Microscopy Methods and Techniques

Flashcards covering key terms and concepts related to microscopy methods and techniques for exam preparation.

Antonie Van Leeuwenhoek

Pioneer of microscopy who contributed to the discovery of microorganisms.

Ernst Abbe

Scientist who developed the mathematical foundation for optical microscopy and described aberration, diffraction, and coma.

Nobel Prize in Chemistry 2014

Awarded to Eric Betzig, Stefan W. Hell, and William E. Moerner for surpassing the resolution limits of optical microscopy.

Bright Field Microscopy

Basic microscopy technique using transmitted light to illuminate specimens.

Confocal Microscopy

Technique using lasers and pinholes to produce high-resolution images from a focused depth.

Fluorescence Microscopy

Technique that uses fluorescent markers to visualize specific biological structures.

Differential Interference Contrast Microscopy (DIC)

Technique that enhances the contrast in transparent samples by exploiting optical interferences and providing them with 3D images

Phase Contrast Microscopy

Technique used to enhance contrast of transparent and colorless specimens.

Vital Dyes

Dyes used in vital fluorescence microscopy to stain live cells.

3D Imaging Technique

Advanced method for visualizing internal structures in tissues, often using X-ray tomography.

Cryostat

Device used for cutting ultra-thin sections of tissue that has been frozen.

Monoclonal Antibodies

cheap antibodies produced from a single clone of cells, specific to one antigen.

Polyclonal Antibodies

cheap antibodies produced by different B cell lineages in the body.

FRET (Förster Resonance Energy Transfer)

Technique that detects interactions between two living fluorescent molecules, specifically their ligand and receptor interaction

FRAP (Fluorescence Recovery After Photobleaching)

a flourescent technique that assesses the dynamics and movements of membrane proteins after it has been photobleached

Electroporation

opens the cell’s pores so that proteins can enter

Two-Photon Microscopy

Technique allowing deeper tissue imaging with lower phototoxicity using two-photon excitation.

Super Resolution Microscopy

Group of techniques that surpass the diffraction limit to achieve higher resolution images.

bright field microscopy steps

1. fixate tissue with formaldehyde since it cross links proteins

2. dehydrate to remove water

3. replace water with ethanol

4. replace the ethanol with xylene

5. place sample in paraffin which is wax

6. cut sections of wax tissue

7. mount sections on scale

8. add water

the cell theory

1. all organisms are made up of cells

2. each cell has independednt properties but the organisms must work togehter

3. the smallest cell is the smallest unit of life

4. cells arise from other cells

bright light microscopy is used by

pathologists to view dead cells

paraffin cuts

sections via wax

cytosections/ cryostat has poor

morphology but processes very fast

mohs surgery uses

cryostat

abbes resolution equation determines

the theoretical limit of resolution or how much we are able to see

phase contrast microscopy uses

constructive or destructive interference to manipulate light for higher contrasts

phase contract microscopy looks

at living cells rather than fixated cells

normanski or differential interference contrast microscopy

looks at living cells and provides a 3-D image of the cells

normanski/ dic is used in

sing;e cell electrophysiology which measures membrane potentials of a cell

patch-clamp

measures in inside out (clamp end is inside) and outside out (clamp end is outside the cell) ion flow through channels

phase contrast microscopy does not use

DYES to stain different parts of the cell since they want to examine living UNSTAINED cells

dye most commonly used: hematoxylin and eosin

hematoxylin stains the nucleus while eosin stains everything else

contrast microscopy then manipulates light

ie: phase microscopes.

dark filed microscopy is used mainly by

microbiologist

dark field microscopy

darkens the backdrop and illuminates the image of unalice cells for a higher contrast, rather than resolution

polarizing light microscopy

used by neurobiologists and muscle cell biologists

single cell electrophisiology

implaes the cell and loos at the change in the membrane

polarizing light microscopy

directs light into one direction to look at highly ordered parallel structures

spinning disk confocal microscopy

a specific type of microscope that is better for viewing living cells since it uses less photobleaching than point- scanning. it is also faster and uses less heat

• lazers pointing though a spinning disk with pinholes in it.

• can reveal changes in the E.R

point-scanning confocal microscopy

type of confocal microscope that is associated with a higher amount of photobleaching

confocal microscopy

uses lasers as the main source of light

makes stereo images

can use only dye

good for multiple labeling

confocal microscopy also

has a pinhole which allows the lazer to point and focus on one specific area

with confocal microscopy

specimens are image spot by spot so that all of the cells groves can be seen

vivascope uses

confocal microscopy to remove light and reveal skin cancers

vivascope is

used mained by dermatologists so that they can see skin biopsies and bedside testing

FRAP

refers to fluorescence recovery after photobleaching

FRAP measures

how fast proteins move along the cell membrane; exchange rates or membrane fluidity

fluorescence microscopy uses

lasers against a dark background to excite fluoropores which causes the specimen to emit a light at lower wavelengths

vital fluorescent dyes: mitotracekr red, rhodamine 123, JC-1

all track mitochondrial activity

JC-1

red/orange= a healthy mitochondria and a higher proton motive force

green = an unhealthy mitochondria with a low proton motive force

live-dead assay

calcein Am (green) and propidium iodide (red) - rupture and stain

FLUO3 -AM

tracks calcium levels in cells

red= high calcium

yellow = medium calcium

blue = low calcium

fluorescence immunocytochemistry

uses fluorochromosomes with antibodies to detect the locateion of POI

fluorochromes are

fluorescent dyes

antigens

create an immune response (the place of interest)

epitope

the domain of an antigen to which the antibody binds

affinity is the

strength of binding

limitations to fluorescence immunocytochemisty

1. has a high specificity

2. has a high affinity

3. can only bind one epitope

4. and uses fluorescence

specificity

an antibody binding to your antigen only and no other antigens

ELISA

uses antibodies to quantify a protein of interest

the indirect technique of ELISA is preferred because

the affinity (the strength of binding) is increased

the direct ELISA technique

directly couples fluorescent antibodies to the proteins

indiret ELISA technique

uses secondary antibodies to bind other antibodies and then bind to the protein

microspectofluoromtry/ plate reading spectrofluorometer

a quantitative assessment of fluorescence

TIRF microscopy (total internal reflection fluorescence microscopy)

creates high contrast- low light backgrounds to reveal the thinner regions of the cell (like plasma membrane)

fluorescence immunoctyochemisty steps

1. prepare sample

2. inclubate with a primary antibody and wash away the unbound antibody

3. incubate with fluorochrome- conjugated secondary antibody and wash away unbound antibody

indirect antibodies have:

primary antibodies which bind first, secondary anibodies which bind to the primary antibody and then fluorophones which are at the ends of the secondary antibody.

polyclonal antibodies

are antibodies that are capable of binding to multiple epitopes on the multiple antigens which means it has LOW SPECIFICITY

polyclonal antibody process

1. inject rabbit with antigen and the body produces antibodies or B-cells

2. the abtibodies can then be collected to make an antiserum

monoclonal antibodies

are made by from cultured cell lines and can treat cancer

monoclonal antibodies have

have higher power affinity and specificity since you can only bind to one epitope.

monoclonal antibodies allow you to

generate antibodies in unlimited amounts and you can even freeze clones to be used later

mAb’s work by binding to

the spike protein preventing the virus from binding to the ACE2 receptor

L - SOMA (liquid-injecting self-orienting millimeter-scale applicator)

a self-injecting capsule that is swallowed and then effectively inject a liquid medicine directly into the stomach wall-

• delivers MONOCLONAL ANTIBODIES to help fight cancer, rheumatoid arthritis, Crohn’s disease.

apoptosis

genetically programmed cell death through shrinkage. done to eliminate damaged, aged, or cells that aren’t needed

causes of apoptosis

chemotherapy, radiation, T-cells (cells that identify tumor cells)

necrosis is

refers to cell explosion due to disease, injury, or failure of blood supply

GFP was invented by

Roger Tsien and utilizes green fluorescent proteins

GFP can reveal

protein expression inside an entire organism as well as cells, adding a chimeric gene for GFP into the organism will illuminate it to be green

there are two types od GFP reporters

1. continuous

2. regulated

continuous gfp reporters

are always turned on

regulated gfp reporters

only fluoresces when a specific gene is expressed

FRET (forster resonance energy transfer)

shows a fluoresing yellow when the protein binds

can also be used to look at enzyme activation too.

FRET biosensors: Calmodulin

detects changes in intracellular calcium over time; a calcium binding protein

FRET biosensors: Ionomycin

pokes hols through the membrane for higher calcium flow.

audioradiography

uses radioactive probes to track cell changes and routes

FISH (fluorescence in sity hybridization)

can track gene sequence changes on a chromosome like MRNA changes or telomeres

intracellular injection technique ONE: single cell intracellular injection

uses luciver yellow to track neurons and is required by somatic cellular nuclear transfer

intracellular injection technique TWO: electroporation

opens up pores for things to enter the cell membrane, is used to place molecules of interest into a cell

intracellular injection technique THREE: liposomes/nanoparticles

closed capsules that carry components inside and can be directed to move components; used in MRNA vaccines,

intracellular injection technique FOUR: viral transfection

is good for research NOT clinical, consists of a retrovirus inserting genes into a genome.

TEM: transmission electron microscopy

transmits electrons through a microscope, IS DIFFRACTION LIMITED, uses thinly sectioned cells

high voltage electron microscopy HVEM

a combination of TEM and SEM better used for imaging thick sections

scanning electron microscopy SEM

surface imaging after scanning samples, the greater voltage means an increase in resolution

plastic thin sectioning

1. fixation using gluteraldehyde which cross links proteins or oso4 to cross link phospholipids

2. dehydration

3. infiltration using epoxy plastic

4. ultramicrotone

5. mounting specimen on mesh

6. use dye like uranium or lead

freeze fracture microscopy

freezing and craaking open the outer membrane to show the membrane face

cryoelectron microscopy

can image proteins, molecules, etc in native form down to the atomic level (3D printed image)

electron tomography

is an extension of TEM that uses TEM to collect 2D images at different angles but reconstructs them into a 3D image.

laser capture microdissection microscopy