesicles) with unprecedented detail * detailed imaging of structures in cell division (mitotic spindle, chromosomes, chromatin) * nanomedicine

Question 79

What is laser capture microdissection microscope and how does it work?

Answer 79

* highly precise to isolate cells/regions of tissue from a heterogenous sample * laser removes specimen of cell/cells 7.5 to 30 um for focused analysis like jigsaw puzzle

Question 80

What research questions does laser capture microdissection attempt to address?

Answer 80

* capture of distinct populations of cells or individual cell from a tissue section, which can then be subjected to further molecular analysis * preserves DNA, RNA and protein content of isolated cells * dissectiing specific neurons, glial cells, or regions of brain tissue

Question 81

What is atomic force microscopy and how does it work?

Answer 81

* not common * no lenses * scanned proximity probe microscope, uses sharp tip that interacts w sample's atomic forces

Question 82

What research questions does atomic force microscopy attempt to address?

Answer 82

* 3D topographical imaging * biophysics and cell biology to study surfaces of molecules * cell adhesion due to chemical interactions between tip and sample

Question 83

What is scanning tunneling microscopy and how does it work?

Answer 83

* uses sharp metal tip and quantum tunneling * scanned proximity probe microscope for looing at surfaces

Question 84

What research questions does scanning tunneling microscopy address?

Answer 84

* imaging individual atoms on surfaces so exact arrangement of atoms in materials can be investigated * chemical reaction on surfaces at atomic level * quantum physics

Question 85

What is two photon microscopy and how does it work?

Answer 85

* fluorescence technique that allows for deep tissue imaging in vivo animals with reduced phtobleaching * two low-energy photons are absorbed stimultaneously by the fluorophore

Question 86

What research questions does two photon microscopy attempt to address?

Answer 86

* imaging deep structures within living tisues (like the brain) * structure and function of neurons * tumor growth in live animal models * high resolution images with minimal photodamage enabling reserachers to explore cellular and molecular processes within living organisms

Question 87

What is deconvolution microscopy and how does it work?

Answer 87

* uses mathematical algorithm to create a 3-D view of fluorescently stained cells * improves resolution and clarity of images obtained through optical microscopy

Question 88

What research questions does deconvolution microscopy attempt to address?

Answer 88

* visualizing fine cellular structures with higher clarity- allows reserachers to study the morphology and sdistribution of celular structures that may be blurred or indistinguishable in conventional images * tissue architechture * improving clarity of fluorescent signals and more accurate quantitative analysis of fluorescence intensity and spatial distribution