Microscopy Flashcards
1
Q
What is Standard Brightfield Microscope?
A
- Main components: light source, condenser lens, stage (holding specimen), objective and ocular (projection) lenses, and ‘detector’ (eye)
- Light diffracted by specimen and undiffracted light (field of view) focused by objective lens
- If object is lighter, the light has come through
- Image is usually captured by video camera
- More sensitive to low light intensities – living cells can be viewed with limited photo (light) damage
- Record image as a digital file – different light intensities converted into 2D array of numbers(quantified)
- Easily manipulate digital images using various computer software programs
- Deconvolution – designed to remove background and out of focus light (yields greater contrast and clarity)
2
Q
What is brightfield microscopy?
A
- Magnification: primary purpose of microscopy is to generate a magnified, high-quality view of the specimen
- Overall magnification = objective lens x ocular lens
- What about the quality of the image? Does continuing to enlarge the image continue to provide more details?
- No – ‘empty magnification’
- Resolution: the minimum distance that can separate two points that still remain identifiable as separate points
- The ability to distinguish two close objects as separate entities
- Most important aspect of today’s microscope
3
Q
What is resolution?
A
- Revolving power of microscope depends on two main factors:
- 1) Wavelength of illumination light
- 2) Numerical aperture (NA) – light gathering qualities of the objective lens and the specimen mounting medium
- Resolution (distance in nm) = (0.61 x wavelength) / NA
- How is the resolution of a microscope maximized?
- Use a shorter wavelength of illuminating light
- Red light = 0.61 x 700 nm/1 (air) = 427 nm
- Blue light = 0.61 x 400 nm/1 (air) = 244 nm
- Increase the NA – alter the mounting medium (air –> oil)
- Blue light = 0.61 x 400 nm / 1.4 (oil) = 174 nm
- Limit of resolution for most standard brightfield and CLSM is 200 nm
- Can only observe larger organelles (nuclei, mitochondria, chloroplasts)
- Limits of resolution:
- Human eye 0.1 nm
- Standard brightfield and fluorescence microscopy (CLSM) 200 nm (500x)
- Super resolution CLSM 20 nm (5000x)
- Electron microscope 0.02 nm (250000x)
- EM uses electrons (= 0.0045 nm) rather than photons – lower wavelength yields higher resolution
4
Q
Limitations of Brightfield Microscopy
A
- Major limitation of brightfield microscopy is a specimen’s poor contrast (lack of structural/cellular details)
- Specimens usually ‘fixed’ (formaldehyde fixation – cross links amino groups on adjacent proteins/nucleic acids), embedded (in plastic of wax) for support, then (thick), sectioned with a microtome, and stained with a molecule- specific dye(s)
- Con: fixation results in cell death, embedding and sectioning can lead to structural ‘artifacts’ and limited (molecule specific stains)
- Fixed means: add a dye and need to keep in one spot, a chemical will fix it
5
Q
What is fluorescence microscopy?
A
- Microscopy technique for visualizing fluorescent molecules in living (or fixed) specimens
- Relies on endogenous fluorescence in specimen, applied fluorescent dyes or dye – conjugated antibodies (immunofluorescence), and/or autoflourescent proteins
- Pro: provide increased contrast and allows for the study of structure(s) and (when not fixed) dynamic processes in living cells, and in 3D
- Con: out of focus fluorescence from (thick) specimen results in a ‘blurred’ image
- Various fluorescence microscopy methods: confocal laser- scanning microscopy (CLSM)
- Similar set up to a standard brightfield light microscope, but with a few additional features
- Scanning head containing one or more lasers of a certain wavelength of light ‘excite’ and focus through the specimen, obtaining a detailed, 3D image
- Specimen can be ‘fixed’ or living
- Endogenous molecules in fixed specimen can be visualized by autofluorescence (chlorophyll) and/or using applied dyes or antibodies – immunofluorescence microscopy
6
Q
What are some principles of fluorescence?
A
- Complex
- Certain atoms can absorb a photon of a certain wavelength (e.g. blue light)
- Atom’s electron becomes ‘excited’ and moves up to higher energy state
- ‘Excited’ electron is highly unstable
- Loses energy and returns to a ground state by emitting a photon with lower energy (longer wavelength) (e.g. red light)
- ‘Emitting’ electron has lower energy (longer wavelength) because some of its energy was initially lost as heat
7
Q
What is Indirect immunofluorescence microscopy for the detection of specific proteins?
A
- Protein(s) of interest detected ‘indirectly’ by a secondary antibody linked to a fluorescent dye, which recognizes primary antibody
- Protocol involves several steps:
- Specimen fixed: cellular components (proteins) immobilized
- Cellular membranes (lipids) permeabilized with detergent to allow entry of applied antibodies
- Specimen (cells or tissue section) mounted on slide
- Application of primary antibody(s) that specifically recognizes the target protein/antigen(s)
- Application of a secondary antibody (raised again primary antibody host animal) covalently linked to a fluorescent dye
- Secondary antibody can also be linked to colloidal gold particles for immuno-electron microscopy or to an enzyme (alkaline phosphatase) for colorimetric detection
- Several secondary antibodies bind primary antibody – application of signal for better detection by microscopy
- Two (or more) proteins can be visualized by (double) immunofluorescence microscopy
- Different proteins detected with antibodies raised in different animals (e.g. rabbit and chicken primary antibodies + goat anti-rabbit and sheep anti-chicken secondary antibodies)
- Immunolabelling of proteins can also be combined with applied fluorescent dyes
8
Q
What is confocal laser scanning?
A
- Specimen viewed with CLSM is usually living (not necessary to ‘fix’ specimen)
- Allows for dynamic biological/cellular processes to be viewed live – via endogenous autofluorescence, vital fluorescent dye, or an ectopically expressed fluorescent fusion protein
- Lasers can penetrate into thicker living specimens (compared to standard light microscopy)
- Specimen rapidly scanned with point laser light at a specific excitation wavelength (based on fluorescence of molecule(s) being detected)
- Emitted fluorescent light from only a single layer (focal plane) within the specimen is focused through the pinhole and then collected/viewed
- All out of focus fluorescence from the specimen (emitted light from above and below the focal plane) is excluded)
- Yields an individual 2D z section or ‘optical slice’ of the specimen that are less ‘blurry’ than images obtained with standard fluorescence microscopy
- Individual z sections collected at different depths in the sample and combined (i.e., stacked together serially) to form a z stack and generate a 3D image
- Several limitations to CLSM:
- Rapid, but cannot capture very dynamic cellular processes
- Point laser light can photo bleach fluorescent molecules (no longer fluorescent) and damage live cells by phototoxicity (fluorescent molecules react with molecular oxygen to produce free radicals that damage membranes–> can kill the cell)
- Not efficient for imaging deep into thicker specimens/tissues
- Limited spatial resolution (200 nm) –> limited by wavelength of light
- Super resolution of CLSM:
- 10x better resolution than standard CLSM (20 nm versus 200 nm spatial resolution)
- Several different optical techniques used to achieve super resolution
9
Q
What is super resolution CLSM?
A
- Different techniques (SIM and STED) involve specimen illumination with a combination of laser light with different wavelengths, angles, and/or beam widths
- Resulting collection of images are combined and computationally processed for increased resolution (less ‘blurry’ images) –> better resolutions
- Especially useful for visualizing intracellular structures
- Cons: specimen scanning is time intensive and not efficient for capturing dynamic (cellular) processes and imaging deeper into specimens
- Cons: very expensive, not good for live, fast processes, not good for thick cells
10
Q
What is light sheet flouresnce microscopy?
A
- New spatiotemporal fluorescence microscopy technique
- Allows for the rapid visualization of cellular structures and dynamics in larger, living specimens (whole cells) in both 3D and real time
- Laser(s) rapidly moves across specimen from the side and emitted fluorescence detected by a second (detection) objective at a right angle to the illumination objective
- Yields a ‘sheet’ of light
- Specimen rapidly imaged plane by plane producing 100’s of sheets/sec
- Resulting images are combined to produce a 3D image over time
- Use with multiple, fluorescent-protein tagged organelle markers to visualize the complexity (dynamics) of organelle- organelle interactions in a living cell
- Provide a cell wide (systems biology) and quantitative map of each organelle’s morphology, numbers, movements, speeds, positions and inter-organelle contacts in 3D and over time, and in response to drugs, pathogens, stress, and a better understanding of the biology in the cell –> understand cell as a whole and how all organelles interact
11
Q
What is transmission electron microscopy?
A
- Used to generate high-magnification and high resolution (up to 0.02 nm) views of internal (intracellular) structures)
- Image formed from a high-velocity beam of electrons that are transmitted through (or reflected off) a specimen
- Used to detect super detail
- Not used for highly dynamic events, but rather super detail
- Wavelength of electrons is very low, so gives higher resolution, different from wavelength of light
12
Q
What are TEM structural features?
A
- Hollow cylindrical chamber
- Under vacuum- living specimens cannot be imaged by TEM
- Electron gun (tungsten filament)
- Applied voltage generates a fine electron beam from the top to the bottom of the chamber
- Increased voltage = increased electron speed, increased specimen penetration, decreased wavelength, increased resolution
- Electromagnetic lenses (magnets) –> focus of electrons
- Equivalent to optical lenses in light microscope
- Alter beam of electrons (by adjusting voltage) to allow for control of focus and magnification
- Detector – image captured by a digital camera and displayed on computer screen
- How is an image formed from a beam of electrons?
- When electrons strike the specimen, some are ‘scattered’ (diffracted) and others are not
- ‘scattering’ due to the selectively electron dense properties of the specimen generated by negative staining
- Macromolecules and membranes stained with heavy metals – become electron dense and prevent electrons from being transmitted to the camera – seen as black areas
- Electrons that pass easily through unstained areas in specimen and appear as white/gray areas
- Specimen preparation for TEM is very important
- Electrons pass through or not, depends on preparation
- Less dense: lighter
- More dense: darker
13
Q
What is the sample preparation for TEM?
A
- Several different ways to prepare a specimen for TEM
- Most common is chemical fixation – e.g. specimen is placed in glutaraldehyde
- Aldehyde groups rapidly react with amino groups in proteins and other cellular macromolecules (e.g. nucleic acids) to form an insoluble ‘cross-linked’ immobile network
- Can sometimes lead to artifacts
- Kill cells slowly (30 sec to 10 mins)
- Does not bind to and immobilize all cellular molecules (lipids)
- Lipids may continue to move and thereby alter the shape, dimensions, and distribution of cellular structures
- Called an artifact, when living and fixed same cell look different, even though same cell
- Cryofixation: specimen is flash frozen using liquid nitrogen and high pressure
- Immobilizes in milliseconds all cellular molecules (including lipids & water) simultaneously
- Results in fewer fixation artifacts
- But, technically demanding and requires very expensive equipment
- Still can produce artifacts, but generally less and considered “better”
- Do not want too frozen
- Go with what think is living cell
- Chemically and cryo- fixed samples can be incubated with specific antibodies
- Immuno-electron microscopy
- Analogous to immuno- fluorescence microscopy
- Antibody linked to protein A coated gold particles (electron dense, everywhere there is this particle, image will be very dark)
- Allow for protein localization at ultrastructural level
- Specimen stained with osmium tetroxide (need to stain)
- Reacts selectively with lipids (fatty acids) in all cellular membranes making them electron dense and visible by TEM
- Specimen dehydrated in ethanol series – removes water from sample
- Specimen embedded in plastic epoxy resin (mechanical support for sectioning)
- Infiltrates into the sample and polymerizes upon heating – provides mechanical support to sample during sectioning
- Sample cut into thick sections (blocks) with a knife/razor blade
- The block is cute into thin sections (<100 nm width) with an ultra microtome
- Cuts very thin which is needed to view image of TEM
- Contains a small glass or diamond knife –> use plastic s support
- Generate serial cuts
- Thin sections through specimen collected serially and placed on a grid (for support)
- Post-staining of thin section with heavy metals
- e.g. uranyl acetate binds to most cellular macromolecules giving greater electron density and increased contrast (need this for electrons to be diffracted to see the image)
- Remember: Less dense: lighter, more dense: darker, but this is relative to the image, so depending on where cut, cells could give completely different image, than the same image of the same cell
- 2D image: thin section of part of cell, shows ultra-detail
14
Q
What is Cyroelectron tomography?
A
- TEM technique that provides ultrastructural details in 3D
- Specimen (cell or isolated organelle) is cryofixed
- Specimen is flash frozen using liquid nitrogen and high pressure (con: sample must be thin)
- Viewed with electron microscope equipped with a special stage – keeps specimen frozen (no heavy metal staining required) and tilts (stepwise) around axis perpendicular to electron beam
- Yields series of 2D images that are merged into 3D images – tomogram
- Tilted images: very detailed and put tilted images together to make 3D images
- Computer can go through series of titled images (incredibly detailed) and put together to make a video
