Adaptive Optics (AO)
Adaptive Optics is a technique used to correct wavefront distortions in optical systems. It was developed originally for telescopes that had difficulty resolving tiny points of light from far-away galaxies, but is now being applied to microscopy, with equally stunning results. AO systems can detect wavefront distortion and correct it with a deformable mirror. Wavefront distortions result when the diffraction of light waves collected in a curved lens cause blurry artifacts.
for more details and sample images.
The Airy disk is the image of a point source of light recorded by an ideal microscope. Because light is a wave and diffracts around surfaces, the image of a very small object will be limited to a disc with a diameter of roughly one-half the wavelength of the light. The wavelength of green light is around 540nm, so the image of a small object will be about 270nm in size even if the object is much smaller than this! Any two points closer together than 270 nm could not be distinguished from one another because of the overlapping Airy disks. Because light is a wave with peaks and valleys, the Airy disk also has a series of small ripples that extend out from the center — similar to what you see when you drop a pebble into a lake.
Alexa Fluor dyes
The Alexa Fluor dyes are a type of fluorescent dye used in biological imaging which produce exceptionally bright and photostable conjugates, compared with other fluorescent dyes. The team will use fluorescent dyes as an intermediate step (before using Quantum dots) – and will use Alexa 488 and Alexa 660. These dyes are made by Invitrogen, and you can learn more here.
An optical aberration that occurs in optical systems, for example, a microscope. It means that the rays in two perpendicular planes have different foci (they don’t focus at the same spot, their sharp focus will be at different distances). Peter’s lab is planning to use astigmatism for 3-D imaging to determine the depth of features.
Each neuron has only one axon. An axon is a long, thin, threadlike extension of a cell body that carries impulses away from the cell body. An axon of one neuron may have enough branches to make contact with as many as 1000 other neurons. In the human nervous system, the axon can extend more than a meter.
The long, thin axon of a neuron presents some logistical challenges challenge because protein synthesis occurs almost primarily in the neuronal cell body. Axonal transport is the method by which neurons actively transport cargo within the axons (in both directions) for their survival and function. Individual cargoes are moved by molecular motors along microtubles (see diagram below). The molecular motor kinesin is responsible for the transport towards the distal end. In the other direction, dynein is the molecular motor that is responsible for the transport.
Defects of this process are strongly implicated in many aging-related human neurodegenerative diseases. Axonal transport also provides a good model to study general intracellular transport because of its simple and well defined geometry.
In a material, the energy difference between its non-conductive state and its conductive state. There is virtually no bandgap in most metals, but a very large one in an insulator (dielectric). In a semiconductor, the bandgap is small. Technically, the bandgap is the energy it takes to move electrons from the valence band to the conduction band.
Quantum dot “bluing” is the shift in emission of a quantum dot towards shorter wavelengths (towards the blue part of the spectrum). Upon steady illumination, the quantum dots undergo photo-oxidation and the CdSe core decreases in size as SeO2 evaporates from the surface. The change in the quantum dot’s core size changes the color of light that is emitted from red, to orange, to green, to blue before photobleaching. This process is stochastic and happens to all the quantum dots in the sample – but not at exactly the same time. The onset of bluing is randomly distributed throughout the sample. Those qualities make it appropriate use in super-resolution imaging.
The microscope is set to detect a “blue-shifted” wavelength below the standard emission wavelength for a QD – in the diagram below the detection window for the red quantum dots is orange. As the sample is illuminated and the cores begin to oxidize, a small subset of the QDs will begin emitting orange light instead of red. These QDs can be detected and localized before they further oxidize and emit green light – outside of the detection window. As the QDs “blue” they shift into the detection window, allowing us to detect and localize the QD for a short period of time before they bleach or further blue and move out of the detection window.
Cell-Penetrating Peptide (CPP)
Cell-penetrating peptides (CPPs) are short peptides (made of amino acids) that help cells take up various molecular cargo, like nanosized particles or quantum dots. They are connected to their cargo by a chemical bond and either penetrate or translocate the cell membrane, bringing their cargo into the cell with them. They act as molecular delivery vehicles.
Central Nervous System (CNS)
The part of the nervous system that consists of the brain and spinal cord.
Confocal microscopy is an optical imaging technique that lets us visualize deep within living cells and tissues with greater resolution and contrast than conventional microscopes. This type of microscope collects sharply defined optical sections (2-D images) at varying depths from which 3-D renderings (see Z-stack) can be created. The key to this technique is that it uses point illumination and a spatial pinhole to filter out/eliminate out-of-focus light in thick specimens. Since only one point in the sample is illuminated at a time, 2D or 3D imaging requires scanning over a regular pattern in the specimen.
Dendrites are threadlike extensions of the cell body. They are usually short and have many branches. Dendrites receive and carry impulses toward the cell body. Some neurons may have many dendrites, others may have only one dendrite, while some have no dendrites.
Diffraction is the slight bending of light as it passes around the edge of an object. The amount of bending depends on the relative size of the wavelength of light to the size of the opening. If the opening is much larger than the light’s wavelength, the bending will be almost unnoticeable. However, if the two are closer in size or equal, the amount of bending is considerable, and easily seen with the naked eye.
In semiconductor industry, doping intentionally introduces impurity into a semiconductor material to achieve new electronic properties. It has been found that tiny amounts of impurities can cause large changes to electronic properties. These changes are the basis of a wide spectrum of microelectronics elements.
A fruit fly – an important model organism in scientific research. Ge Yang’s lab is using the common species Drosophila melanogaster.
A duty cycle is the time that an entity spends in an active state (for example how long the fluorophore is “on” or emitting light) as a fraction of the total time. STORM microscopy requires fluorophores that emit light only a small fraction of the time (small/low duty cycle).
A fish that was born 5 days ago. “Dpf” stands for “days post fertilization.” The day of fertilization is day 0. Usually, zebrafish used for experiments are 5dpf.
Fluorescence is the emission of visible light by a substance that has absorbed either visible light or electromagnetic radiation of other wavelengths (the term was coined in 1852 by George Stokes). Some naturally forming minerals exhibit this property. Biologists have used fluorescent dyes (Texas Red and Alexa Fluor dyes for examples) to aid in imaging biological structures. Invitrogen, a company that makes fluorescent dyes, has an introductory tutorial on fluorescence
at this website (click on the green #1 screen).
The use of fluorescent quantum dots is a key feature of the QSTORM project. Qdot fluorescence is brighter and longer lasting than that of dyes. In most cases, the light emitted by a fluorescing substance is of a lower energy level than the light absorbed and thus of a longer wavelength. It is not uncommon for the absorption range to be in the wavelengths shorter than visible light, such as ultraviolet – while the emission glows with a strong color. For instance, the spectra of the absorption range of the QSTORM model Qdot (Invitrogen’s Qdot® 525) peaks below 325 nm while the emission peaks at 525 nm, a very bright green indeed.
A component of a molecule which causes a molecule to be fluorescent. It is a functional group in a molecule which will absorb energy of a specific wavelength and re-emit energy at a different (but equally specific) wavelength. The amount and wavelength of the emitted energy depend on both the fluorophore and the chemical environment of the fluorophore.
Fluorescence Resonance Energy Transfer (also called Forster Resonance Energy Transfer) is a quenching mechanism that decreases fluorescence of a fluorophore. Discovered by a German scientist, Theodor Forster, FRET describes the energy transfer between two fluorophores – which is highly dependent on the distance between the donor and the acceptor – when they are in close proximity, the donor in the excited state can transfer energy directly to the acceptor without releasing a photon.
This is the science of harnessing an organism’s immune system to precisely locate or target delivery to particular molecules – called antigens – inside or on the surface of particular cells. Primary antibodies bind to antigens, while secondary antibodies bind to the primary antibodies. Researchers can conjugate (attach) therapeutic agents to the primary or secondary antibodies -as well as fluorophores (light-emitters) – that then render the targeted structures visible to optical microscopes.
Work that is conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms.
Work that is conducted with living organisms in their normal, intact state.
Inside the cell. Also means existing within the cells.
A device that emits light (electromagnetic radiation) through a process of optical amplification based on the stimulated emission of photons. Laser light is very different from normal light. Laser light has the following properties:
– The light released is monochromatic. It contains one specific wavelength of light (one specific color). The wavelength of light is determined by the amount of energy released when the electron drops to a lower orbit.
– The light released is coherent. It is “organized” — each photon moves in step with the others. This means that all of the photons have wave fronts that launch in unison.
– The light is very directional. A laser light has a very tight beam and is very strong and concentrated. A flashlight, on the other hand, releases light in many directions, and the light is very weak and diffuse.
A micelle is a nano-sized core-shell structure assembled by multiple amphiphilic molecules. Each amphiphilic molecule has a hydrophilic (water-loving) segment and a hydrophobic (water-hating) segment. In water, to minimize the total energy of the system, a certain number of amphiphilic molecules spontaneously assemble together to form a core-shell structure, i.e. a micelle, with the shell composed of the hydrophilic segments of the amphiphilic molecules and the core composed of the hydrophobic segments. In the schematic, green spheres represent the hydrophilic segments of amphiphilic molecules and green lines represent the hydrophobic segments of amphiphilic molecules. Because the core of a micelle is hydrophobic, thermodynamics dictates that it can encapsulate hydrophobic objects, such as anticancer drugs (for applications in drug delivery), oils (for applications in petroleum industry and dish washing), and nanocrystals (gray spheres in the schematic, for biological imaging).
Using a glass micropipette (needle with diameter of 0.5-5 microns) to insert substances into a single living cell. A simple mechanical process where the needle penetrates the cell membrane, injects the desired material into the cell (or sub-cellular compartment) and the needle is removed.
Microtubules are a component of a cell’s cytoskeleton. These very thin, but long rope-like polymers of tubulin can grow as long as 25 micrometers with an outer diameter of only about 25 nm. Microtubules are important for maintaining cell structure, providing platforms for intracellular transport, forming the spindle during mitosis, as well as other cellular processes. There are many proteins that bind to the microtubule, including motor proteins such as kinesin and dynein.
Mn-doped Quantum Dots
These quantum dots are ZnSe-based (Zinc-Selenide) QDs, rather than the CdSe-based QDs from Invitrogen. They are doped with Manganese in order to make them photoswitchable. These QDs are activated by a blue laser, emit yellow light, and can be quenched with a red laser. The mechanism for the fluorescence modulation (switching on and off) relies only on internal electronic transitions within Mn-doped ZnSe quantum dots. This light-driven modulation was first reported by Stefan Hell’s group (Published by Irvine, et. al. (Angew. Chem. Int. Ed. 14/2008)).
A basic unit of a muscle. Muscles are composed of tubular cells called myocytes or myofibers. Myofibers are composed of tubular myofibrils. Myofibrils are composed of long proteins such as actin, myosin, and titin, and other proteins that hold them together. These proteins are organized into thin filaments and thick filaments, which repeat along the length of the myofibril in sections called sarcomeres. Muscles contract by sliding the thin (actin) and thick (myosin) filaments along each other.
The filaments of myofibrils constructed from proteins. The principal types of muscle are striated muscle, obliquely striated muscle and smooth muscle. Various arrangements of myofilaments create different muscles. Striated muscle has transverse bands of filaments. In obliquely striated muscle, the filaments are staggered,and smooth muscle has irregular arrangements of filaments.
The segment in between the myotomes (muscle segments) of a fish. It is the site where the myotomes attach, and is composed of collagen, which is the main connective tissue in fishes.
A tiny, microscopic particle with at least one dimension less than 100 nm.
A term referring to the sub-field of microscopy that attains nanometer-scale resolution.
Photo-Activated Localization Microscopy (PALM) is a super-resolution microscopy technique with resolution up to 10x greater than traditional microscopy techniques. PALM is similar to STORM imaging – it sequentially images photoswitchable fluorophores (fluorescence can be turned on/off with light) that are spread to distances greater than the diffraction limit. The fluorophores are tagged in the cell at a high density, but only a small subset of the fluorophores are activated at any given time. When activated, each fluorophore gives off light – due to the diffraction limit, the image captured is a disk of light 200-250nm (airy disk) and the center of each point of light is localized. The fluorophores are then quenched (turned off), and another subset of fluorophores are activated, then localized, then quenched. The process is repeated many times. Raw data consist of an image stack typically containing thousands of individual frames, each featuring a subset of fluorophores present in the specimen. When combined, the resulting image localizes structures in the cell well below the diffraction-limit.
The Zeiss website describes PALM imaging in greater detail here.
PC12 is a cell line derived from the rat adrenal medulla. PC12 cells stop dividing and terminally differentiate when treated with nerve growth factor. This makes PC12 cells useful as a model system for neuronal differentiation. Ge Yang is using PC12 cells in some of his imaging studies.
Photobleaching is the photochemical destruction of a fluorophore. When it’s in the excited state, the fluorophore is unstable and high-intensity illumination can sometimes cause the fluorophore to change its structure so that it no longer fluoresces. This is a challenge for fluorescence microscopy, because over time, the fluorescence in a sample fades and the fluorophores no longer emit light even when excited by the required light energy.
Quantum dots don’t photobleach the way fluorescent dyes do, so they are considered photostable which makes them more desirable for biological imaging.
Any of several molecular species that can be activated by light; they are used especially in biochemistry to attach a molecule to a biologically active compound and then study its behavior once activated.
A particle representing the basic unit of light, and all other forms of electromagnetic radiation. It is considered a discrete particle having zero mass, no electric charge, and can have interactions over indefinitely long distances. Best explained by quantum mechanics, they exhibit properties of both waves and particles.
Principle Investigator (PI)
The lead scientist or engineer for a particular well-defined science or research project, such as a laboratory study or clinical trial.
A long, hip flexor muscle (the same muscle in a ‘filet mignon’ cut of meat). Beth Brainerd’s lab will provide labeled rabbit psoas muscle cells for imaging in this project.
quantum dots (QDs, Qdots)
A quantum dot is a tiny nanoscale semiconducting crystal whose electronic characteristics are closely related to the size and shape of the individual crystal – its excitons are confined in all 3 dimensions. When excited by an energy/light source, quantum dots fluoresce – they emit light. The color of the light depends on the size of the quantum dot – larger crystals emit red light, smaller crystals emit blue light. Generally, the smaller the size of the crystal, the larger the band gap, the greater the difference in energy between the bands, therefore more energy is needed to excite the dot, and as a result, more energy is released when the crystal returns to its resting state. Possible applications include: biological imaging, transistors, solar cells, LEDs, and diode lasers. One particular benefit to using quantum dots over fluorescent dyes in biological imaging is that they are much brighter and don’t photobleach/fade over time.
The preliminary work in QSTORM uses quantum dots from Invitrogen. These quantum dots are on the nanometer scale (roughly protein-sized) and have a core of semiconductor material (cadmium mixed with selenium or tellurium), which has been coated with an additional semiconductor shell (zinc sulfide). They also have a polymer coating to limit its toxicity and protect the cell. It can also be coupled with biomolecules which lets the quantum dots bind to targets of interest. You can find out more about the structure of Invitrogen’s Qdot nanocrystals here.
The basic unit of a muscle. They are composed of long, fibrous proteins that slide past each other when the muscles contract and relax.
(From Wikipedia) STED stands for STimulated Emission Depletion microscopy. It is a type of super-resolution fluoresence microscopy that uses the de-excitation of fluorescent dyes to overcome the resolution limited imposed by diffraction. STED microscopy uses fluorescent dyes to label specific sites of a sample. These fluorophores are excited by certain wavelengths of light. From the excited state, the fluorophore can spontaneously relax to the ground state and a photon is emitted. Instead of spontaneous relaxation and fluorescence emission, a molecule can also return to its ground state by stimulated emission. If an excited fluorophore is irradiated with light of similar wavelength compared to the fluorescence light, it can immediately return to the ground state and emits a photon of exactly the same wavelength and momentum of the light used. So, the fluorophore can be switched off by additional irradiation of a red-shifted ‘de-excitation’ beam.
In a STED microscope the excited molecules in the outer rim of the excitation spot are additionally switched off by stimulated emission. Therefore a second, red-shifted ‘de-excitation’ laser beam is focused into the sample whose wavefront is altered so that a ring-like intensity profile is achieved. While this light distribution with a dark spot in its center is itself diffraction-limited, it features at least some intensity near the focus and is zero only at the very center. Therefore, using intense depletion light causes almost all of the excited molecules to return to the ground state, leaving only the region of the sample very close to the center of the excitation spot excited. Fluorescence from the remaining excited dye molecules is then detected by the microscope.
A stochastic process is one whose behavior is non-deterministic, in that a system’s subsequent state is determined both by the process’s predictable actions and by a random element.
A super-resolution imaging technique – STochastic Optical Reconstruction Microscopy.
As the Howard Hughes Medical Institute describes it, “To create an image with STORM, researchers label the molecules they want to study with fluorescent probes, and then use a burst of light to activate the fluorescence in a small percentage of labeled molecules. The microscope captures an image of the fluorescing probes. The technique is designed to activate a sufficiently low percentage of the probes to allow the image of each fluorescing molecule to be seen separately. This allows the molecules to be localized individually. The process is repeated many times, capturing a different subset of molecules with each iteration. A final compilation of the images shows each molecule in its precise location in the cell with nanometer accuracy.” The STORM process also can be used to produce reconstructed images in three dimensions.
The Strehl Ratio is a figure of merit for the microscope image quality. The Strehl Ratio is defined as the relative peak intensity for the image of a point source (the Point Spread Function or Airy Disk) compared to an ideal system with the same numerical aperture. If a system has no aberrations, the Strehl ratio will be 1. As the aberrations get worse, the Strehl ratio goes down. A Strehl ratio greater than about 0.7 indicates a well-corrected system.
Techniques that improve the resolution of current microscopy. It will allow us to identify and distinguish between much smaller objects than we can currently. The previous limit for optical flouresence microscopy was approx. 200nm, but the super-resolution techniques have reported resolution to 20 nm (for static) and 60 nm for dynamic resolution.
switchable quantum dots
A switchable QD is one that fluorescence can be controlled externally. i.e. it can be turned on/off by pH or by light.
Tau proteins are proteins that stabilize microtubules. They are abundant in neurons of the central nervous system. Tau is not present in dendrites and is active primarily in the distal portions of axons. Ge Yang’s lab will label tau protein in neurons for imaging.
A red fluorescent dye, used in histology for staining cell specimens, for sorting cells with fluorescent-activated cell sorting machines, and in fluorescence microscopy applications.
Transmission Electron Microscope
TEM operates on the same basic principles as the light microscope but uses electrons instead of light. What you can see with a light microscope is limited by the wavelength of light. TEMs use electrons as “light source” and their much lower wavelength makes it possible to get a resolution a thousand times better than with a light microscope.
A small membrane-enclosed sack that can store or transport substances. Vesicles can form naturally because of the properties of lipid membranes, or they may be prepared. Artificially prepared vesicles are known as liposomes. Most vesicles have specialized functions depending on what materials they contain.
A tropical freshwater fish belonging to the minnow family. It is an important model organism in scientific research.
An ordinary two-dimensional image gives us width and height, or the x-axis and y-axis at one particular focal plane, which is normally determined by the depth of field allowed by light and lens capability. However, if one were trying to produce a set of layered images of a 3D object, with high resolution at each specific focal plane on the z-axis (depth), one would produce a “z-stack” of images at sequential z-axis values. Such techniques are common in biological imaging, where researchers are seeking to explore structure and function at high resolution inside tiny biological substructures. By viewing such still image z-stacks, of microscopy images, for example, in a program like QuickTime, one can achieve a kind of flip-frame animated tour through a biological structure from one surface, through successive cross-sections, to the opposite surface. The z-stack below shows zebrafish muscle fibers under a 60x objective.