Microscopy Flashcards
what is microscopy used for
It’s when we use microscopes to view objects/specimens that are not visible to the naked eye.
What are the fundamental parts of a microscope?
NOTE: This is the same in EVERY microscope
(5)
detector
objective
specimen
light conditioning system
light source
fundamental parts of a microscope
function of detector
allows us to see the result of what we are looking at (e.g. naked eye, camera, photomultiplier that transfers info to computer)
example of detector used?
PMT - photo multiplier
fundamental parts of a microscope
function of objective
like a magnifying glass (can go through air, liquid) to zoom in
what are the components of the light conditioning system
Kohler illumination
phase ring
Wollaston prism and polarisers
filter cubes for fluorescence
function of light conditioning system
allows choice of how you view specimen:
- choose specific wavelength etc
Describe the placement of the specimen in light microscopy
It is mounted on the glass slide and covered by a cover slip
The sample is surrounded by embedding medium
We can observe specimens that are dead or alive.
what is used in the investigation of live specimen?
what is essential in this method to keep the specimen alive?
live imaging boxes
CO2 and temperature control are ESSENTIAL when you want to keep a sample alive
describe how live imaging boxes can be used to upkeep a living specimen during microscopy
- control the temperature and CO2 to keep the sample alive
- keep conditions as constant as possible
live imaging boxes
how is the temperature kept in control?
It involves the use of an incubator box AND precision air heater to ensure temperature of the specimen and the microscope remain equilibrated and tightly controlled
live imaging boxes
how is the CO2 kept in control
- the controller is used to adjust air flow and CO2 percentage
- an air tight table top encloses the live cell culture devices – it’s used in very small samples as the box too big; it helps us better to control conditions in microenvironment, e.g. cells.
experimental timescale
what are the 2 problems with Long experiments
for example observing the development of a zebrafish embryo
- Problems maintaining stability and viability of the samples
- Requires complex systems that allow you to see different positions of the samples overtime
experimental timescale
what is the 1 problem with short experiments
for example viewing microtubule based movement
Shorter experiments require a higher level of resolution and acquisition time (faster capturing of images)
in microscopy, there is something known as the ‘triangle of frustration’.
There needs to be a compromise between 3 factors when investigating. What are the 3 factors?
temporal resolution
spatial resolution
sensitivity
triangle of frustration
what is meant by temporal resolution
how long and how fast images need to be taken
triangle of frustration
what is meant by spatial resolution?
when is high resolution needed?
pixel number
high resolution needed if trying to look at how the particle specifically looks
high resolution NOT needed if trying to look at movement of particle
triangle of frustration
what is meant by sensitivity
the ability to pick up image in lower light conditions (quality of image)
what are some markers on an objective (magnifier)
- coverslip thickness
- immersion medium
- magnification
- numerical aperture
- application
- working distance (mm)
what is meant by:
magnification
numerical aperture
magnification:
how many times the objective magnifies the image
numerical aperture:
a measure of the objective’s ability to gather light and resolve fine specimen detail at a fixed object distance (concentrates light in a specific way to give a crisper image)
How does the aperture of the objective determine the resolution?
The higher the numerical aperture (NA), the better the resolution power of the objective (the more detail can be seen).
the numerical aperture of the objective determines the resolution
what can light microscopy be used for
It can view samples ranging from tissues to cells.
how can the light in light microscopy be modified?
The full light can be modified through rings and filters – it doesn’t alter wavelength, but instead the way it goes through.
LIGHT MICROSCOPY
what is meant by..
- BF
- DIC
- Ph
BF = colour brightfield
DIC = differential interference contrast
Ph = phase contrast
LIGHT MICROSCOPY
difference between what is viewed in…
- BF
- DIC
- Ph
BF = no filter
DIC = able to contrast background and sample (has some 3 dimensionality)
Ph = useful for tissue and cells that are changing shape. Enable observations on whether sample is changing shape.
Describe what light microscopy can be used to view (3)
- Histology
- Phase contrast- cell morphology
- Time-lapse (heart cell differentiation, cell migration)
Describe the histology you can observe using light microscopy.
Histology allows you to view the whole tissue and shows you geographically what is happening in the tissue, HOWEVER, it does NOT tell you what each individual cell is doing
how can you see what each cell is doing in more detail when observing histology using light microscopy
- immunohistochemistry
2. laser capture microdissection
describe what immunohistochemistry is
You can use antibodies to find where your protein of interest is found (specific antibody will interact with antigen on cell)
describe what laser capture microdissection is
remove area of interest using laser that cuts through selected area
Describe what happens in phase contrast which allows you to view cells
You can select which intensity of light goes through the sample – wavelength is not changed, but how much is reflected and how much is refracted is.
It’s important when looking at where cells or tissue stays
what method is used in the time-lapse aspect of light microscopy
The box life imaging method is used (control CO2 and temperature).
give 2 examples of what can be observed when using the time lapse aspect of light microscopy
Heart cell differentiation can be observed.
Cell migration, e.g. crawling leukocyte chasing bacteria, can also be observed.
describe the basis of electron microscopy and the differences that can be seen
An electron source is used instead of light – a beam of electrons go to sample, and gives us a dark image of the areas sampled.
If an area is more dense, the electrons have more difficulty passing through the structure meaning it has a darker appearance.
difference between TEM and SEM
TRANSMISSION EM –
this is not 3D; a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through
SCANNING EM –
the sample is treated with specific reagents, then we scan a beam of electrons through sample at a particular angle, this creates a 3D image
Describe fluorescence microscopy
- what light source is used
- what is the ocular
Here, we are controlling the wavelength of light – selecting if light going through is red, green, etc.
We are able to modify proteins in the sample to respond to specific wavelengths.
ocular:
- eyes
- camera
- PTM
Different between light microscopy (bright field) and fluorescence microscopy
The placement of the light source
Describe the mechanism of a fluorescence microscope
- what is fluorescence?
- describe the process
Fluorescence is the ability of certain types of molecules to absorb light, become excited and generate energy, and emit energy.
The specimen is exposed to light; it then absorbs light (excitation), and releases energy (emission).
It emits light at a specific wavelength. Several rounds of this cycle will eventually lead to energy loss and the molecule getting destroyed, leading to no more fluorescence.
What is stokes shift?
Stokes shift is the difference between the wavelength of excitation and the wavelength of the emission
Note: the excitation wavelength is ALWAYS smaller than the emission wavelength
You’ll have 2 peaks: the excitation peak (energy given to molecule), and the emission peak (molecule releases energy) .
Due to energy loss, the emitted light is shifted to longer wavelength relative to the excitation light.
Describe photobleaching.
High intensity illumination may destroy fluorophores and cause them to permanently lose their ability to emit light.
You should work with reduced light intensity, use shorter exposure times or use anti-bleach in mounting media to avoid this.
Fluorescent proteins (eg- GFP) can be fused with other proteins and introduced in cells via transfection. This allows the live study of fluorescent tags in living cells/organisms. What are two ways in which we can achieve that?
- using antibodies
- protein fusion (tag the gene)
Describe using antibodies to fuse fluorescent proteins
antibodies can recognise specific antigen proteins and can then attach and give off colour
using antibodies to fuse fluorescent proteins
+ and -
The advantage: it’s 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).
The disadvantage: it can only be applied to a fixed sample if you want antigen to bind to sub-cellular structures (as the antibody is big and can’t go past cell membrane).
Describe protein fusion with fusing fluorescent proteins.
You use plasmids to incorporate a gene that causes fluorescence.
It’s incorporated into undifferentiated ES (embryonic stem) cells, which can eventually form fibroblasts.
protein fusion with fusing fluorescent proteins
+ and -
The advantage: it can be used in live cells.
The disadvantage: it lacks specificity, and you don’t know if the molecule can be kept (cell can undergo apoptosis if they recognise the inserted gene is exogenous).
Describe confocal light microscopy.
- process
- light source?
If we are able to control the depth that the source of light was going through sample, we’d be able to see through thinner portions of sample.
Thus, this involves stopping the beam of light at certain levels, so you can see different levels of sample, e.g. in seeing an epithelial cell (seeing specifically only apical or basolateral side).
The light source is a laser – so we are able to control how it goes through sample.
confocal light microscopy
+ and - (compared to wide field)
The advantage: better Z resolution, better specifics as we can see through several layers; it also allows live imaging (control temp and CO2).
The disadvantage: only a small volume can be visualised by confocal microscopes at once bigger volumes more time consuming, as more sampling and image reassembling is needed (widefield is better for bigger volumes!).
What can we use confocal light microscopy to view?
• Developmental dispersal of drosophila haemocytes
View the tissue and cellular localisation
• Microtubule dynamics using GFP-tubulin
• Vesicle transport through microtubules using GFP-kinesin
• Look at the cell surface
• 3D reconstruction
Comparison of neoplastic to non neoplastic
