Introduction To Microscopy Flashcards
1
Q
What is microscopy?
A
Using microscopes to view objects specimens that are not visible to the naked eye.
2
Q
State the major components of the microscope?
A
- Light source (halogen, XBO)
- Light conditioning system
- Optimises beam of light to sample and avoids reflections so best possible image can be produced.
- Specimen (cover glass)
- Light transmits here
- Objective (immersion medium)
- Magnifies or makes image bigger
5. Detector via eyes or computer
3
Q
Describe what a light microscopic specimen is normally kept on?
A
- Cover glass with glass slide
- Samples is surrounded by an embedding medium (might contain anti-bleach agent)
4
Q
What is used for life imaging and why?
A
- Life imaging - alive samples over a period of time
- The Box (custom design of openings and doors for sample) is used
- Must make sure temperature + CO2 atmosphere is maintained
- Small changes in atmosphere leads to changes in extension or contraction of microscope stand changing plane of focus - so micrscope doesn’t remaine equilibrated
- Controller is used for air flow adjustment and control of CO2 %
- If sample is very small, use air tight table top over live cell culutre which keeps conditions constant in cell
5
Q
Describe problems that can arise using alive sample in terms of experimental timescales?
A
- Depending on what you are looking at, there will be limitations on the images you are looking at depending on the length of timeframe
- Short timeframe (smaller organelles) - artifacts occuring in 4D imaging
- Artefacts = something observed in a scientific investigation or experiment that is not naturally present but occurs as a result of the preparative or investigative procedure
- Long timeframe (development) - stability, viability and multi-position timelapse
6
Q
Describe the ‘triangle of frustration’ of signal detection?
A
- For long processes: Prioritise temporal resolution
- For short processes/ very low signal: prioritise sensitivity/binning
7
Q
What is binning?
A
- Binning is a technique to boost camera frame rate and dynamic range whilst reducing noise by sacrificing resolution
- It is often used for high speed fluorescence time- lapse experiments
- Exposure time is the length of time the camera collects light from your sample and there are tradeoffs between exposure time, image brightness, and phototoxicity, especially for live-cell imaging.
8
Q
Describe spatial resolution?
A
- Resolution is proportional to pixel area
- The smaller the pixel area, the higher the resolution
- Resolution = sharpness of image
- Pixel area determines the detail of the image
9
Q
Describe intensity resolution?
A
- The Greater the info. (bits) -> increased intensity -> The greater the resolution of pictures
- Number of bits represents each pixel within the image
10
Q
State the various markings found on objectives?
VD
A
- Magnification
- Application
- Lens-image distance/coverslip thickness
- Numerical aperture/immersion medium
- Working distance
- Working distance = max. distance objective can go from sample
11
Q
Describe what the objective resolution power is dependent on?
A
- The aperture (measure of its ability to gather light and resolve fine specimen detail at a fixed object distance) of the objective determines the resolution (detail)
- The higher the numerical aperture the better the resolution power of the objective.
- Resolution has nothing to do with magnification
- Magnification is only about making the image bigger, not clearer
12
Q
What is light microscopy used for and state 3 types?
A
- It is used to illuminate and magnify an image
- 3 types - colour Brightfield, Phase-contrast + differential interference contrast (DIC)
- Fundamental setup always the same
- DIC, Phase & colour brightful
- VD
13
Q
State 3 uses of light microscopy?
A
- Histology
- Phase contrast- cell morphology
- Time-lapse (heart cell differentiation, cell migration)
14
Q
State pros and cons of studying histology using microscopy?
A
- Pros: Can identify areas of interest within sample
- Cons: Can’t see detail within cells
- Can use antibodies to identify protein of interest, but again limited via fixed cell and low detail
15
Q
How is phase contrast studied by light microscopy?
A
- Phase contrast microscopy of fibroblasts cultured on:
- (a) intact collagen;
- (b) denatured collagen.
- Allows to compare between two individual times of how cells looked
- Limitations: Fixed timeline of both cultures so can’t define how cells underwent changes
- Can’t track the process of how changes occured
