microscopy Flashcards
What is microscopy?
- using microscope to view objects/specimens that are not visible to the naked eye.
- Hooke, 1665
What are parts of the microscope?
- detector (PMT, CCD)
- objective (+/- immersion medium)
- specimen (cover glass)
- Light conditioning system
- kohler illumination
- phase ring
- wollaston prism and plarizers
- filter cubes (for fluorescence) - light source (halogen, XBO)
How are specimen in light microscope kept alive?
“the box” controls the conditions to keep specimen alive:
maintenance of CO2 atmosphere , air tight table top.
-O2
-temp
What are problems with experimental time scales?
- Difference of seconds cause :
- artefacts in multichannel/4D imaging - stability, viability, possibility of multi-position timelapse.
Define triangle of frustration
- temporal resolution
- low exposure time,
- low pixel number
- binning - spatial resolution
- high pixel number
- no binning - sensitivity
- high exposure time
- binning
All detections have their benefits and limitations, what is best depends on the application requirement.
Define binning
a technique to boost camera frame rate and dynamics, whilst reducing noise by sacrificing resolution .
What are markings on objectives?
- magnification : 100x
- application : DICH
- coverslip thickness
- working distance
- Numerical aperture/ immersion medium (oil)
What is objective resolution?
- Aperture of the objective determines resolution.
- The higher the numerical aperture the better the resolution power of the objective.
What is the difference between magnification and resolution?
resolution does NOT = magnification
- Magnification = ability to enlarge the sample
- Resolution = ability to clearly distinguish between two points.
What do we use light microscopy for?
- histology - using antibodies
- phase contrast -cell morphology
- time -lapse (heart cell differentiation, cell migration)
What is the limitation of light microscopy histology?
-gives general pattern but not enough detail.
What is the limitation of phase contrast -cell morphology?
-we can only see a fixed timeline and we can’t define how these cells have gone from intact collagen to denatured collagen.
What are 2 types of electron microscopy?
- Transmission EM (2D)
2.Scanning EM (3D)
=> hit specimen with beams of electron
=> areas that are more dense (have microtubules), harder for electrons to pass through so appear as darker spots.
Outline the fluorescence absorption and emission cycle.
- when molecule absorbs light at particular wavelength it will be excited and reach higher level of energy
- the high level of energy wont be maintained for too long so releases energy.
- At lower energy light is emitted.
What is stroke shift?
- due to energy loss the emitted light is shifted to longer wavelength relative to the excitation light.
- difference in energy between ye lowest energy of absorbence and highest energy of emission = stroke shift.
- emitted light wavelength is always longer wavelength than excitation wavelength.
What is photobleaching?
- bleaching of fluorochromes
- due to high intensity illumination the fluorophores might lose permanently their ability to emit light.
How can you overcome photobleaching?
- create an environment where we can work with reduced excitation light intensities
- use shorter exposure
- use anti-bleaching in our mount.
What are Green fluorescent proteins (GFPs) and what are they used for?
- proteins naturally found in light-producing cells of cnidarins
- GFPs can be fused with other proteins and introduced in cells via transfection. This allows live study of fluorescent tags in living cells
What are 2 ways fluorescence can be used as tags?
- attached to antibodies
2. fused with protein
What is the problem with using GFP attached to antibodies?
- you can only visualise those specimens which are fixed so NOT alive
- alternative way to to visualise live specimen is by using plasmid constructions
compare widefield and confocal microscopy
+ve: higher z- resolution and reduced out -of-focus- blur make confocal pictures crisper and clearer
-ve : only a small volume can be visualised by confocal microscopes at once. Bigger volumes need time consuming sampling and image reassembling.
What are examples of intracellular live imaging?
- microtubule dynamics (GFP-tubulin)
2. vesicle transport through microtubules (GFP-kinesin)
What is the limitation of time lapse light microscopy?
Can be hard to differentiate between cardiomyocytes like cells from adipocytes
