5 - microscopy Flashcards
(20 cards)
1
Q
what are the main components of a standard brightfield microscope?
A
- main components: light source, condenser lens, stage, objective and ocular/projection lenses, detector
2
Q
how does a standard brightfield microscope work?
A
- light diffracted by specimen and undiffracted light focused by objective lens
- produces the image we can see with our eyes
- image usually captured by video camera
- more sensitive to low light intensities
- living cells can be viewed with limited photo/light damage
- record image as digital file
- light intensities quantified by 2D array of numbers
- easily manipulate digital images using various software
- e.g. deconvolution is used to remove background and out of focus light
- greater contrast and clarity
- specimens are usually fixed (e.g. formaldehyde causes cross linking), embedded in something for support, then sectioned with a microtome, and stained with a molecule specific dye
3
Q
what is the purpose of microscopy?
A
- primary purpose of microscopy is to generate a magnified, high quality view of specimen
- overall magnification = objective lens x ocular lens
- empty magnification: enlarging an image but not gaining more details from it
4
Q
what is a major limitation of the standard brightfield microscope?
A
- major limitation is specimen’s poor contrast
- lack of detail
5
Q
what is resolution?
A
- resolution: the minimum distance that can separate two points that still remain identifiable as separate points
- most important aspect of today’s microscope
- resolving power depends on two main factors
- wavelength of illumination (ƛ)
- numerical aperture (NA)
- light gathering qualities of the objective lens and the specimen mounting medium
- resolution (nm) = (0.61 x ƛ)/NA
6
Q
how is resolution maximized?
A
- resolution is maximized by using a shorter wavelength of illuminating light
- blue (400 nm) instead of red (700 nm)
- also maximized by increasing the numerical aperture by altering the mounting medium
- using oil instead of air
7
Q
where is resolution limited?
A
- limit of resolution for most standard brightfield and CLSM is about 200 nm
- can only observe larger organelles
8
Q
how do electron microscopes work?
A
- electron microscopes use electrons with a wavelength of 0.0045 nm instead of photons which yields a higher resolution
9
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 dyed conjugated antibodies (immunofluorescence), or autofluorescent proteins
10
Q
what are the pros and cons of fluorescence microscopy?
A
- pro: provides more contrast and lets you study of structures and dynamic processes in living cells
- con: out of focus fluorescence from sectioned/thick specimen results in a blurred image
11
Q
what are the principles of fluorescence?
A
- principles of fluorescence are a complex process
- certain atoms can absorb a photon of a certain wavelength
- atom’s electron becomes excited and moves up to a higher energy state
- excited electron is highly unstable
- loses energy and returns to a ground state by emitting a photon with lower energy
- emitting electron has lower energy/longer wavelength bc some energy was lost as heat
12
Q
what are the various fluorescence microscopy methods?
A
- confocal laser scanning microscopy (CLSM)
- similar set up to a standard brightfield light microscope but more features
- scanning head has one or more leasers of a certain wavelength that excite and focus through the specimen to get a detailed 3D image
- specimen can be fixed or living
- endogenous molecules in fixed specimen can be visualized by autofluorescence or using applied dyes or antibodies
- immunofluorescence microscopy
13
Q
what is indirect immunofluorescence microscopy?
A
- protein of interest is detected indirectly by a secondary antibody linked to a fluorescent dye which recognizes the primary antibody
14
Q
what are the steps of indirect immunofluorescence microscopy?
A
- specimen is fixed: cellular components are immobilized
- cellular membranes are permeabilized with detergent to allow entry of applied antibodies
- specimen is mounted on slide
- primary antibody that recognized the target protein is applied
- secondary antibody against the primary antibody is covalently linked to a fluorescent dye
- several secondary antibodies bind primary antibody
- - amplifies signal for better detection by microscopy
15
Q
what is double immunofluorescence microscopy?
A
- two or more proteins can be visualized by double immunofluorescence microscopy
- different proteins are detected with antibodies raised in different animals
- immunolabelling of proteins can also be combined with applied fluorescent dyes
16
Q
what is confocal laser scanning microscopy?
A
- specimen viewed with CLSM is usually living
- allows for cellular processes to be viewed live
- lasers can penetrate into thicker living specimens
- specimen is rapidly scanned with point laser light at a specific excitation wavelength
- based on fluorescence of molecules 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 is excluded
- does not pass through the pinhole
- yields an individual 2D z section/optical slice of the specimen that is less blurry than images from standard fluorescence microscopy
- individual z sections are collected at different depths in the sample and combine to form a z stack
- generates a 3D image
17
Q
what are the limitations of CLSM?
A
- rapid but can’t capture very dynamic processes
- point laser light can photobleach fluorescent molecules so they are no longer fluorescent and damage live cells by phototoxicity
- excited fluorescent molecules react with molecular oxygen to produce free radicals that damage membranes
- not efficient for imaging deep into thicker specimens/tissues
- limited spatial resolution
18
Q
what is super resolution CLSM?
A
- 10x better than standard CLSM
- 20 nm spatial resolution vs 200 nm
- different optical techniques for super resolution
- different techniques involve specimen illumination with a combination of laser light with different wavelengths, angles, and beam widths
- useful for visualizing smaller, intracellular structures
19
Q
what are the limitations of super resolution CLSM?
A
- specimen scanning is time intensive and not efficient for capturing dynamic processes and imaging deeper into specimens
20
Q
what is light sheet fluorescence microscopy?
A
- allows for rapid visualization of cellular structures and dynamics in larger, living specimens in 3D real time
- lasers rapidly moves across specimen from the side and emitted fluorescence is detected by a second detection objective at a right angle to the illumination objective
- makes a sheet of light
- specimen rapidly imaged plane by plane which produces hundreds of sheets per second
- resulting images are combined to produce a 3D image over time
- used with multiple organelle markers tagged with autofluorescent protein to visualize the complexity of organelle to organelle interactions in a live cell
- provides a cell wide and quantitative map of each organelle’s morphology, numbers, movements, speeds, positions, and interorganelle contacts in 3D time and in response to stimulants
