3. Microscopy Flashcards Preview

Clinical Pathology > 3. Microscopy > Flashcards

Flashcards in 3. Microscopy Deck (24)
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1
Q

What is microscopy?

A

Allows us to view objects/specimens that are not visible to the naked eye.

2
Q

What is the basic microscope apparatus composed of?

A
  • DETECTOR (PMT, CCD) – allows us to see result what we are looking at (e.g. naked eye, camera, photomultiplier that transfers info to computer)
  • OBJECTIVE (± immersion medium) – like a magnifying glass (can go through air, liquid) to zoom in
  • SPECIMEN (cover glass)
  • LIGHT CONDITIONING SYSTEM (Kohler illumination, phase ring, Wollaston prism and polarisers, filter cubes for fluorescence) – do we want whole length, choosing specific wavelength, reflecting light, etc
  • LIGHT SOURCE (Halogen, XBO, etc)
3
Q

What order does the apparatus travel from eye detector to the light source?

A

Eye /detector -> objective to zoom in -> specimen -> light conditioning system -> light source

4
Q

What is the light microscopic specimen composed of?

A
  • Cover glass (0.17mm)
  • Sample surrounded by embedding medium (might contain anti-bleach agent)
  • Glass slide

• Microscopes, regardless of complexity, are built with the same parts

5
Q

What is life imaging? – The box

A

• Live imaging boxes used in investigation of live specimen – control of temperature and CO2 to keep sample alive and conditions for microscope as constant as possible
• Tightly controlled conditions to keep specimen alive
- Involves use of incubator box combined with a precision air heater to ensure temperature of specimen and microscope remain equilibrated and tightly controlled.
• Even small changes in ambient temperature can lead to thermal extension/contraction of microscope stand, stage and objective -> changes plane of focus

6
Q

What is the box and the cube?

A
  • The box: - Custom design for the individual microscopy setup. Intricate system of openings and doors for comfortable access to microscope controls and specimen.
  • The cube: - Highest quality fan; controller cube with external, low-vibration and low-noise design.
7
Q

How is CO2 levels maintained in the box?

A
  • Controller used to adjust air flow and CO2 percentage
  • An air tight table top encloses the live cell culture devices – used in very small samples as box too big -> better to control conditions in microenvironment, e.g. cells
8
Q

What are important factors in various experimental timescales?

A
  • Important to consider when looking at cells/structures over time – e.g. cytoskeleton, cell motility, cell differentiation and development (the timescale getting longer respectively)
  • Requires higher level of resolution and acquisition time (faster capturing of images)
  • So, system must be designed to ensure viability of sample for the amount of time needed
9
Q

What is the “triangle of frustration”?

A

• Compromise between three factors – so consider what you are trying to investigate!
1. Temporal resolution – how long and how fast images need to be taken
2. Spatial resolution – pixel number (bigger cube -> image taken faster but lower quality -> low resolution OR smaller cube -> image taken slower but higher quality -> high resolution)
• Consider what you are trying to investigate and compromise
• E.g. if main aim is to look at movement of particle, high resolution not needed; but if looking at how particle looks, then needed.
3. Sensitivity – ability to pick up image in lower light conditions (quality of image)
• So temporal resolution -> time; spatial resolution and sensitivity -> quality

10
Q

What is intensity resolution?

A

Intensity resolution – how finely a system can represent or distinguish differences of intensity, usually expressed as a number levels or a number of bits

11
Q

What do the marking on objectives mean?

A
  • Magnification
  • Application
  • Coverslip thickness
  • Numerical aperture – gives resolution power (not the same as magnification) ~ The higher the numerical aperture, the better the resolution power of the objective.
  • Working distance = how far from the sample the objective lens can go
  • Immersion medium where and which medium the objective goes
12
Q

What are the components of a light microscope?

A
  • Ocular
  • Objectives
  • Sample
  • Condenser
  • Light source
13
Q

How can full light be modified in a light microscope?

A

• Full light can be modified through rings and filters – doesn’t alter wavelength, but instead the way it goes through

  • Is it more condensed, more reflected, less reflected, etc.
  • BF (bright field) – no filter
  • DIC (Differential interference contrast) – able to contrast background and sample (has some 3 dimensionality) ~ you condense the light to a smaller area
  • Ph (phase contrast) – full ring with a little circumference that goes around the ring and this is where the light goes through ~ useful for tissue and cells that are changing shape, create refringement area to enable observations on whether sample is changing shape
14
Q

What is a light microscope used for?

A

A) HISTOLOGY

  1. Laser capture microdissection
    - Once area is detected, able to remove area of interest using laser that cuts through selected area (e.g. separating stroma and epithelium)
    - Advantage: area of interest can be cut out and reused for other investigations (can be re-dyed with other dyes, etc)
  2. Immunohistochemistry
    - Additional technique that allows us to identify presence or absence of protein of interest in sample (as histological sample itself only gives us an idea of distribution)
    - Identification of pattern of protein in tissue or cell
    - Using antibodies

B) PHASE CONTRAST – cell morphology
• Select which intensity of light goes through sample – wavelength not changed, but how much is reflected and how much is refracted
• Important when looking at where cells or tissue stays
• E.g. phase contrast microscopy culture on intact and denatured collagen (see image) -> cells change shape trying to align to collagen
• Contrast allows us to see this!

C) TIME-LAPSE
• Box life imaging method used (control CO2 and temperature)
• Heart cell differentiation
• Cell migration, e.g. crawling leukocyte chasing bacteria

15
Q

What is electron microscopy? – different types

A

• Electron source instead of light – beam of electrons go to sample, gives us dark image of areas sampled
1. TRANSMISSION EM – not 3D, beam of electrons transmitted through ultra-thin specimen, interacting with specimen as it passes through
2. SCANNING EM – sample treated with specific reagents, scan a beam of electrons through sample -> creates 3D image (key difference with transmission!)
• Disadvantage: cannot use a live sample

16
Q

How does fluorescence microscopy work?

A
  • Controlling the wavelength of light – selecting if light going through is red, green, etc
  • Series of mirrors allows you to modify light to ensure you see a particular wavelength
  • Able to modify proteins in sample to respond to specific wavelengths
  • Structure similar, but light source is fluorescent light source.
  • Ocular can be eyes, camera or photomultiplier
17
Q

What is the mechanism of fluorescence microscopy?

A
  • Specimen exposed to light (ground state) -> absorbs light (excitation) -> releases energy (emission)
  • Light is absorbed by fluorophores at a particular wavelength
  • Emits light at a specific wavelength
  • Several rounds of this cycle will eventually lead to energy loss and molecule gets destroyed -> no fluorescence
18
Q

What is stokes shift?

A
  • There are 2 peaks: excitation peak (energy given to molecule), emission peak (molecule releases energy)
  • Stokes shift is the difference between the 2 wavelengths
  • Excitation peak is always at a lower wavelength compared to the emission peak
  • Due to energy loss, the emitted light is shifted to longer wavelength relative to the excitation light
19
Q

What is photobleaching?

A
  • High intensity illumination may destroy fluorophores and cause them to permanently lose their ability to emit light
  • Work with reduced light intensity, use shorter exposure times or use anti-bleach in mounting media
20
Q

What are fluorescence proteins and how do they work? (2 ways)

A

• Fluorescent proteins can be fused with other proteins and introduced in cells via transfection. This allows live study of fluorescent tags in living cells/organisms

  1. ANTIBODIES
  2. PROTEIN FUSHION (tags)
21
Q

Expand on the use of antibodies in fluorescent proteins..

A
  • Molecules normally able to recognise specific proteins (antigens) – can attach to molecule that gives off colour (fluorophore)
  • Advantage: Specific (can know exactly where fluorescence is, so antibody with specific fluorescent marker can bind to target molecule in wanted area, e.g. protein in nucleus)
  • Disadvantage: Can only be applied to fixed sample if you want antigen to bind to subcellular structures (as AB big and can’t go past cell membrane)
22
Q

Expand on the use of protein fusion in fluorescent proteins.

A
  • Use plasmids to incorporate gene that causes fluorescence
  • Incorporate into undifferentiated ES cells, which can eventually form fibroblasts
  • Advantage: can be used in live cells
  • Disadvantage: lacks specificity? and don’t know if molecule can be kept (cell can undergo apoptosis if recognises inserted gene is exogenous)
23
Q

Compare confocal and widefield microscopy

A

CONFOCAL:
• Stopping beam of light at certain levels -> can see different levels of sample, e.g. in seeing an epithelial cell (seeing specifically only apical or basolateral side)
• Light source is laser – able to control how it goes through sample
(+) better Z resolution, better specifics as can see through several layers, also allows live imaging (control temp and CO2) ~ higher z resolution allows a crisper and a clearer image.
(-) only small volume can be visualised by confocal microscopes at once -> bigger volumes more time consuming, as more sampling and image reassembling needed (Widefield better for bigger volumes!)
• With widefield, you can see the whole cell and cannot focus on a particular area.

24
Q

Applications of fluorescent microscopy

A
  • Tissue and cellular localisation
  • Intracellular live imagine – microtubule dynamics, vesicle transport through microtubules
  • Colocalisation
  • You can create 3D images out of the single layer images

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