A Introduction To Microscopy Flashcards
What is microscopy?
Using microscope to view objects/specimens that are not visible to naked eye.
What are the essential parts of the microscope?
Detector
Objective
Specimen (cover glass)
Light conditioning system (aim to concentrate beam of light towards sample, avoid reflection and filter types of light)
Light source
How is a light microscope specimen prepared?
Cover glass
Sample surrounded by embedding medium
Glass slide
How can live specimens be viewed under a microscope?
Have a box or cube for the specimen
Small changes in temperature lead to thermal extension or contraction in the microscope stand,stage and objective - changing plane of focus
Incubator box and precision air heater ensures that the temperature of specimen and microscope remain equilibrated and tightly controlled
How is a CO2 atmosphere maintained in a live specimen box?
- controller allows to control airflow + CO2%
OR
- a air tight table top encloses the liver cell culture device
What is the problem with observing short and fast events under microscopy?
Artefacts in multichannel/4D imaging
What is the problem with observing longer processes under a microscope?
- stability, visibility
What is the “triangle of frustration”?
Temporal resolution - sensitivity - spatial resolution
What is the relationship between pixel area and resolution?
The higher the resolution the smaller the pixel area
What is the relationship between intensity levels and resolution?
With greater intensity levels and greater bits we will have greater resolution.
What do the markings on objectives of a microscope mean?
Applications :DIC
Working distance (mm) :WD
Cover slip thickness: -
Numerical aperture/immersion medium: 1.30 Oil
What is the relation between the numerical aperture and resolution?
The higher the numerical aperture the greater the resolution power of the objective.
What is light microscopy?
Uses light and lenses to illuminate and magnify
Has a:
- light source
- condenser
- sample
- objectives
What are the 3 types of light microscopy?
DIC
Phase
Colour brightfield
What is the advantages and disadvantages of using full light microscopy in histology?
Advantages - Have a overview of the tissue and identify areas of interest
Disadvantages - unable to see how the cells interact with each other
How are proteins of interest found under a light microscope for histology?
Using antibodies to identify and “dye” specific cells
How can light microscopy be used to analyse cell morphology?
Microscopy of fibroblasts cultured in:
Intact collagen
Denatures collagen
Can observe structural changes
When can light microscopy be used for time lapses?
Heart cell differentiation
Differentiation of cardiomyocyte-like cells derived from adipocytes
Or in cell migration
What is electron microscopy?
Scanning and transmission
Components:
- electron source
- electron beam
- specimens
- electromagnetic lense
- Viewing screen
What is fluorescence microscopy?
Components:
- fluorescence source of light
- fluorescence filter cube
Explain the absorption and emission of fluorescence light
- Excitation:
Molecules absorb light and her excited - Loss of energy: (STOKES SHIFT)
Molecules loose energy - Emission:
Molecule emits light as it looses energy
What is the stokes shift?
Due to the energy loss the emitted light is shifted to a longer wave length relative to the excitation light.
Absorption always has a slightly lower wavelength than emission
What is photo bleaching?
Bleaching of fluorochromes due to high intensity illumination the fluorochromes might loose permanently they’re ability to emit light
Name some common fluorochromes in light microscopy
ultraviolet/blue:
DAPI
Blue/green:
FITC
GFP
Cy2
Green/red:
TRITC
Cy3
Red/infrared:
Cy5
Where are GFP fluorescent proteins found?
Naturally found in light-producing cells of cnidarians
How to tag cells with fluorescence?
Antibodies vs protein infusion
Plasmid construction inserted into cells and fluorescent proteins expressed
What are the two different fluorescence microscopes?
Confocal:
Laser
Detector pinhole aperture
Dichroic mirror
Photoplier detector (detects light emitted by fluorescence)
Widefeild:
Original set up
What is the difference between widefeild and confocal fluorescence microscopy?
Confocal advantages:
higher resolution and reduced out of focus blur make images more crisper and clearer
Confocal imitations:
Only a small volume can be visualised by confocal microscopes at once
Bigger volumes needed time consuming sampling and image reassembling
What can be observed using a confocal microscope?
Vaccinia virus uses actin tails just after crossing the plasma membrane of infected cells
