Visualizing Cells
stochastic optical reconstruction microscopy/photoactivated localization microscopy
Uses lasers to sequentially switch on a subset of fluorescent molecules in a specimen continuing photoactivatable or photoswitchable fluorescent labels which are activated and imaged before bleaching quenches them. Switching on and off can be done over and over.
light microscope
a class of microscope that uses visible light to create an image. A limit to its resolution is that the range of visibility was the same as the wavelength for visible light
structured illumination microscopy
a fluorescence imaging method with a resolution of about 100 nm (twice the resolution of conventional bright field and confocal microscopy). Can be used to create 3D data. Combines the pattern of an unknown structure and the pattern of a known structure resulting in a somewhat moire pattern containing more information.
immunogold electron microscopy
a thin section is first incubated with a specific antibody and then a second antibody to which a colloidal gold particle has been attached and can be seen as a black dot in the electron microscope. Low sensitivity method, can only detect antigens at the surface of the section.
numerical aperture
affects the light gathering ability of the lens and is related both to the angle of the cone of light that can enter it and to the refractive index of the medium the lens is operating in (the wider the microscope opens its "eye" the sharper it can see).
Negative staining
allows finer detail to be seen. The molecules are supported on a thin film of carbon and mixed with a solution of a heavy-metal salt such as uranyl acetate. Especially seful for viewing large macromolecular aggregates such as viruses or ribosomes.
Transmission electron microscope
analogous to light microscopy, requires detailed sample preparation, used to visualize intracellular structures.
antibodies in microscopy
antibodies are labeled with electron dense particles and purified from antiserum to remove all nonspecific antibodies. Antibodies are used as probes to detect and assay specific molecules in cells.
objective lens
collects a cone of light rays to create an image
image processing
computer based techniques in microscopy that process digital images in order to extract latent information. Enables compensation for some optical faults in microscopes, enhanced contrast to improve detection of small differences in light intensity, and subtraction of background irregularities in the optical system.
second barrier filter in a fluorescence microscop
cuts out unwanted fluorescent signals, passing the specific green fluorescein emission between 520 and 560 nm
superresolution
describes several approaches in light microscopy that bypass the limit imposed by the diffraction of light and successfully allow objects as small as 20 nm to be imaged and clearly resolved.
noise
describes the random variability by which specimens are obscured. This happens by a statistical fluctuations in the numbers of particles detected in each region which give the image a speckled appearance and limit its precision.
attachment of a sensitive digital camera to a light microscope
detect light by means of charge-coupled devices or high sensitivity complementary metal-oxide semiconductor sensors similar to those found in digital cameras. Allow for the observation of cells for long periods at very low light levels thereby avoiding the damaging effects of prolonged bright light and heat. Especially important for viewing fluorescent molecules in living cells.
Gaussian distribution
distribution of light has a width at half-maximum height under ideal conditions at about 200 nm. two points that are closer together will become hard to resolve because their images overlap too much.
confocal fluorescence microscope
fluorescent specimen is illuminated with a focused point of light from a pinhole. Emitted fluorescent light from in focus point is focused at pinhole and reaches detector. Emitted light from out-of-focus point is out of focus at pinhole and is largely excluded by the detector.
condenser lens
focuses a cone of light rays onto each point of the specimen
interference between light waves
if two trans of waves reaching the same point are in phase with each other (crest with crest and trough with trough) then they will reinforce each other so as to increase brightness. If they are out of phase and interfere with each other they will cancel each other out either partly or entirely.
ways in which contrast in a specimen can be generated
incident light (white) through a stained section of cell, oblique incident light, incident light (white) through an unstained cell.
Green fluorescent protein (GFP)
isolated from jellyfish. The protein is encoded by a single gene which can be cloned and produced into cells of other species. Undergoes a self-catalyzed post-translational modification to generate an efficient fluorochrome, shielded within the interior of a barrel-like protein which will now fluoresce when illuminated appropriately with blue light. Used as a fluorescent probe to monitor gene expression.
first barrier filter of a fluorescence microscope
lets through only blue light with a wavelength between 450 and 490 nm
bright field microscope
normal light microscope in which the image is obtained by simple transmission of light through the object being viewed
Differences between optical and electron microscopy
optical microscopes use an optical lens, have low magnification, don't require a vacuum for operation, have small depth of field, and are fairly inexpensive; electron microscopes use magnetic lenses, have high magnification, require a vacuum for operation, have large depth of field, and fairly expensive.
cell doctrine
proposed in 1838 by Schleiden and Schwann; The proposal that all living organisms are composed of one or more cells and that all cells arise from the division of other living cells
Atomic force microscopy (AFM)
provides a method to manipulate individual molecules themselves. The tip of the AFM probe has a highly precise positioning system that allows it to be moved over very small distances. It is also able to collect information about a variety of forces that it encounters as they are felt by the tip moving close to or touching the surface.
beam-spitting mirror in a fluorescence microscope
reflects light below 510 nm but transmits light above 510 nm
fluorescence microscope
similar to an ordinary light microscope except that the illuminating light, from a very powerful source, is passed through two sets of filters - one to filter the light before it reaches the specimen and one to filter the light obtained from the specimen. Often used to detect specific proteins or other molecules in cells and tissues.
Optical Microscopy
specifically labels and produces images of individual cell components and the reconstruction of their 3D architecture. One advantage is that the light is regularly nondestructive. Allows the position of molecules to be mapped.
Electron microscope tomography
technique for viewing 3D specimens in the electron microscope in which multiple views are taken from different directions by tilting the specimen holder.
Point-spread function
the distribution of light intensity within the 3D, blurred image that is formed when a single point source of light is brought to a focus with a lens.
limit of resolution
the limiting separation at which two objects appear distinct; depends on both the wavelength of the light and numerical aperture of the lens system used.
image deconvolution
the process of reversing the optical distortion that takes place in an optical microscope, electron microscope, telescope, or other imaging instrument, thus creating clearer images. It is usually done in the digital domain by a software algorithm, as part of a suite of microscope image processing techniques. Deconvolution is also practical to sharpen images that suffer from fast motion or jiggles during capturing
refractive index
the ratio of the speed of light in a vacuum to the speed of light in a particular transparent medium
quantum dots
tiny particles of cadmium selenide, a semiconductor, with a coating to make them water-soluble. They can e coupled to protein molecules such as antibodies or streptavidin and when introduced into a cell, will bind to a target protein of interest. Different sizes will emit different colors but they are all excited by the same blue light and can keep shining for weeks.
preparation of tissues for microscopy
tissues are often frozen or embedded into a supporting medium and then cut into very thin transparent slices called sections and treated with a fixative to preserve the cells within the tissue. The sections are then stained with organic dyes that have some specific affinity for particular subcellular components.
light waves
travel through an optical system by many slightly different routes so that they interfere with one another and cause optical diffraction effects
Scanning electron microscope
type of electron microscope that produces an image of the surface of an object. Usually smaller and cheaper than a transmission electron microscope. Uses electrons that are scattered or emitted from the specimen's surface.
phase contrast microscope
type of light microscope that exploits the interference effects that occur when light passes through material of different refractive indices. Used to view living cells.
differential interference contrast microscope
type of light microscope that exploits the interference effects that occur when light passes through parts of a cell of different refractive indices. Used to view unstained living cells.
confocal microscope
type of light microscope that produces a clear image of a given plane within a solid object. It uses a laser beam as a pinpoint source of illumination and scans across the plane to produce a two dimensional optical section.
dark field microscopy
type of light microscopy in which oblique rays of light focused on the specimen do not enter the objective lens, but light that is scattered by components in the living cell can be collected to produce a bright image on a dark background.
indirect immunocytochemistry
uses a primary antibody that binds to a specific antigen but is unlabeled and adds to it a labeled secondary antibody that binds to the primary antibody and creates a stronger signal.
Total Internal Reflection Fluorescence Microscopy (TIRF)
uses excitatory laser light to illuminate the cover-slip surface at the critical angle at which all the light is reflected by the glass-water interface. some electromagnetic energy extends a short distance across the interface as an evanescent wave that excites just those molecules that are attached to the cover slip or are very close to its surface. Allows single molecules to visualized under certain conditions, passive method.
cryoelectron microscopy
A thin layer of an aqueous suspension of virus or purified molecular complex is prepared on a microscope grid and is then rapidly frozen by being plunged into a coolant. Produces an image from the macromolecular structure itself.
stimulated emission depletion microscopy
Like confocal microscopy, STED uses very short pulses of laser light to cause molecules in a specimen to fluoresce. The first pulse is immediately followed by a ring shaped depletion pulse, which causes stimulated emission, moving the electrons from the excited state, which causes fluorescence to occur, to a lower energy state before they can fluoresce. The interaction of the two pulses effectively results in a smaller spot size that could be achieved with a conventional microscope.
oblique incident light
Method of generating contrast in a specimen. In the dark field microscope, oblique rays of light focused on the specimen don't enter the objective lens but the light that is scattered by components in the living cell can be collected to produce a bright image on a dark background.
incident light (white) through an unstained cell
Method of generating contrast in a specimen. Light passing through the unstained living cell experiences very little change in amplitude and the structural details can't be seen even if the image is highly magnified. The phase of the light however is altered by its passage through either thicker or denser parts of the cell, and small phase differences may be made visible by exploiting interference effects using a phase contrast or a differential interference contrast microscope.
incident light (white) through a stained section of cell
Method of generating contrast in a specimen. The stained portion of the cell will absorb light of some wavelengths which depends on the stain but will allow other wavelengths to pass through it. A colored image of the cell is there obtained that is visible in the normal bright field light microscope.