Microscopy

Question 1

What is microscopy?

Answer 1

Microscopes have been used for hundreds of years to view objects/specimens that are not visible to the naked eye.

Question 2

Describe the parts of the microscope

Answer 2

• Every microscope has a light source
  o This can be sunlight in a basic microscope, or bulbs such as halogen bulbs
• We can then modify this light (light conditioning system)
  o This is how we allow the light to reach the specimen
   There are different ways of doing this
  • Kohler illumination
  • Phase ring
  • Wollaston prism and polarizers
  • Filter cubes (for fluorescence)
• Then we have the specimen (cover glass)
• The objective (the magnifying glass)
• Lastly, we need a detector
  o The simplest one being our eyes
  o But you can also have cameras, photomultiplier tubes (PMTs)
   PMTs increase the signal and transform it into data that can be read by a computer

Question 3

What are the constants of the light microscope?

Answer 3

• Cover glass – 0.17mm (very thin)  specific constant width!!, to go on top of specimen, must allow light to pass through  keep solid specimens flat and liquid specimens into a flat layer with even thickness
• Sample surrounded by embedding medium (might contain anti-bleach agent) – holds specimen in place between cover and glass slide.
• Glass slide – use to place specimen on top, must allow light to pass through

Question 4

What is live image?

Answer 4

Viewing of a specimen that is live

Question 5

What conditions need to be met for live imaging?

Answer 5

o So, for live imaging, we need to create an environment for the microscope that allows us to keep the temperature and level of oxygen constant/at the level we want.
o Also, the physics of the microscope can vary with temperature
 You need to create an environment that prevents the objective lens from changing focus. This keeps microscope stable

Question 6

What does the box and cube do?

Answer 6

The box:

o Even small changes in ambient temperature can lead to thermal extension/contraction of microscope stand, stage and objective  changes plane of focus
o Involves use of incubator box combined with a precision air heater to ensure temperature of specimen and microscope remain equilibrated and tightly controlled.

o On one side, it prevents temperatures from changing a lot
o It also allows us to keep a certain amount of carbon dioxide and oxygen to keep the sample alive.

Cube:

Fan for low noise

Question 7

What is the normal CO2 atmosphere?

Answer 7

o Normally you would keep the sample at 37 degrees and no more than 5-10% maximum of CO2

Question 8

Define temporal resolution, spatial resolution and sensitivity

Answer 8

• Compromise between three factors, so consider what you are trying to investigate. All detectors have benefits and limitations. What is best, depends on the application requirements.
• Temporal resolution – how long and how fast images need to be taken
• 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.
• Sensitivity – ability to pick up image in lower light conditions (quality of image)
• So temporal resolution  time; spatial resolution and sensitivity  quality
• If you want a higher resolution  has to be more information per unit
  o Large pixels  low resolution
  o Small pixels  high resolution
• The same principle applies to intensity
  o If you are only able to choose between black and white  define less
   Define less in comparison to if you can differentiate with grey’s as well.
  • Known as intensity resolution - how finely a system can represent or distinguish differences of intensity, usually expressed as a number levels or a number of bits.

Question 9

Define objectives

Answer 9

Magnifying glasses

Question 10

Define numerical aperture

Answer 10

o Numerical Aperture
 Nothing to do with magnification
 It is the ability of the objective to resolve two points that are very close together
 Different values of aperture  different values of resolution  how crisp is the image?
 The higher the numerical aperture  the better the resolution power of the objective
 Resolution IS NOT THE SAME as magnification

Question 11

Define working distance

Answer 11

o Working distance (mm)

 E.g. 0.20mm  this objective can work 0.20mm from the sample

Question 12

Define immersion medium

Answer 12

o Immersion medium
 Light travels differently through different mediums
• E.g. if the objective says oil but you put it in water, you will not be able to focus

Question 13

There are rings and diaphragms that allow us to modify how light it reaches the sample

Define brightfield

DIC

Phase contrast

Answer 13

• Brightfield  all the light reaches the sample, no filter.
  Condensing the amount of light that goes through, which gives some 3D properties. Oberserving a sample
• DIC  condense the light through a smaller area. Able to contrast background and sample, allows for 3D
• Phase contrast  uses a phase ring
  o The ring is a full ring and there is only a little circumference that goes around the ring  this is where the light can go through.
• In all these cases, you are mainly playing with the contrast
  o The more shades of grey, the less contrast you have  you can see things in general, but not in detail
  o As you reduce the number of greys  less detail but more definition

Question 14

Why do we use light microscopy in histology?

Answer 14

1) Histology  looking at sections of a tissue.
o Chemicals that react with different parts of the tissue – chemicals that have affinity for basic and acidic components allows you to distinguish the different parts of a tissue
o Pros
 Gives you a general idea of the tissue
 Allows you to distinguish the different parts of a tissue
o Cons
 Lacks details, hard to distinguish between cells
o You can use modifications of the colour brightfield microscopy
 Laser capture microdissection  a laser can define an area that you want to get rid of in the sample image.
• You can see how the limits of the other area are organised.
• Advantage: area of interest can be cut out and reused for other investigations (can be re-dyed with other dyes, etc)
- You might want to know where a protein of interest is localised
o For this you would use an immunology approach
 Take specific antibodies against the protein of interest and see how they look
 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

Question 15

What is phrase contrast in microscopy?

Answer 15

o Select which intensity of light goes through sample – wavelength not changed, but how much is reflected and how much is refracted
o Important when looking at where cells or tissue stays
o Reduce the amount of grey’s  either black or white
 Can use phase contrast to see e.g. how fibroblasts are behaving in their substrate (collagen)
• Intact collagen vs Denatured collagen
o Fibroblasts start to collapse when you denature the collagen
• You could use normal light microscopy to see this, however phase contrast helps you to see the margin of the cells and the areas where the collagen has been denatured

Question 16

What is time lapse microscopy?

Answer 16

o Box life imaging method used (control CO2 and temperature)
o Colour brightfield microscopy can be used to see life events as well
 E.g. heart cell differentiation
o Can do this in a time course manner  taking a point and fixing the image, then looking at it
o Or just put the cells under a microscope and monitor them periodically.
o Cell migration can be seen via time-lapse microscopy
 Take images after specific time intervals and then put them together

Question 17

Describe the structure of an EM

Answer 17

• The basic structure is the same
  o But the light source is now an electron source. The electrons reach the specimen. Beam of electrons go to sample, gives us dark image of areas sampled.
  o We need a way to transform the electrons into images
   We collect the energy of the electrons and transform it into an image that can be seen through the eyepieces and the computer
  o This lets us see this at the ultrastructure level

Question 18

Whats the difference between a transmission EM and scanning EM?

Answer 18

• Transmission EM – not 3D, beam of electrons transmitted through ultra-thin specimen, interacting with specimen as it passes through
• 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

Question 19

How does a fluorescence microscopy work?

Answer 19

• Controlling the wavelength of light – selecting if light going through is red, green, etc
• 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

Question 20

What is the mechanism of absorption and emission of a fluorescence microscope

Answer 20

o Specimen exposed to light (ground state)  absorbs light  excitation  releases energy (emission)
o Emits light at a specific wavelength
o Several rounds of this cycle will eventually lead to energy loss and molecule gets destroyed  no fluorescence
o Chemicals that react with different parts of the tissue – chemicals that have affinity for basic and acidic components allows you to distinguish the different parts of a tissue.

Question 21

What is stokes shift?

Answer 21

• 2 peaks: excitation peak (energy given to molecule), emission peak (molecule releases energy)
• Due to energy loss, the emitted light is shifted to longer wavelength relative to the excitation light
  o The difference between the excitation and emission wavelengths is known as the Stokes shift
  o Excitation always happens at the lower wavelength than the emission

Question 22

What is photobleaching?

Answer 22

• The amount of light that you put in the excitation may break the continuous cycle above
  o Known as Photobleaching
• The fluorescence can eventually disappear
• Bleaching of fluorochromes: due to high intensity illumination the fluorophores might loose permanently their ability to emit light.
• Work with reduced excitation light intensities or gray filters, use shorter exposure times/higher gain settings and longer intervals during time lapse studies; use anti-bleach in your mounting media.

Question 23

What is the range of a FM?

Answer 23

UV to infra red

Question 24

• 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

How can we do this?

Answer 24

1) Antibodies
- 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)

2) Protein fusion (tag the gene)
- 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)

Question 25

What is GFP?

Answer 25

• The first protein used for this purpose
• Fluorescent proteins can be fused with other proteins and introduced in cells via transfection
• This allows the live study of fluorescent tags in living cells

Question 26

How does Fluorescence work?

Answer 26

• You use antibodies to see a protein of interest
• Secondary antibodies recognise the primary antibody.
• The secondary antibody has a fluorophore that that is excited and then emits at a particular wavelength, so you can see your protein of interest in colour
• But what if you want to see something that is alive?
  o Generate tools (plasmid)
   The plasmid has a sequence of the protein linked to the sequence of the fluorophore.
  • When this plasmid goes inside of a cell and is expressed, you will see the protein in that particular colour.

Question 27

Compare confocal and widefield

Answer 27

• The structure is the same
  o The light source for a confocal microscope is a laser that can emit the wavelength.
• When the sample receives the laser, it will get excited and emit at a specific wavelength
  o It will be enhanced by a photomultiplier and then you will be able to see it on a computer screen.
• 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
• Advantage: better Z resolution, better specifics as can see through several layers, also allows live imaging (control temp and CO2). Confocal images give a higher resolution and reduces out-of-focus blur, the images are crisper and clearer
• Disadvantage: 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!) However, only small volumes can be visualised by confocal microscopes. Bigger volumes need time consuming sampling and image resembling.