Lecture 4 Reading Chapter 9 visualizing cells Flashcards
Scope of light microscope
0.4-0.7 micrometers
Cell doctrine
All plants and animal tissues are aggregated of individual cells. Schleiden and Schwann, 1838
Optical diffraction effects
Light waves travel through different routes, and not in a straight line, so they interfere with each other
In phase
Interference makes things brighter
Out of phase
Light waves interfere with each other in such a way as to cancel each other partly or entirely
Micrometer
10^-6 m
Nanometer
10^-9 meters
Angstrom
10^-10 meters
Limit of resolution
The limiting separation at which two objects appear distinct. Depends on wavelength of light and numerical aperture of lens system used.
How does numerical aperture work
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 I
Refractive index
Ratio of the speed of light in a vacuum to the speed of light in a particular transparent medium
How can contrast in a specimen be generated
Light microscopes with special optical systems
Bright field microscope
Light passing through a cell in culture forms the image directly
Dark field microscopy
Exploits the fact that light rays can be scattered in all directions by small objects in their path. Bright image against a black background
How does light change as it passes through a cell
The phase of the light wave is changed according to the cell’s refractive index.
How do phase contrast microscope and differential interference contrast microscope work
Increase phase differences caused by cell’s refractive index so that waves are more nearly out of phase, producing amplitude differences when the sets of waves recombine, thereby creating an image of the cell’s structure
How do we increase our ability to observe cells
We can attach a sensitive digital camera to a microscope, the camera detects light by means of charged coupled devices(CCDs) or high sensitivity complementary metal-oxide semiconductors (CMOs) sensors. These sensors are ten times more sensitive than naked human eye and can detect 100 times more intensity levels
How do we prepare tissue for microscopy
Fix and section tissue, then freeze or embed
Sections
Very thin transparent slices
Fixative
Forms covalent bonds with the free amino groups of proteins, cross linking the, so that they are stabilized and locked in position
Usual embedding medium for tissue
Waxes or resins
Three main approaches to working with thin tissue sections that reveal differences in types of molecules that are present
Sections can be stained with organic dyesthat have some specific affinity for particular subcellular components
Sectioned tissues can be used to visualize specific patterns of differential gene expression
Using fluorescent probes and markers
How do fluorescent molecules absorb and emit light
Absorb light at one wavelength and emit it at a longer wavelength.
Fluorescence microscope
Tool to visualize fluorescent dyes for staining cells. Illuminating light 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,
When is fluorescence microscopy used
Ro detect specific proteins or other molecules in cells and tissues
How are antibodies used with fluorescent dyes
They are coupled. The. Antibody molecules serve as highly specific and versatile staining reagents that bind selectively to the particular macromolecules they recognize in cells or in the extracellular matrix.
What two dyes are used with antibodies frequently
Fluorescein , emits intense green fluorescence when excited with blue light
Rhodamine, emits deep red fluorescence when excited with green-yellow light
Quantum dots
Tiny crystals of semiconductor metal that can be excited to fluoresce by a broad spectrum of blue light
How are antibodies prepared for microscopy
Either purified from antiserum so as to remove all nonspecific antibodies, or they are specific monoclonal antibodies that only recognize the target molecule
Indirect immunocytochemistry
Using an unlabeled primary antibody and then detecting it with a group of labeled secondary antibodies that bind to it
Two ways to solve out of focus problem
Give many sectional views of optical sections
Image deconvolution
Image deconvolution
Computer calculates what blurring would have done and reverses
Point spread function
Blurred image of a point source
Confocal microscope
Manipulates light before it is measures. Analog technique.
Generally used with fluorescent optics. Focused on single point not ph whole object
Thick specimens are viewed with
Multi photon microscopes, with two-photon effect.
Green fluorescent protein (GFP)
Isolated from a jellyfish. Encoded by a single that can be cloned and introduced into cells of other species
How is GFP used usually
As a reporter molecule, a fluorescent probe to monitor gene expression
How do we use fluorescent proteins to uncover kinetic properties of a cell
Fluorescence resonance energy transfer (FRET)
Photoactivation
Fluorescence recovery after photo bleaching (FRAP)
FRET
Teo molecules of interest are each labeled with a different fluorochrome, chosen so that the emission spectrum of one fluorochrome, the donor, overlaps with the absorption spectrum of the other, the acceptor.
Photoactivation
Fluorescence tagging technique that allows detailed observations of proteins within cells. Involves synthesizing an inactive form of the fluorescent molecule of interest, introducing it into the cell, and then activating it suddenly at a chosen site in the cell by focusing a spot light on it.
Caged molecules
Inactive photosensitive precursors
Fluorescence recovery after photo bleaching (FRAP)
One uses a strong beam of light from a laser to extinguish the GFP fluorescence in a specified region of the cell, after which one can analyze the way in which remaining unbleached fluorescent protein molecules move into the bleached area as a function of time.
Why are micro electrodes not good at measuring changing ion concentrations
They reveal ion concentration only at one point of cell
Ion sensitive indicators
Suited to record rapid and transient changes in ion concentration. Have light emission that reflects local concentration of ion.
Aequorin
Luminescent protein. Emits blue light in presence of Ca2+, and responds to changes in concentration in the range of 0.5-10 micrometers.
Genetically encoded fluorescent indicators
Bind ion tightly. Are excited by or emit light at slightly different wavelengths when they are free of io than when bound. By measuring the ratio of fluorescence intensity at two excitation or emission wavelengths, one can determine concentration ratio of ion bound indicator to the free indicator and get a measurement of ion.
Why can’t single fluorescent molecules be reliably detected in ordinary microscopes
Limitation arises from the strong background due to light emitted or scattered by out-of-focus molecules. This tends to blot out the fluorescence from the particular molecule of interest.
Total internal reflection fluorescence (TIRF) microscopy
Because of total internal reflection, light does not enter sample, so majority of fluorescent molecules are not illuminated. Electromagnetic energy does extend, as an evanescence field, for a short distance beyond the surface of the cover slip and into the specimen, allowing just those molecules in the layer closest to the surface to become excited.
Atomic force microscopy (AFM)
Provides a method to manipulate individual molecules. With its tip, probe can collect data on the variety of forces it encounters.
Approaches in light microscopy that bypass resolution imposed by diffraction of light. Superresolution approaches
Structured illumination microscopy (SIM)
Stimulated emission depletion microscopy (STED)
Photo activated localization microscopy (PALM)
Structured illumination microscopy SIM
Fluorescence imaging method with resolution of about 100 nm.uses grated or structured pattern of light to illuminate the sample. Illuminating grid and sample features combine into an interference pattern, from which the original high resolution contributions to the image beyond the classical resolution can be measures. Imaging is repeated several times and then combined mathematically to create an enhanced image.
Point spread function
Distribution of light intensity within the three dimensional, blurred image that is formed when a single point source of light is brought to a focus with a lens.
Stimulated emission depletion microscopy DTED
Increasing resolution by switching all the fluorescent molecules at the periphery of the blurry excitation spot back to their ground state, where they no longer fluoresce in their normal way. Diffraction limit breached because technique ensures that similar but very closely spaced molecules are in one of two different states, either fluorescing or dark.
Photo activated localization microscopy (PALM)
Labels are activated by illumination with near UV light. A small subset of molecules is modified so that they fluoresce when exposed to an excitation beam at another wavelength. These switch and another subset is activates. The photons of each subset given off are recorded and then aggregated into an image.
Preparing a specimen for electron microscopy
Living tissue is killed and preserved by fixation, usually with glutaraldehyde and osmium tetroxide, which binds to and stabilizes lipid bilayers as well as proteins. Cut into extremely thin sections.
What does image clarity depend on in an electron micrograph?
A range of contrasting electron densities. Impregnate tissues with salts of heavy metals.
Immunoglobulins electron microscopy
Incubate a thin section with a specific primary antibody, and then with a secondary antibody to which a colloidal gold particle has been attached.
Creating a third dimension view of a specimen
Have views of the same specimen from many different directions
Electron microscope tomography
Specimen holder is tilted to arrive at a 3d view
Scanning electron microscope (SEM)
Directly produces an image of the three dimensional structure of the surface of a specimen. Uses electrons that are scattered or emitted from the specimen’s surface.
Negative staining
Allows finer detail of macromolecules to be seen. Molecules are supported on a thin film of carbon and mixed with a solution of heavy metal salt. Creates reverse or negative image of molecule.
