Lecture 2 - Microscopy Flashcards
What is used to illuminate specimen in a microscope?
condenser
What is used to detect light in a microscope?
objective
What is magnification?
the extent to which the image of the specimen (viewed under microscope) is enlarged
What happens if specimen is placed in the path of light/electron beam? (beach ball altering the motion of the rope)
specimen changes the physical characteristics of a beam to create an image for human eye/photographic detector
Why does phase shift happen?
due to difference in refractive index
phase shift is undetected by eye; but can be converted to amplitude shift in phase-contrast miscroscope
Why does amplitude change (change in brightness) happen?
due to light absorbance (for unstained biological specimen - minor)
What is refractive index?
measure of change in the velocity (quickness of motion) of light as it passes from one medium (any material substance which can propagate waves or energy) to another
n=c/v
ratio of speed of light in vacuum (c) to the speed of light in that medium (v)
What is diffraction?
bending of a wave around the edges of an opening or an obstacle
the image you see when you look at a specimen through a series of lenses is really just a pattern of either additive or canceling interference of the waves that went through the lenses, a phenomenon known as diffraction.
What are the 3 elements always needed to form an image?
- source of illumination
- specimen
- system of lenses
Why very small objects can be seen only by electron microscopy?
the wavelengths of electrons are very much shorter than those of photons. Thus, objects such as viruses and ribosomes are too small to perturb the waveform of photons, but they can read-ily interact with electrons.
What is interference?
the process by which two or more waves combine to reinforce or cancel one another, pro-ducing a wave equal to the sum of the two combining waves
What is focal length?
the distance between the mid-line of the lens and the point at which rays passing through the lens converge to a focus
The focal length is determined by the index of refraction of the lens itself, the me-dium in which it is immersed, and the geometry of the lens
What is angular aperture?
the half-angle α of the cone of light entering the objective lens of the microscope from the specimen
a measure of how much of the illumination that leaves the specimen actually passes through the lens
What is resolution?
the minimum distance two points can be apart and yet still remain identifiable as separate points when viewed through the microscope: the higher the resolution, the smaller the objects that can be distinguished using a particular lens
factors affecting resolution:
light properties (the wavelength of the light used to illuminate the specimen) lens properties (the angular aperture, aberrations) the refractive index of the medium surrounding the specimen
Why some microscope lenses are designed to be used with a layer of immersion oil between the lens and the specimen?
Immersion oil has a higher refractive index than air and therefore allows the lens to receive more of the light transmitted through the specimen.
r=200nm with an oil immersion lens
r=300nm in air
How does airy disc appear?
as a bright point of light around which concentric ringes/ripples are seen
What determines the diffraction pattern? (airy disc size)
wavelength of light and the size of aperture (circular opening) through which the light passes
light waves of the same wavelength and frequency arriving simultaneously at the same point
interfere with one another
light waves in phase combine
constructive interference - amplitude increases - perceived brightness increases
light waves out of phase
cancel each other partly - destructive interference - amplitude decreases - brightness decreases
3 characteristics of Brightfield microscopy
- transparency of most of biological specimens
- need to use dyes
- need to fix (chemically preserve) and stain => dead samples
How to observe living cells directly?
- Phase-contrast microscopy
- Differential interference contrast microscopy
- Fluorescence microscopy
How does phase-contrast microscopy work?
converts phase difference into brightness difference
How does differential interference contrast (DIC) microscopy work?
polarized light is used
How does fluorescent dye work?
the absorption of light by a mol-ecule and ends with emission of light with a longer wavelength
What is fluorescent probe?
molecule capable of emitting fluorescent light to indicate the presence of a specific molecule/ion
used for immunostaining
What is immunostaining?
technique based on ability of antibodies to recognize and bind to specific molecules
How does indirect immunofluorescence happen?
a tissue or cell is treated with an antibody (primary) that is not labeled with dye; this antibody attaches to specific antogenic sites within tissue/cell; then the secondary antobody (labeled with a fluorescent dye) is added
The difference of confocal miscroscopy
light miscroscope that employs a laser beam minimizing blurring excluding out-of-focus light from an image of a single plane
before the detector - second pinhole - at a position where rays from illuminated point come to focus
How does stimulated emission depletion (STED) work?
uses very short pulses of laser light to cause molecules in a specimen to fluorescence.
The 1st pulse -> ring-shaped depletion pulse (moves electrons from the excited state to a lower energy state before fluorescencing, thus reducing the amount of spontaneous fluorescent emission)
What is the difference between PALM and STORM?
PALM uses photo switchable/convertible fluorescent proteins (FPs), whereas STORM uses organic dyes as fluorescent probes for imaging.
How does STORM microscopy work?
single fluorophores are being imaged (when it is important to count the exact number of molecules in a given structure)
a target structure is activated by a subset of probes (fluorescent labels). After enough fluorophors have been localized, high-resolution image occures
What is the practical resolution limit of electron microscopy?
0.1 nm
