Microscopy Flashcards
What is the use of microscopes?
Microscopes used to view objects/specimens that are not visible to the naked eye.
Invented by eyeglass makers in 1590s
What are the characteristic parts of a microscope?
- Light source
- Detector
- Objective
- Specimen
- Light conditioning system
Describe the types of detector used in microscopes
Photomultiplier tubes (PMT), CCD
PMTs increase signal and transform it into data read by a computer
What is the purpose of the light conditioning system?
Light conditioning system allows light to reach specimen
Name different light conditioning systems
- Kӧhler illumination
- Phase ring
- Wollaston prism and polarizers
- Filter cubes (for fluorescence
What are the different light sources for microscopes?
Halogen bulb, sunlight, lasers etc.
How do we ensure all microscopes work to a standard?
To ensure correct functioning of the objectives and to quantify what we see there needs to be constants in place
What is the approximate thickness of a light microscope cover glass?
Cover glass (0.17- 0.18 mm) - anything thicker = objective has to be corrected
What are samples embedded in for light microscope use?
Sample surrounded by embedding medium (might contain anti-bleach agent)
on a glass slide
What does the box in light microscopes enable?
Allows for life imaging as prevents focus instability
What is the effect of temperature on light microscopes?
Small changes in ambient temperature => thermal expansion / contraction in:
- microscope stand
- stage
- objective
> thereby changing the plane of focus
How is temperature regulated in light microscopes?
An incubator box combined w/ a precision air heater ensures specimen and microscope temperature remain equilibrated and tightly controlled
Describe the light microscope box structure
> Custom design for individual microscopy setup
Intricate system of openings and doors for comfortable access to microscope controls and specimen
Describe the structure of the light microscope incubator cube
Highest quality fan, controller cube with external, low vibration and low-nose design
What is the role of the light microscope cube?
One side prevents drastic temperature changes and also maintains CO₂ / O₂ levels to keep specimen alive
How is the airflow regulated in the light microscope?
A controller allows adjustment of air flow and %CO₂
Possibility of guiding gas stream through bottle of water to diminish loss of humidity
How is the live cell culture device enclosed in a light microscope?
Airtight table top encloses the live cell culture device
What time considerations are required when deciding what microscope / sample to use?
Need to consider the time required and how to preserve the sample for that long
Describe the different experimental timescales of viewing different samples
Development of zebrafish embryo requires hours and even more complex animals may need days
To see how a microtubule extends / shrinks only requires seconds
What are the inconveniences of having longer timescales?
In longer timescales: - Stability - Viability - Possibility - Multi-portion timelapse require different complex algorithms
What problems need to be overcome when using short experimental timescales?
For shorter timescales:
- Ensure magnifier used has ability to capture imaging
quickly
- Need to distinguish environment from sample
- Artefacts in multichannel / 4D imaging
What is the triangle of frustration?
3 factors of microscope use that are compromised between depending on what we need to investigate
What are the 3 factors of the triangle of frustration?
- Spatial resolution
- Sensitivity
- Temporal Resolution
What is temporal resolution?
How long and how fast images need to be taken
What is sensitivity?
The ability to pick up image in lower light conditions (quality of image)
What is spatial resolution?
The pixel no.
> bigger cube image taken faster but lower quality low resolution
> smaller cube image taken slower but higher quality high resolution)
How does time taken effect image resolution?
To get a high resolution image, microscope requires more time to collect data and CPU to read it
How long does it take for a microscope to produce a low resolution image?
Microscope can generate a low resolution image very quickly
Why may we sacrifice high resolution when capturing an image?
In fast occurring processes, capturing an image can be hard as everytime you look at the image it will have changed
Need system that captures images fast: Sacrifice resolution to capture image
Why are non-living specimens usually captured in higher resolution?
In non-living specimens, there is no time limit so can spend longer on an image and gain higher resolution
What is a microscope objective?
An objective is a series of high quality pieces of glass placed in a particular way, allowing us to see magnified images
What is the magnification?
The magnification tells us how much bigger the image will be seen compared to original
Can be colour coded or written as number (e.g. 40x)
What is an objective application?
Certain objectives are more suitable for certain applications over others - objective will be marked for it’s suitable applications
Why may an immersion medium be required?
Immersion medium may be needed (e.g. water, oil) to see specimen - looking through air won’t produce an image and vice versa
What is a working distance of an objective?
Distance from sample the objective can work at (e.g. 0.20 mm)
What markings are present on an objective?
- Magnification
- Application
- Lens - image distance
- Cover slip thickness (mm)
- Working Distance (mm)
- Numerical aperture
- Immersion medium
What is the role of the objective aperture?
The aperture of the objective determines the resolution
How can we determine resolution from numerical apertures?
The higher the numerical aperture the better the resolution power of the objective
What is the relationship between resolution and magnification?
RESOLUTION ≠ MAGNIFICATION
What features allows an image to be seen using a light microscope?
Light and Lenses to illuminate and magnify
Describe the fundamental Setup of a light microscope
Always contain:
- Light source
- Trebles
- Rings / Lens to modify light source if needed
- Objective magnifies image
- Viewed through ocular
What are the different types of light microscopy techniques?
DIC
Brightfield
Phase Contrast
What is brightfield light microscopy?
Brightfield - all light reaches sample
What is DIC microscopy?
DIC - condenses light through a smaller area
Allows for 3D
Describe phase contrast light microscopy
Phase contrast - uses a phase ring with a small circumference where light can go through.
What are the pros of using light microscopy for histology?
Pros: Gain general idea of tissue
What is the disadvantage of light microscopy for histology?
Cons: Lacks details, hard to distinguish between cells
How is light microscopy used to identify ovulation site in an ovary?
Use chemicals that react with particular parts of tissue based mainly within pH range of sample
What enables the chemicals to stick to the different types of tissues?
There are chemicals that have a higher affinity for basic tissues and others have a higher affinity for acidic tissues
What is immunohistochemistry?
Protein of interest is found using antibodies produced against an exogenous agent with high affinities for particular antigens
What is the use of immunohistochemistry?
allows us to see specific proteins
How does phase contrast work?
Reduces the amount of grey’s (black or white) - increasing contrast to see cell margins
What is a major use of brightfield?
Colour brightfield used to see life events
e.g. Heart Cell Differentiation
Differentiation of cardiomyocyte-like cells derived from adipocytes.
How are samples maintained in colour brightfield microscopy of life imaging?
Need to control and regulate temperature and CO₂ to keep sample alive
What are the 2 methods of life imaging ?
- Take fixed images over a course of time
2. View under microscope and monitor periodically to see development
What cell feature is viewed using time-lapse microscopy?
Cell migration also viewed via time-lapse microscopy
> takes images after specific time intervals and collates them together
How do (~smallpox) virus infected cells migrate?
When the cells are infected they migrate towards empty area where they extend and send viral particles to one another
How did using time-lapse microscopy enable us to prevent viral spread?
Allowed us to remove specific gene from infected cells prevented migration and spread of infection
Describe the light source in e- microscopes
The light source in electron microscopy is a beam of electrons and not light
Describe transmission electron microscopes
TEM not 3D, beam of e- transmitted through ultra-thin specimen, interacting with specimen as it passes through (Uranyl acetate used as a stain)
Describe an EM image
e- can’t pass through denser areas show up darker and lighter areas are less dense
How is the image detected in EM?
e- eventually end up at a viewing screen connected to a camera / PMTs to view higher resolution images
Describe the use of scanning electron microscopes
sample treated with specific reagents, scans a beam of electrons through sample creates 3D images (key difference with transmission!)
How do fluorescence microscopes create fluorescence?
Uses a particular wavelength of light; fluorescence light
How do fluorescence microscopes produce an image?
- Light modified through various filters
- Specimen exposed to light
- Specimen absorbs light (excitation) and releases energy
(emission) - Emission is taken up to the oculars
How is fluorescent image detected in a fluorescent microscope?
PMTs transform the fluorescent image into computer data that is safe to view
What is fluorescence?
Fluorescence is the ability of certain types of molecules to absorb light and generate energy
Describe the mechanism of fluorescence
Specimen exposed to light absorbs light (excitation) releases energy (emission)
Why is fluorescence a finite feature?
Several rounds of this cycle will eventually lead to energy loss and molecule gets destroyed no fluorescence
What is stokes shift?
Difference between wavelength used to excite molecule of interest, and emission wavelength of molecule after energy release
Describe the wavelengths of emission and excitation
Excitation < emission wavelength always bigger
Why is emission always larger than excitation?
Due to energy loss the emitted light is shifted to longer wavelengths relative to the excitation light
Why may photo-bleaching occur?
Bleaching of fluorochrome; due to high intensity illumination
Causes fluorochrome to lose permanently their ability to emit light
How can photo-bleaching be prevented?
→Work w/ 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 mounting media
Describe the variety of fluorochromes available currently
Now have lots of fluorochromes able to emit and absorb most wavelengths of light spectrum
Give an example of a fluorescent protein
GFP: These proteins are naturally found in light-producing cells of cnidarians
How can fluorescent proteins be used for live imaging?
Fluorescent proteins can be fused with other proteins and introduced in cells via transfection
Allows live study of fluorescent tags in living cells / organisms
Why are GFP better than antibodies?
Can tag GOI with GFP gene via electroporation; allow slive study of fluorescent tags in living cells
When is a confocal fluorescent microscope used?
To view specific portions of a sample a confocal (can be fluorescence) microscope is used.
How does a confocal fluorescent microscope setup differ?
The microscope setup is the same, except for a laser light source
Outline the mechanism of a confocal fluorescent microscope
- Laser emits particular wavelength (can have specific
lasers for different wavelengths) - Laser travels through different modifiers to reach
sample - Sample excited due to fluorophores reaching high
energy - When fluorophores lose energy they emit a certain
wavelength collected by a PMT → CPU → digital image
How do wide field and confocal microscopes differ?
The difference between confocal and wide field microscopes is the detector pinhole - metal with a hole in
What is the purpose of the detector pinhole in fluorescent microscopes?
Of all light emitted by sample, only the light passing through pinhole will reach PMTs to form image - helps remove background and allows us to see very specific areas of sample
Why are confocal images clearer than wide field?
Higher z-resolution and reduced out-of-focus-blur make confocal pictures crisper and clearer
Describe the sample size seen by confocal microscopes
Only a small volume can be visualised by confocal microscopes at once
What is the limitation of sampling bigger volumes?
Bigger volumes need time consuming sampling and image reassembling
