L2 - Imaging the Cell

Background Knowledge in Microscopy:

Anton van Leeuwenhoek(1632-1723):

• Built many simple, single lens microscopes
• First to observe living protozoa and bacteria which he called “animalcules”
• Went on to visualize human red blood cells and sperm
• With great skill at grinding lenses, naturally acute eyesight and lots of patience he was able to achieve a magnification of 200X.

Resolution:

• the ability to distinguish between two very closely positioned object as separate entities,
  • distance resolved between 2 points,
• smaller resolution is better.
• To lower the resolution:
  • Better objective,
  • Oil and water for higher n
  • Lower angle.
• 

Wavelength Spectrum used in Microscopy:

Modern Compound Microscope:

Bright-field Microscopy:

Properties of Bright-field Microscopy:

• typical light microscope magnification is 40 to 1000X
• only structures with a high refractive index (ability to bend light) are observable
• refractive property of the specimen allow us to see with microscopes.

Structure of Bright-field Microscopy:

• light source
• condenser lens to focus light on specimen
• objective lens to collect light after it has passed through specimen
• ocular or eyepiece lens to focus image onto eye
• 

Resolution of Microscopes:

• a conventional light microscope usually cannot resolve objects/cellular features that are less than ~0.2 mM apart.
• 
• Resolution = D (of Bright Field Microscope):
  • 
  • 0.61 = natural wavelength of light passing through the specimen
  • λ: wavelength of light
  • N sinα: numerical aperture(higher is better)
  • N: refractive index of medium between the specimen and the objective lens
  • α:1/2 angle of light entering objective
  • The limit of resolution is 0.2 mm=200 nm

Phase Contrast Microscopy:

Obtaining contrast in light microscopy by exploiting changes in the phase of light:

• Refractive ability = ability to bend light
  • Nucleus has dense amino acid, slows down light the most.
  • 

Interference of Light:

• certain parts of the cell (i.e. nucleus) refract light more than other parts
• cellular constituents with high refractive properties can slow the passage of a light beam by a quarter wavelength (~1/4λ)
  • two waves out of phase interfere and cancel each other to be dimmer.
  • 

Properties of Phase Contrast Microscope:

• Used to examine live “unstained” cells
  • Live or fixed
• Small differences in refractive index & thickness within the cell are further exploited and converted into contrast visible to the eye
  • allows us to see more details

Mechanism of Phase Contrast Microscope:

• interference of two phases dims light as the two slowed light rays cancel each other
• un-refracted light show the bright background
  • light is superposed on the porifera of the cells,
• fast and slow light create a stronger light around the cells as they create a phase
  • discern cellular data for both live and dead cells
• 

Differential Interference Contrast Microscopy (Normarski microscopy):

Properties of Differential Interference Contrast Microscopy:

• Small differences in refractive index & thickness within the cell are converted into contrast visible to the eye
  • $polarizers$ separates light
  • 
• Defines the outline of large organelles such as nucleus and vacuole and provides better detail of cell edge
  • Sharper image than Phase Contract
  • “shadow” primarily represents a difference in refractive index of a specimen rather than its topography (note that AFM visualizes the topography of a cell).

Phase Contrast vs DIC:

• Interfering with phase vs polar reform
• 2d vs 3d topographical shape
  • 3D feature is not shadow, but instead a difference in refractive index of a specimen
• DIC has a sharper image
• 

Fluorescence Microscopy:

Fluorescence Microscopy:

Properties of Fluorescence Microscopy:

• can be used for live cells
• uses a property of certain molecules to fluoresce,
  • i.e. to emit visible light when they absorb light at a specific wavelength (e.g. invisible UV light).
• location of fluorescent dyes or fluorescent protein molecules can be imaged.
• can visualize more than one protein or cell structure:
  • %%Green – Tubulin%%
  • ==Red – Mitochondria==
  • ^^Blue- Nuclei^^
• Fluorochromes dyes:
  • Absorb energy kicking electrons (e-) into a higher orbital (unstable)
  • When excited by a certain wavelength of light, it kicks off the out e- of the atoms to an upper energy level, then emits a light at lower energy level as it falls back to ground state.
  • 
  • Stoke shift:
  • Difference in color of excitation and emission light
  • 

Fluorescence Imaging:

• Light source is very intense, more powerful, broader spectrum of light
• Light pathway of Fluorescence Microscopy:
  • both excitation (%%green%%) and emission (==red==) light goes through objective.
  • No light passes through the cell specimen
  • cell specimen is labelled with fluorescent dyes that have specific excitation and emission wavelengths.
  • light source (excitation) = strong, intense, short wavelength
  • light output (emission) = weaker, longer wavelength

Structure of Fluroscence Microscopy:

• 
• Excitation Filter:
  • Only allows light with certain wavelength.
• Dichroic mirror:
  • Only reflects light with lower wavelength,
  • Allows lights with longer wavelength to pass.
• Emission Filter:
  • Only allows lights with certain wavelength.
• Use appropriate filters for the specific fluorochrome to optimize fluorescence images

The many colours of fluorochromes:

• Signals are bright on a black background.
• A variety of fluorochromes exist with different excitation and emission wavelengths that allow labelling of more than one protein or organelle at the same time
• Chemicals linked to fluorochromes are available to stain cell structures and organelles
  • Attempt to pick fluorochromes with distinct emission colour for clear presentation
  • ^^DAPI to stain nuclei blue^^,
  • ==Mitotracker Red or Rhodamine-labeled phalloidin to stain mitochondria or actin filaments red== respectively).
• a dye can be conjugated with antibodies  to localize any molecules of your interest in cells (immunofluorescent staining).
  • Antibodies with fluorescent property can be used to target specific protein

Immunofluorescence Microscopy:

Monoclonal Antibody Production:

• Helps to scale up the antibody for researchers and clinical use:
• HAT medium
  • (a selection medium) is toxic for myeloma cells, which have mutation of specific genes.
  • Kills all of the unfused myeloma cells as they are transformed and $lack of a series of genes$
• Hybrid cells
  • survive in HAT medium because they obtain a missing gene from spleen cells,
  • $Hybrid cells are immortal like myeloma cells and produce antibodies of the spleen cell.$
  • Spleen cells + transformed cell = cells without missing genes that can make antibody for antigen X

Properties of Immunofluorescence Microscopy:

• Restricted to fixed dead cells
• Antibodies are made to specific proteins (i.e. microtubules)

Preparation of Immunofluorescence Microscopy:

1. Fixation

• Freezing, a snapshot preserving all the ultrastructure in the dead cell

1. Primary antibody binding

• Antibodies are added to cells fixed on a slide which bind the specific protein they were designed to recognize
  • Invisible

1. Fluorochrome conjugated secondary antibody bonding

• Secondary antibodies with attached fluorochromes are added and bind the primary antibody
• 
• Each fluorochrome has a unique excitation and emission wavelength that can be detected with appropriate filters in the microscope.

Dual Label Fluorescence Microscopy:

• Use appropriate microscope filter set for each fluorochrome then digitally overlay images
  • Immunofluorescence is only performed on fixed (i.e. dead) cells.
  • Combination of Antibodies and stains
  • 

Fluorescent Imaging in Live Cells: Green Fluorescent Protein (GFP):

Fluorescent Protein

• Green Fluorescent Protein (GFP):
  • Derived from a naturally occurring protein
  • found in a bioluminescent jellyfish
  • contains a short sequence of amino acids (chromophore) that
  • fluoresce when excited with blue light
• Fluorescent proteins come in many different flavors:
  • By mutating various amino acids in GFP, new types of fluorescent proteins were created with different excitation and emission profiles
  • All have their own excitation and emission profile
  • 

Mechanism of Fluorescent Imaging

• Gene isolated and heavily modified to encode for a protein with properties ideally suited for live cell fluorescent imaging
  • GFP-Fusion proteins allow fluorescent imaging in live cells:
  • emitting green light in live cells to identify protein of interest
  • 

Two-Photon Excitation Microscopy for Imaging Deep into Tissue Samples:

Mechanism of Two-Photon Excitation Microscopy

• Certain fluorochromes can be excited by either by a single photon or by two photons at half the energy
  • 1 *** 488 nm or 2 * 960 nm – will generate the same emission wavelength
• Two-photon microscopy can be used to explore much thicker samples (longer wavelength)
  • Because only fluorochromes in the focal plane are excited,
  • there is no need for a pinhole to exclude out-of-focus light.
  • 

Mini Scope

• Attach the scope to the head of the animal
• Fibre optic cables
• Allow the ability to visualize with two fluoroscope (two photon microscopy)
• 
• 

Fluorescence resonance energy transfer (FRET) to measure protein interactions in live cell:

Mechanism of FRET:

• If no protein interaction occurs then excitation of cyan fluorescent protein (CFP) will only result in cyan fluorescence (480 nm)
• If protein interaction occurs then excitation of CFP will result in yellow fluorescence (535 nm)
• In picture, protein interaction detected at front of migrating cell \n
  • When protein/enzyme X and Y interact with each other:
  • CFP is brought close enough for the emission of CFP to excite YFP
  • Filters for the fluorescent microscope:
    • Excitation filter for CFP
    • Emission filter for YFP

Excitation wavelength is always shorter than emission wavelength

FRET Biosensors:

• Used in live cells and real time to monitor and observe protein, kinase activities
• CYP and YFP physically linked by large protein domains (i.e. Sensor and Ligand domains) that prevent close interaction between fluorescent proteins
• Phosphorylation of sensor domain allows ligand binding domain to interact and bring CFP and YFP close together to permit a FRET signal
• A link group of protein
  • Adding P makes ligand domain recognizable by the sensor domain

Laser Microscopy:

Laser Scanning Confocal Microscope:

Properties of Laser Scanning Confocal Microscope:

• Exceptional clarity
• 3D reconstruction
• 

Principle of Confocal Microscopy:

• designed in a way that only allows light from in-focus points to reach the detector
  • fluorescent light is emitted after laser shines on the dyed specimen
  • emission from in-focus focuses reaches the detector
  • emission from out-of-focus focuses is largely excluded from detector

Image Acquisition:

• A mitotic fertilized egg from a sea urchin:
• 
• The mitotic spindle composed of tubulin is blurred because fluorescence is detected from above and below the focal plane.
• Detecting fluorescence only from the focal plane produces a sharp image (thin optical section).

Deconvolution Microscopy:

• A computationally intensive math procedure to remove fluorescence contributed from out-of-focus parts of the stained sample
  • considers so-called point spread function which determines the degree of blurriness by comparison to a reference set of tiny fluorescent beads
  • Images are taken at different focal planes (called a $Z-stack$) by means of a precisely controlled robotic microscope stage
  • 
  • Programmed to captured the bottom layer of the cell of the layer.
• $Subtracting off the light (fuzzy part) of the computer scanning that is off focus by comparing the layer (Z-stacks) above and below$
  • Layer below might be in excited state and emit some light too
  • With point spread computer algorithms.
• %%Quick scan, do not cause photon toxicity as the time is shorter%%

Electron Microscopy:

Electron Microscopy vs Fluorescence Microscopy:

• EM provides better resolution than FM.
• 
• EM, however, needs ==fixed and sectioned samples== or @@metal-coated samples@@,
  • living cells cannot be imaged.

Electron Microscopy Essentials:

• wire filament is an electron source & when it’s heated, electrons accelerate towards anode
  • Beam of electron
• a magnetic (not glass) condenser focuses electrons on specimen
  • Directs the beam of electron
• specimen is stained with electron-dense heavy metals (lead, uranium, osmium tetroxide)

Types of Electron Microscopy:

Transmission Electron Microscope

• thin layers, section of cells, for details
  • samples are stained
  • Whenever the beam hits parts of heavy metal, heavy metal stain, interfere with the transmission of electron
  • Some will bounce away once it hits heavy metal and shows black on the image as lack of electron

Scanning Electron Microscope

• Uniformly cover everything in metal
  • Structure is preserved
• Imaging based on the degree of electron reflection,
  • Electron beam reflected by specimen to the detector
  • computer generates the image

Transimission Electron Microscopy:

Resolution of TEM:

• very fine “D”
• 
• there is no n, because nothing will interfere with the electron beam
  • no “N” as light is replaced by electrons in a vacuum
• sin α is now α since electron scatter is almost 0
• Theoretical resolution is 0.005 nm; the effective resolution is $0.1 nm$ (2000X greater than light microscopy).
• 

Sample preparation for TEM:

1. Fixing the cell (chemically)
   • into plastic-embedded specimen and on copper grid
   • with aldehyde then dehydrated
   • 
2. Stain it with heavy metal
   • and treat with stains
   • 

• \

Imaging with TEM:

• electrons hit specimen but deflect due to metals deposited on organelles,
• unobstructed electrons are focused by lenses onto a phosphorescent screen,
• crystals in the screen, excited by the electrons, give off energy as visible light,
• B&W image is made up of shadows where electrons failed to penetrate.

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