Microscopy I Flashcards
How do we know what we know about cells
Lm and em
Biochemical techniques
Genetic techniques - yeast
Combos of any of these
When lm important
Most important until 1950s - bc not much biochem
When EM important
Dominant from 1950-70s
WHAT dominated from 80s-now
Combo of biochem and yeast genetics - dominated from 1980s - present
What is making comeback
Light microscopy
Bc new ability to follow dynamics of proteins in living cells
Antony van Leeuwenhoek
1632-1723
Made microscopes - powerful magnifying glass
Little animals - rotifers, multicellular animals but small
Visualized yeaSt
Robert Hooke
1635-1703
Invited compound microscope- multiple lenses, easier to make
Contemporary of van Leeuwenhoek but English
Gave name cells to statures he saw in cork and in wood
Spread idea of microscopy
Describe cell = generally
Bacterial cell = 1-2microns
Polarized epithelial cell = 15 microns, bigger
Describe micronscale - image
Light micropscpae =
Antibody staining
See 15 microns, cell, nucleus has no mcirotubuels, see microtubuels outside nucleus
Many parts of cell = too small, cannot see details
Large cell = 30microns, Rbcs = 4micro
100microns = human hair, so large cell = 1/3 of human hair on avg
Describe nanometers scale
Electron microscope - cannot see with lm
Higher resolution = can visualize protein molecules and diff aas
Axons of neurons (diameter = 1 micron), see myelin, more prep for em samples
Microtubule = 24nanometer diameter- diffusion of light makes it look thicker than is
But if 2 objects = less than 250 nanometers = cannot resolve, images blend together
Limited by resultion of light =lm
CANNOT SEE THIS WITH LM
Describe pm of cell- em image
Bilayer = 7-8nm typically, depends on where cell is (1 nm = 1/1000 of micron)
Can see the 2 layers with em
Hydrophobia inside,2 fatty acid chains, integral membrane proteins embedded in membrane
Cannot see head groups - but just see 2 layers and space inside
Mid 1950s, better now
What can we see with naked eye
1cm-less than 1 mm
1/4 mm or bigger, 250microns (1mm = 1000 microns)
What can we see with lm
Large range - 1/4mm to less than 1 micron
Plant cell,animal cell (10-30microns), bacterium
For internal stature - shapes outside
What can we see with em
Em = 1/4 mm to o.1nm = atom
Can see v small things, plant cell, animal cell,virus, ribosome, globular protein, small molecules, atom
Get good internal structure
What are issues with lm
Resolution limited by diffraction of light - as long as not closer than 0.25 microns/250nm
What are issues with em
Energy to sample and cook in process - poor contrast, heavy metal staining = problems
More elaborate sample prep - cannot be done for live cells
Micrometer, nanometer, Angstrom units
Um = micrometer = 10^-6m
Nm =nanometer = 10^-9 m
A = Angstrom unit = 10^-10 m
Describe light microscopy
In bio labs
Compound microscopes used
Flourecnse microscopes used too
Now common = new kind fluorescence microscope = Confocal microscope
Describe conventional transmission light microscope
For histology
To see sample = light goes through it= some absorption or something else that created contrast
Illuminator, mirror = reflects light
Stage position adjustment = light absorption/contrast
Condenser = sends light through it then can see specimens
Name all the types of lm
Transmitted light = brightfield, phase contrast
Fluorescence
Confocal = specialized kind fo Fluroesnce micropsopy - deals with large or 3d samples
Super resolution - recent, Much better resolution
Are cells opaque
Most cell = transparent
Very thin
How to make cells visible
Normal - brightfield* illumination = cells v haard to see
To make visible = staining with dyes (hematoxylin followed by eosin)
Or use special optics
Name special optics
GOOD FOR LIVE CELLS
Phase contrast*
Differential interference contrast = dic/nomarksi
Dark field - illuminated from side, look at scattterd light
Name types of samples
Cells in tissues
Tissue culture cells
Live vs fixed cells
Describe cells in tissues
Usually fixed, embedded in paraffin wax or plastic or freezed = usually for thicker samples
Describe tissue culture cells
Usually shaped like fried eggs - very flat
Much easier to work than tissueswith but usually not normal - less representative of body
Describe live vs fixed cells
Tissue culture cells = can be fixed or alive - actual intact tissues hard to work with alive (formaldehyde) - cannot section and must keep alive but sometimes still done
Which cells are easier to deal with
Tissue culture cells
Describe resp epi - lm
Get cells in correct 3d representation
Cannot do this with tissue culture - bc lose 3d relationships
Cells in tissues
Describe bright field
No staining
Can see lysosomes, nucleus’s endosomes
Describe phase contrast
No staining
Works for live cells
Describe dark field
Lysosomes around nucleus
Packed with protein - scatters light, so v visible with dark field
Advantages of methods to observe unstained live cells
Allow prolonged observation of live cells
Movements in cell division and of intracellular structures can be studied
Particularly if filmed - microcinematography - recorded on videotape or camera
How are live cells microscoped
Inverted microscopes used =
Objective - comes from below, coverslip in bottom of dish - keep dry from media
What is used for cell biological research
Tissue culture cells
Bc = microscopy of cells in tissue culture = v easy
What is tissue culture
Cells grown outside organism
Describe organ culture
Done sometimes
Perfume with solution, tricky tho
Keep most of physiological conditions from a. Living organism
Describe explanations culture
Host - dissect - fine chop organ, and do primary explant culture- lay on coverslip on media solution - can be grown
Easier to maintain in culture - and still presents 3d cell organization
Describe primary cells
Use protease = trypsin to digest and separate tissues = grow and maintain on dish, new cells = grow up in incubator
Describe continuous cell lines
Primary cell culture - must be able to go through mitosis
Digress connections with substrates
1st passage - 1:3 split ratio - vary depends on cell type - can keep going, could be 1:20 or more = can repeat this process
But after 30-40 times = will have crisis, telomeres shorten
Describe immortalized cell lines
No crisis, no death
How do cells grow
Grow only in monolayer culture due to contact inhibition properties
What exhibits contact inhibition
Tissue culture cells obtained from primary cultures = will eexhbit
= form single monolayer on a plate - will not continue to grow once space filled
What lacks contact inhibition
Cancer cells (hela cells) lack it
= will continue to grow In tissue culture and pile up on each other
= transformed cells
Oncogenes - lost ability to regulate growth, can also transform in culture with virsues
Define primary cells
Same cells obtained from source
Not immortalized or transformerd
(But primary cells obtained from a cancer are usually transformed cells)
Define non immortalized cell line
Primary cells = reproduce in tissue culture
Can be grown for many generations in tissue culture but not indefinitely
Define immortalized cell line
Mutations = like Telomerase expressed = allow for indefinite growth in tissue culture
Cells otherwise retain good behaviour = contact inhibition
Define transformed cell line
Cells lose contact inhibition and have other abnormalities - like abnormal mitosis
Equivalent to cancer cells, almost always immortalized
Easy to grow- used in labs a lot
How can cell lines become transformed
Other cell lines can become transformed = through mutations or through use of viruses or introduced dna to express oncogenes
Name the 3 things needed for maintaining cells in culture
Artificial medium
Temperature control
Sterile environment - Prevent external contamination
Describe artificial medium
Physiological ph = 7.4
Nutrients- aas, vitamins, salts
Glucose
Serum - growth factors
Antibiotics - option
What is needed for physiological ph
Carbonate buffer, co2 gas, ph indicator (phenol red - turns yellow bc cell produces waste)
Describe why we put antibiotics in cell culture
Cells divide every 24 hrs - but bacteria divides very fast - so put antibiotics in case external contamination
Describe temp of cell culture
37 degrees Celsius
Humified environment
Describe fluorescent molecules - fluorescence microscopy
Shine one colour and get another off it
Fluorescent molecules - like fluoresciene (green) or rhodamine (red)- excited by green light, absorb light of high energy (short wavelength, blue = flouresces green) and emits light at lower energy - longer wavelength
Describe fluorescent microscopy - method
Tissue and cells are irritated with a blue violet or ultraviolet light so that emission is in visible part of spectrum - could also use infrared waves
Describe fluorescent microscopy - results
Fluorescent structures appear as bright an coloured on black background
Describe fluorescent microscopy - sensitivity
Sensitivity of method very high
Describe early fluorescence microscopes
Not her great
See something fluoresce
Gas/liquid excitation filter = blocks uv rays - need filter to get ride of excitation light - must be efficient, and selective but hard to fine one
Carbon arc lam = provides uv rays
Describe epi florescence microscope
Blue light to excite - selective filter
Beam splitting mirror = reflects blue and transmitts green
Then blue hits sample, objective lens - fluorescence excitation from objective lense and also the condenser
2nd filter barrier - then green light goes through to eyeball
Describe locating modules with fluorescence - gen
Cells have some slight natural flureosnce - auto fluorescence - not useful most of Tim e exception = chlorophyll
Need techniques to label proteins of interest with Florenscnet molecule
Name the 3 ways to label proteins
1- Chemically label protein outside cell and add it - doesn’t always work
2- Label antibody against protein and stain cell - but cell must be formaldehyde fixed and permeabilized
3- Fuse protein of interest with gfp and expressed (recomb gene in living cell)
Describe micro injection
Immuno + gfp used
Controlled = used to potion needle
Foot pedal = produce gas pressure behind needle and once on cell = can inject, 50% chance of killing cell
