Unit 2.1 Flashcards
most common microscope in use today:
the compound microscope
the compound microscope contains:
several lenses that magnify the image of a specimen
objective lens
picks up the light transmitted by the specimen and focus it on the focal plane of the objective lens, creating a magnified image
condenser lens:
focuses the light onto the specimen no magnification
iris diaphragm
restricts the amount of light entering the lens
occular lens (eyepiece)
The image on the objective focal plane is further magnified by the ocular lens, or eyepiece, and projects it onto the human eye
If the objective lens magnifies 100-fold (a 100X lens) and the ocular lens magnifies 10-fold (a 10X lens), the final magnification will be:
100 x 10 = 1000-fold
the most important property of the microscope is not its magnification but its
resolving power (resolution)
Resolution (D) is the ability to
See two nearby points as distinct images. The smaller the value of D, the better the resolution.
Resolution (D) is determined by
The objective lens and its ability to gather the “cone of light” coming from the specimen. The light comes into the objective lens as a cone due to diffraction by the specimen.
the smaller the value of D, the _
better the resolution
a represents
half the angle of the cone of light
lambda is:
the wavelength of incident light in nm
n is:
the refractive index of the medium between the
specimen and the objective
The lower the wavelength, the
better the resolution.
The shortest wavelength visible light is
450 nm (blue).
A fundamental limitation on all microscopes:
a given type of radiation cannot be used to probe details smaller than its own wavelength (l).
A fundamental limitation on all microscopes: a given type of radiation cannot be used to probe details smaller than its own wavelength (l).
We can partially circumvent this limitation by increasing
a, which will decrease D.
The best objectives have an a value of
70°
Thus as a increases, the denominator:
ncreases, lowering the value of D).
A fundamental limitation on all microscopes: a given type of radiation cannot be used to probe details smaller than its own wavelength (l).
Another way to circumvent this limitation:
increase the refractive index of the medium between the specimen and the objective lens (n)
No matter how many times the image is magnified, the light microscope can never resolve objects that are less than
~ 0.2 μm in size
Three common types of light microscopy:
(1) Brightfield
(2) Phase contrast
(3) Differential interference contract (DIC) or nomarski interference
Brightfield light microscopy:
no contrast other than natural is provided, image is projected on a background of the cone of light that enters the objective lens.
Phase contrast light microscopy:
as light passes through a sample it is slowed in a medium of higher refractive index. The refracted and unrefracted light are recombined to form the image. If light is out of phase it will be less bright, in phase it will be more bright. Requires a phase plate to be inserted into the microscope after light passes through the objective lens.
Differential interference contrast (DIC) or Nomarski interference light microscopy
based on interference between polarized light and the medium (refractive index) through which it travels. Method of choice for thicker samples.
Transmission electron microscopes (TEMs) use
electrons instead of light to form images.
Comparison between TEM and light microscope (5):
- Tem is LARGER (>2m) compared to a light microscope (30cm)
- Tem uses ELECTRONS instead of light
3.Beams of electrons are projected DOWNWARDS while light is projected upwards - TEM uses ELECTROMAGNETIC COILS to focus the beam of electrons and to magnify the image while light microscopy uses glass lenses
- TEM is maintained in a VACCUM wwhile the light microscope operatesin air
TEM has ___ more resolution compared to a light microscope
1000 fold
Minimum resolvable
by electron microscope
0.2 nm
–> to view molecules
minimum resolvable by light microscope:
200 nm
–> to view organelles
Fluorescence microscopy
Fluorescent molecules absorb light at one wavelength (the excitation wavelength) and emit light (fluoresce) at another, longer wavelength (the emission wavelength)
Fluorescence is a three-stage process
Excitation: A photon of energy is supplied by an external source (e.g. a laser) and absorbed by a fluorescent molecule creating an excited state (S1’)
Energy dissipation: The energy of S1’ is partially dissipated, yielding a relaxed state (S1)
Fluorescence emission: A photon of energy is emitted, returning the fluorescent molecule to its ground state (S0)
Fluorescein emits
green light when activated by light of the appropriate wavelength
Tetramethylrhodamine emits
red light when activated by light of the appropriate wavelength
Confocal microscopy improves
the image of a conventional fluorescence microscope
how does confocal microscopy enhance image:
Conventional fluorescence microscopy generates a blurry image because light from above and below the plane of focus are also collected.
Confocal microscopy focuses a single point of light at a specific depth in the specimen.
Immunofluorescence microscopy used to
Revealing specific proteins in fixed cells -
In immunofluorescence microscopy: the use of secondary antibodies:
results in the amplification of the fluorescent signal, thereby increasing the sensitivity of immunofluorescence microscopy
What does fixation of a sample do?
“locks” the proteins in place while preserving cell architecture.
There are two main types of fixation:
(1) cross linking
(2) precipitation:
Cross-linking
reagents such as paraformaldehyde or
glutaraldehyde. Glutaraldehyde autofluoresces (green
emission) and may interfere with fluorescence signal.
Precipitation
using a cold organic solutions such as
acetone or methanol. Dehydrates the sample and can result in changes to cell architecture
In immunofluorescence microscopy:Once the sample is fixed, the membranes will need to be
permeabilized to allow for entry of the antibodies. This is accomplished by organic solutions (precipitation fixation accomplishes this simultaneously) or by treatment with detergent such as Triton X-100.
Considerations when using multiple primary antibodies for immunofluorescence microscopy
- Make sure the host species (species in which the antibodies are raised – rabbit, mouse, human) for the primary antibodies are different. Otherwise, the secondary antibodies will bind to both primary antibodies and you will not be able to distinguish your proteins of interest.
- Make sure the fluorescent molecules or proteins on the secondary antibodies emit at a wavelength that is sufficiently different so that you can distinguish the signals.
Revealing specific proteins in living cells
Green fluorescent protein (GFP)
describe GFP
Emits a green fluorescence when exposed to light of the
exciting wavelength.
When “linked” to a protein of interest, GFP (or other fluorescent proteins) can be used to follow the localization and movement of the protein of interest in live cells.
There are now many fluorescent proteins, spanning numerous different wavelengths. This allows researchers to
investigate several proteins in the same cell.
Tissue culture
Cells can be isolated from tissues: 3 steps:
- Disrupt cell-cell contacts with a protease such as trypsin or collagenase, or with EDTA which chelates Ca2+ (required for cell-cell adhesion)
- Plate the cells in a plastic dish. Some cells require a layer of collagen to adhere to the plates, others can adhere to the plastic (adherent cells) while others are will not adhere to the plastic or to an extracellular component (non-adherent cells).
- If there is a mixed population of cells, fluorescence- activated cell sorting (FACS) can be used to separate the population.
Sorting cell types by FACS
An antibody recognizing a protein facing the outside surface of the cell is coupled to a fluorescent dye and suspended in a fluid.
Droplets with single cells pass by a laser to excite the fluorescent dye. If the detector registers fluorescence, the droplet is immediately negatively charged. Otherwise, the drops are not charged.
The droplets are then deflected in an electric field and collected. They are then put into culture on plastic dishes.
confluency
a measure of the surface area occupied by the cells
As the cells grow, they occupy more space on the dish. Once they reach 100% confluency (a measure of the surface area occupied by the cells), they must be passaged. :
. They are removed from the dish with trypsin or EDTA and plated onto a new dish.
Cells that are derived from a tissue are called primary cells. These cells can only be passaged 25-40 times and then
they stop dividing (they become senescent). They do not express telomerase.
Primary cells can be immortalized by adding DNA that expresses
telomerase.
Primary cells can be immortalized by adding DNA that expresses telomerase. The cells are referred to as a
cell line.
Some cancer cells have the ability to grow indefinitely and are referred to as
transformed cell line.
HeLa
human epithelial
293
human kidney
CHO
hamster ovary
MDCK
dog epithelia
COS
monkey kidney
The growth media for cultured cells contains:
- 9 essential amino acids (Phe, Val, Thr, Trp, Ile, Met, Leu, Lys, His)
- Glutamine – non-essential amino acid but used as a nitrogen source
- Vitamins
- Fatty acids
- Glucose
- Serum (non-cellular portion of clotted blood)
Ø Hormones (e.g. insulin)
Ø Transferrin (iron transport) Ø Growth factors
