3. Microscopy Flashcards
What is microscopy?
Allows us to view objects/specimens that are not visible to the naked eye.
What is the basic microscope apparatus composed of?
- 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)
What order does the apparatus travel from eye detector to the light source?
Eye /detector -> objective to zoom in -> specimen -> light conditioning system -> light source
What is the light microscopic specimen composed of?
- 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
What is life imaging? – The box
• 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
What is the box and the cube?
- 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.
How is CO2 levels maintained in the box?
- 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
What are important factors in various experimental timescales?
- 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
What is the “triangle of frustration”?
• 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
What is intensity resolution?
Intensity resolution – how finely a system can represent or distinguish differences of intensity, usually expressed as a number levels or a number of bits
What do the marking on objectives mean?
- 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
What are the components of a light microscope?
- Ocular
- Objectives
- Sample
- Condenser
- Light source
How can full light be modified in a light microscope?
• 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
What is a light microscope used for?
A) HISTOLOGY
- 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) - 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
What is electron microscopy? – different types
• 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
