3. Microscopy

Question 1

What is microscopy?

Answer 1

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

Question 2

What is the basic microscope apparatus composed of?

Answer 2

• 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)

Question 3

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

Answer 3

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

Question 4

What is the light microscopic specimen composed of?

Answer 4

• 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

Question 5

What is life imaging? – The box

Answer 5

• 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

Question 6

What is the box and the cube?

Answer 6

• 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.

Question 7

How is CO2 levels maintained in the box?

Answer 7

• 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

Question 8

What are important factors in various experimental timescales?

Answer 8

• 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

Question 9

What is the “triangle of frustration”?

Answer 9

• 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

Question 10

What is intensity resolution?

Answer 10

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 11

What do the marking on objectives mean?

Answer 11

• 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

Question 12

What are the components of a light microscope?

Answer 12

• Ocular
• Objectives
• Sample
• Condenser
• Light source

Question 13

How can full light be modified in a light microscope?

Answer 13

• 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

Question 14

What is a light microscope used for?

Answer 14

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

Question 15

What is electron microscopy? – different types

Answer 15

• 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

Question 16

How does fluorescence microscopy work?

Answer 16

• 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

Question 17

What is the mechanism of fluorescence microscopy?

Answer 17

• 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

Question 18

What is stokes shift?

Answer 18

• 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

Question 19

What is photobleaching?

Answer 19

• 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

Question 20

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

Answer 20

• 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)

Question 21

Expand on the use of antibodies in fluorescent proteins..

Answer 21

• 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)

Question 22

Expand on the use of protein fusion in fluorescent proteins.

Answer 22

• 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 23

Compare confocal and widefield microscopy

Answer 23

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.

Question 24

Applications of fluorescent microscopy

Answer 24

• 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