measuring size

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MEASURING SIZE Macromolecular Biochemistry Course a.y. 2014-2015

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Techniques that can be used to experimentally determine the size of a macromolecule.

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  • MEASURING SIZEMacromolecular Biochemistry Coursea.y. 2014-2015

  • Size of particle

  • What does it mean size? Difficult to define one size

  • Sphere approximation

  • Parameters used to characterize macromolecules

    (Main parameteres:) Molecular weight Hydrodynamic radius (Stokes radius) Radius of gyration Sedimentation coefficent

    Different methods measure different parameters

  • Hydrodynamic radius (Rh)or Stokes radiusthe radius of a hard sphere that moves in solution in the same way as the molecule of interest

  • Radius of gyration (Rg)root mean square distance of the object's parts from the center of mass

  • Rg/Rh ratioRg/Rh ratio gives an idea of the shape of the macromolecule

  • Sedimentation coefficientcharacterizes the behaviour of a molecule in sedimentation processes

    v : sedimentation ratea : usually r2 (centrifugal acceleration)

  • Analytical ultracentrifugationSedimentation Velocity and Sedimentation EquilibriumHow a particles behave during centrifugation depends on its property

  • Sedimentation velocityBoundary moves along the tube and broadens during time

  • Slope = s2 sCalculation of s

  • Boundary broadening during time

    diffusion coefficient (D)(reflects how the molecole diffuses in solution)

    Using s and D M

    : partial specific volume of the molecule (reciprocal of the density)(measured using techniques like density gradient sedimentation)

  • Equilibrium between diffusion and centrifugal force (low speed)

    Sedimentation equilibrium

  • In a plot log(c) vs r2

    Slope proportional to M(partial specific volume is required)

    Analysis of complex mixtures is difficult (different detection methods)

    Sedimentation equilibrium

  • SedimentationSedimentation Velocity s, D and possibly M ( needed)ADVANTAGEFast ( hours)DISADVANTAGEPolidispersity complicates data analysis

    Sedimentation equilibrium M ( needed)ADVANTAGEPrecise mesurement of MDISADVANTAGELong time ( days)Polysispersity size distribution

  • Light scatteringStatic and Dynamic light scattering (elastic scattering)Due to the interaction of a molecule dipole with light size >

  • Scattering at different angles(size large enough for angular dependence) Static light scattering

  • Sample and blank measurements has to be performed against a reference (usually toluene)Rayleigh ratio

  • function of mass concentration (m/V)function of and n (refractive index)molecular weight of the particlescattering vector : function of radius of gyrationq2 (lenght-2)MRgR (lenght-1)Calculation of MW and Radius of Gyration

  • Dynamic light scatteringScattering during time

  • Fluctuations due to Brownian MotionDynamic light scattering

  • Autocorrelation function

  • Hydrodynamic radius (Stokes radius) : the diamenter of a sphere that moves like the average of your particles does

    Function of T !!! equilibration very importantCalculation of Hydrodynamic radius

  • Static and Dynamic Light scatteringADVANTAGEFastCheap and small instrumentationIn solution (less preparation bias)DISADVATAGEPolydispersity and dust are real problemsThe nature of the scattering pose limit to these techniques (size>10 nm)

  • Small-angle scattering SAXS and SANSX-ray (synchroton)Neutron (nuclear reactor)ADVANTAGEAlso smaller molecules, information about the shapeIn solution (real structure)Dust is not a problemDISADVANTAGELarge and expensive instrumentationTime-consuming (especially SANS)

  • Underling theoryEinsein-kinetic theoryStokes-Einstein equationThere is a relationship between the parameters that we saw before

  • All these tecnique are based on the einstain kinetic theory. The principal equation of this theory is the einstein stokes equation that relate many of the parameter that we saw before

  • Size exclusion chromatography stationary phase mobile phase

  • Calculation of MW

  • Kav= (Ve Vo)/(Vt Vo)Calculation of MW

  • Hydrodynamic chromatography

  • f = 6Rs

    D = kT / f = kT / 6Rs

    Rs = (1 / D) 2.2 x 10 -6 Calculation of Stokes radiusf frictional coefficientRs Stokes radiusD diffusion coefficientk Boltzman's constantT temperature

  • Chromatography

    ADVANTAGE- good separation of large from small molecules with a minimal volume of eluate- preserving the biological activity of the particles to be sapareted- there are short and well define separation times and narrow bands wich leads to good sensitivity- there is no sample loss because solute do not interact with the stationary phase

    DISADVANTAGE- possibility of interaction between the stationary phase and the analyte- only a limited number of bands can be accumulated because the time scale of the chromatogram is short- there has to be a 10% difference in molecular mass to have a good resolution - mass is not measured so much as the hydrodynamic volume of the polymer molecules

  • Transmission electron microscopy a beam of electrons is transmitted through an ultrathin specimen interacting with the specimen as it passes through;

    an image is formed from the interaction of the electrons transmitted through the specimen;

    the image is magnified and focused onto an imaging device or detected by a sensor.

  • Electron microscopyNEGATIVE STAINING

    ROTARY SHADOWING

    CRYO-EM

  • Negative stainingADVANTAGE resolution of 10 Angstrom or less is possible; very fast process; you could analyze sample that can not be visualized in convetional preparation (ex: isolated component from cell fractionation or biomolecule); Semplicity.

    DISADVANTAGE repeatability, greatly varing results both between samples and even on the same grid; toxicity for use of heavy metal .

  • Rotary shadowingADVANTAGESimple preparation of the sample;can visualize single globular domains as small as 10 kDa (3.5 nm diameter) and elongated molecules as thin as 1.5 nm.

    DISADVANTAGE only stable dried organic or inorganic molecule that will not change shape under high vacuum conditions;Only one side of the molecule;Non covalent bound could be disrupted.

  • Cryo-EMADVANTAGEexamination of native and hydrated structural features of biological sample;no stains or chemical fixatives to distort the sample;enables to control the chemical environment so that examination of different functional states of molecules is possible.

    DISADVANTAGE when the sample adheres to the carbon grid, it could stick in a preferential orientation;very low signal to noise ratiomore time consuming to generate samples;sample must be maintained at less than 135C.

  • Measuring sizeCrystallography Molecular dynamicsSequencingMass spectrometry

  • Measuring sizeDifferent techniques give different information about your molecule

    Complementary rather than alternativesThank you!

    **

    A particle is a minute fragment or quantity of matter. The word is used to describe a small localized object to wich can be ascribed several physical or chemical propertiessuch as volume or mass. The concept of particle is particularly useful when modelling nature, as the full treatment of many phenomena is complex. It can be used to make simplifying assumptions concerning the process involved. A macromolecule is a very large molecule commonly created by polimerization of smaller subunits. In biochemistry, macromolecule are biopolymers (nucleic acid, proteins, carbohydrates) and non polymeric molecules with large molecular mass such as lipids and macrocycles.

    Size include SHAPE, VOLUME and WEIGHT. Usually particle are aproximize to an equivalent sphere. But, unless they are perfect sphere they cannot be fully described by a single dimension. *Size include SHAPE, VOLUME and WEIGHT. Usually particle are aproximize to an equivalent sphere. But, unless they are perfect sphere they cannot be fully described by a single dimension. *To Be or Not To Be a Sphere: Of all the three-dimensional particles, the sphere is by far the most important in particle sizing. Why is that? Is it because most particles are spheres? No, though many come close to it (unaggregated latex, monoclonal antibodies, oil-in-water and water-in-oil emulsions, spherical micelles, liposomes, etc.). And still more are nearly so, especially if measurements are averaged over rotationally diffusing particles 1 . Over the timescales of many types of measurements, we are measuring a rotationally averaged size and thus a sphere represents, often, a reasonable approximation. In addition, if highly irregular particles are broken down due abrasion, long ones arebroken down into shorter ones and they become more globular rather than less.*all somehow related***the radius of a sphere that moves like (the average of) your particle(s) does

    Stokes radius is sometimes used synonymously with effective hydrated radius in solution.[1] Hydrodynamic radius, RH, can refer to the Stokes radius of a polymer or other macromolecule.*Radius of gyration or gyradius refer to the distribution of the components of an object around an axis. In terms of mass moment of inertia, it is the perpendicular distance from the axis of rotation to a point mass (of mass, m) that gives an equivalent inertia to the original object(s) (of mass, m). The nature of the object does not affect the concept, which applies equally to a surface, a bulk mass, or an ensemble of points.

    Mathematically the radius of gyration is the root mean square distance of the object's parts from either its center of mass or a given axis, depending on the relevant application.Molecular applications: In polymer physics, the radius of gyration is used to describe the dimensions of a polymer chain.

    (General Physics) a length that represents the distance in a rotating system between the point about which it is rotating and the point to or from which a transfer of energy has the maximum effect. Symbol: k or r. In a system with a moment of inertia I and mass m, k = I/m****j + 2.5 ????????**Exploiting different detection methods : absorbance, interference, fluorescence ...*Exploiting different detection methods : absorbance, interference, fluorescence ...**At different times (the boundary broadens in time)*At different times (the boundary broadens in time)****partial bla bla bla barrato* with other technique*******This fenomenon can be exploited mainly by two different techniques*****rref Rayleigh ratio reference (lenght-1)*rref Rayleigh ratio reference (lenght-1)

    **for diluited solutionsThe square radius of gyration is the average squared distance of any point in the object (polymer coil) from its center of mass*for diluited solutionsThe square radius of gyration is the average squared distance of any point in the object (polymer coil) from its center of mass******only for low concentrations*only for low concentrationsred circle

    **Hydrodynamic radius (Stokes radius) : the diamenter of a sphere that moves like (the average of) your particle(s) does***Second virial coefficient : a measure of the non-ideality of a solution due to protein-solvent and protein-protein interactions ((applying some correction to the theory before)))increment : given or measured experimentally*Second virial coefficient : a measure of the non-ideality of a solution due to protein-solvent and protein-protein interactions ((applying some correction to the theory before)))increment : given or measured experimentally

    **Very large, expensive and time consuming (expecially SANS)**All these tecnique are based on the einstain kinetic theory. The principal equation of this theory is the einstein stokes equation that relate many of the parameter that we saw before**Size Exclusion Chromatography (SEC) is the separation technique based on the molecular size of the components.Separation is achieved by the differential exclusion from the pores of the packing material, of the sample molecules as they pass through a bed of porous particles.The underlying principle of SEC is that particles of different sizes will elute (filter) through a stationary phase at different rates. This results in the separation of a solution of particles based on size. Provided that all the particles are loaded simultaneously or near-simultaneously, particles of the same size should elute together.One requirement for SEC is that the analyte does not interact with the surface of the stationary phases, with differences in elution time between analytes ideally being based solely on the volume, and also to anabling high retension of biomolecular activity.Each size exclusion column has a range of molecular weights that can be separated. The exclusion limit defines the molecular weight at the upper end of the column 'working' range and is where molecules are too large to be trapped in the stationary phase. The lower end of the range is defined by the permeation limit, which defines the molecular weight of a molecules that is small to penetrate all pores of the stationary phase. All molecules below this molecular mass are so small that they elute as a single band.This is usually achieved with an apparatus called a column, which consists of a hollow tube tightly packed with extremely small porous polymer beads designed to have pores of different sizes.Like other forms of chromatography, increasing the column length will enhance the resolution, and increasing the column diameter increases the capacity of the column. Proper column packing is important to maximize resolution: An overpacked column can collapse the pores in the beads, resulting in a loss of resolution. An underpacked column can reduce the relative surface area of the stationary phase accessible to smaller species, resulting in those species spending less time trapped in pores. Unlike affinity chromatography techniques, a solvent head at the top of the column can drastically diminish resolution as the sample diffuses prior to loading, broadening the downstream elution. In simple manual columns, the eluent is collected in constant volumes, known as fractions. More advanced columns overcome this problem by constantly monitoring the eluent. The collected fractions are often examined by spectroscopic techniques to determine the concentration of the particles eluted, like refractive index (RI) and ultraviolet (UV). It is also possible to analyse the eluent flow continuously with RI, LALLS, Multi-Angle Laser Light Scattering MALS, UV, and/or viscosity measurements.*Results from gel filtration are usually expressed as an elution profile or chromatogram that shows the variation in concentration (typically in terms of UV absorbance at A 280 nm) of sample components as they elute from the column in order of their molecular size).Molecules that do not enter the matrix are eluted in the void volume, Vo as they pass directly through the column at the same speed as the flow of buffer. Molecules with partial access to the pores of the matrix elute from the column in order of decreasing size. Molecules with full access to the pores move down the column, but do not separate from each other. These molecules usually elute just before one total column volume, Vt, of buffer has passed through the column.Since symmetrical peaks are common in gel filtration, elution volumes are easily determined. However, Ve does not completely define the behavior of the sample since Ve will vary with the total volume of the packed bed (Vt) and the way in which the column has been packed. The elution of a sample is best characterized by a distribution coefficient (Kd) derived as follows:The volume of the mobile phase (buffer) is equal to the void volume, Vo, i.e. the elution volume of molecules that remain in the buffer because they are larger than the largest pores in the matrix and pass straight through the packed bed. In a well packed column the void volume is approximately 30% of the total column.The volume of the stationary phase, Vs, is equal to Vi, the volume of buffer inside the matrix which is available to very small molecules, i.e. the elution volume of molecules that distribute freely between the mobile and stationary phases minus the void volume.Since, in practice, Vs or Vi are difficult to determine, it is more convenient to substitute the term (Vt Vo). The estimated volume of the stationary phase will therefore include the volume of solid material which forms the matrix.Kd represents the fraction of the stationary phase that is available for diffusion of a given molecular species. The stationary phase Vs can be can be substituted by the term (Vt Vo) in order to obtain a value Kav. Kav= (Ve Vo)/(Vt Vo)Since (Vt Vo) includes the volume of the matrix that is inaccessible to all solute molecules, Kav is not a true partition coefficient. However, for a given medium there is a constant ratio of Kav:Kd which is independent of the nature of the molecule or its concentration. K av is easily determined and, like Kd, defines sample behavior independently of the column dimensions and packing. Other methods of normalizing data give values which vary depending upon how well the column is packed.

    * If standards of a known size are run previously, then a calibration curve can be created to determine the sizes of polymer molecules of interest in the solvent chosen for analysis graph of this relationship is called a calibration curve. Calibration curves are prepared by using molecular weight markers with known weights to find the elution volume and plotting the logarithm of the molecular weight on the vertical axis versus the elution volume on the horizontal axis (retention time). Molecules larger than the pore size pass straight through (are excluded). This is called the exclusion limit. Conversely, molecules below a certain size completely penetrate the pores and tend to elute almost in the same position. This is called the permeation limit. The calibration curve is valid in the range between this exclusion limit and permeation limit. Within this range, the larger the molecule, the sooner it elutes and the smaller the molecule the later it elutes.

    Calibration curves with a steeply sloped portion are obtained using packing materials with large pores intended to measure larger molecules. Curves with more gradual slopes are obtained using packing materials with small pores, intended for measuring smaller molecules.

    The lower the slope, the larger the difference in elution volume between small differences in molecular weight. Therefore, if two columns are available that can measure the target sample molecules; the one with the less sloped calibration curve will provide more accurate molecular weights.

    http://www.shimadzu.com/an/hplc/support/lib/lctalk/55/55intro.html*Hydrodynamic chromatography (HDC) has experienced a resurgence in recent years for particle and polymer characterization, principally because of its coupling to a multiplicity of physical detection methods. When coupled to light scattering (both multiangle static and quasi-elastic), viscometric, and refractometric detectors, HDC can determine the molar mass, size, shape, and structure of colloidal analytes continuously and as a function of one another, all in a single analysis. HDC is a solution-phase separation method that can be performed in an open tube (capillary) or in a column packed with nonporous, inert particles (as we discuss below, packing particles with very small pores, vis-`a-vis the solution size of the analyte, can also be employed). In HDC, sample components are segregated in a size-dependent manner on the basis of preferential sampling of the streamlines of flow in the capillary or in the interstitial medium of the packed column. Such segregation is the first step in characterizing size averages and distributions. Coupling HDC to various detection methods further informs our knowledge of analyte shape and structure and of the dependence of these features on size as a continuous function of the latter.Elution order in HDC is thus the same as in SEC (8): The larger analytes elute ahead of the smaller ones in both cases. The mechanisms of retention of these two techniques are different, however: In SEC, retention is due to preferential sampling of pore volume, whereas in HDC, it is due to preferential sampling of the streamlines of flow (or to a preferential distribution of analyte between fluid mechanicalphases).*Once a calibration curve is prepared, the elution volume for a protein of similar shape, but unknown weight, can be used to determine the MW. The key issue is of similar shape. Generally, the calibration proteins are all globular, and if the unknown protein is also globular, the calibrated gel filtration column does give a good approximation of its molecular weight. The problem is that the shape of an unknown protein is generally unknown. If the unknown protein is elongated, it can easily elute at a position twice the molecular weight of a globular protein. The gel filtration column actually separates proteins not on their molecular weight but on their frictional coefficient. Since the frictional coefficient, f, is not an intuitive parameter, it is usually replaced by the Stokes radius R s . R s is defined as the radius of a smooth sphere that would have the actual f of the protein. This is much more intuitive since it allows one to imagine a real sphere approximately the size of the protein, or somewhat larger if the protein is elongated and has bound water. As mentioned above for Eq. 4.2, Stokes calculated theoretically the frictional coefficient of a smooth sphere to be: f = 6Rs.The Stokes radius R s is larger than R min because it is the radius of a smooth sphere whose f would match the actual f of the protein. It accounts for both the asymmetry of the protein and the shell of bound water. More quantitatively, f/f min =S max /S=R s /R min . A gel filtration column can determine R s relative to the R s of the standard calibration proteins. The R s of these standards was generally determined from experimentally measured diffusion coefficients. Some tabulations of hydrodynamic data list the diffusion coefficient, D, rather than R s , so it is worth knowing the relationship: D = kT / f = kT / 6Rs; where k=1.3810 16 g cm 2 s 2 K 1 is Boltzmans constant and T is the absolute temperature. k is given here in cmg-s units because D is typically expressed in cmg-s; R s will be expressed in centimeter in this equation. Typical proteins have D in the range of 10 6 to 10 7 cm 2 s 1 . Converting to nanometer and for T=300 K and =0.01: Rs = (1 / D) 2.2 x 10 -6 ; where R s is in nanometer and D is in centimeter squared per second. Simply knowing, R s is not very valuable in itself, except for estimating the degree of asymmetry, but this would be the same analysis developed above for S max /S. However, if one determines both R s and S, this permits a direct determination of molecular weight, which cannot be deduced from either one alone. This is described in the next section.**Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through an ultra-thin specimen, interacting with the specimen as it passes through. An image is formed from the interaction of the electrons transmitted through the specimen; the image is magnified and focused onto an imaging device, such as a fluorescent screen, on a layer of photographic film, or to be detected by a sensor such as a CCD camera.TEMs are capable of imaging at a significantly higher resolution than light microscopes, thanks wavelength of electrons. This enables the instrument's user to examine fine detaileven as small as a single column of atoms, which is thousands of times smaller than the smallest resolvable object in a light microscope. TEM forms a major analysis method in a range of scientific fields, in both physical and biological sciences.At smaller magnifications TEM image contrast is due to absorption of electrons in the material, due to the thickness and composition of the material.Electron microscopy can be used to determine the shape and structures of biological molecules that are difficult to study by more traditional structural approaches, such as X-ray crystallography and NMR analysis. However, since biological molecules are composed mainly of low electron scattering atoms and contain large amounts of water, it is necessary to use special preparation techniques to protect the structural integrity of biological specimens in the vacuum of the electron microscope and improve the amount of contrast created by the sample. Negative staining, rotary shadowing and cryo-EM are all powerful techniques that are useful for examining the structures and molecular organization of biological complexes by EM.*Ohi M. EM analysis of protein structure - negative staining, rotary shadowing, and cryoEM. Encyclopedia of Life Sciences. 2009 Dec 12.*Negative stain is another EM technique capable of imaging single protein molecules. It is especially useful for imaging larger molecules with a complex internal structure, which appear only as a large blob in rotary shadowing. Importantly, noncovalent proteinprotein bonds are sometimes disrupted in the rotary shadowing technique (8), but uranyl acetate, in addition to providing high resolution contrast, fixes oligomeric protein structures in a few milliseconds (22)For structural detail to be recorded in a micrograph, it must be faithfully preserved as the specimen is prepared and subsequently exposed to the vacuum and electron beam. The main purpose of negative-staining is to surround or embed the biological object in a suitable electron dense material which provides high contrast and good preservation (Fig. II.39). This method is capable of providing information about structural details often finer than those visible in thin sections, replicas, or shadowed specimens. In addition to the possibility of obtaining a spectacular enhancement of contrast, negative-staining has the advantage of speed and simplicity.The potential value of negative stain microscopy derives from the relative ease with which specimens can be imaged at the molecular level of resolution. The technique has mainly been used to examine particulate (purified) specimens - e.g.. ribosomes, enzyme molecules, viruses, bacteriophages, microtubules, actin filaments, etc. at a resolution of 1.5-2.5 nm. This technique generally allows the shape, size, and the surface structure of the object to be studied as well as provide information about subunit stoichiometries and symmetry in oligomeric complexes. Any surface of the specimen accessible to water can potentially be stained, and thus, that part of the specimen will be imaged at high contrast.In electron microscopy, staining is usually done with heavy metal salts commonly derived from molybdenum, uranium, or tungsten. Heavy ions are used since they will readily interact with the electron beam and produce phase contrast. A small drop of the sample is deposited on the carbon coated grid, allowed to settle for approximately one minute, blotted dry if necessary, and then covered with a small drop of the stain (for example 2% uranyl acetate). After a few seconds, this drop is also blotted dry, and the sample is ready for viewing.*A second method for examining the surface topology and structures of specimens in a TEM employs shadowing techniques. In this case the image contrast is produced by the uneven distribution of fine metal particles. Once again electron dense metals are the coatings of choice and platinum, chromium, palladium, uranium, and gold are some of the more commonly used metals for shadowing. Also, as the name implies information about the surface topology is gained by creating a shadow effect which is directly proportional to the microarchitecture of the specimen. This is accomplished by depositing the coating metal from a low angle (5 - 30 degrees) relative to the general plane of the specimen. The greater the height of portions of the specimen the larger will be the resultant shadow. The contrast difference created by a shadow that is created is opposite to a shadow produced by sunlight.In interpreting a shadowed preparation it is important to know the direction from which metal was deposited. In fact if the angle and direction of the shadowing source are known relative to the specimen the height of the specimen can be calculated using the equation:H = tan O X l Where H = height of specimen, O = angle of shadowing, l = length of shadow, or H = b/c X l Where b = Height from level to source, c = Distance from sample to sourceShadowing may be done from a fixed angle (static shadowing) or on a rotating specimen (rotary shadowing). Rotary shadowing allows one to resolve portions of the specimen that might otherwise have been obscured by the shadow. As with negative staining resolution in the TEM of shadowed specimens is dependent on the grain size of the deposited metal.It is common to deposit the electron dense metal from a predetermined angle to create the shadow effect and then to evaporated from directly above, a fine layer of carbon which does not add much electron opacity but does provide strength to the shadow cast, particularly in regions where no metal was deposited. In terms of resolution shadow casting, especially low angle rotary shadowing, can equal or exceed the resolution capable from negative staining. *Cryo-Electron Microscopy specializes in interpreting and visualizing unstained biological complexes such as viruses, small organelle, and macromolecular biological complexes of 200 kDa or larger preserved in vitreous (i.e. glassy or non-crystalline) ice. The basic goal is to compare other electron microscopy techniques to use cryo-fixation to rapidly freeze the biological sample so as not to destroy its aqueous enviornment. This avoids ultrastructural changes, redistribution of elements, and washing away of substances. Specimens frozen in vitreous ice show a structure similar to the liquid state, or the native state. The near native imaging conditions allows three dimensional reconstruction of the cellular machinery. Using state of the art computer controlled, automated microscopes, image reconstruction software, and visualization tools, sub-nanometer resolution structures of large biological complexes can be achieved. In Cryo-Electron Microscopy, an electron beam, a stream of high energy particles bombards the sample. The image that is viewed is a result of the interaction of the sample with this beam. Most of the electrons that form the high resolution image appear due to elastic scattering, where only their trajectory has been changed, but their energy is unaffected. However, a small fraction of the electrons transfer some of their energy to the sample. This energy accumulates and can break apart molecular bonds, destroying the sample after some time. ADVANTAGE Allows the examination of native and hydrated structural features of the biological sample. The sample is always in solution and never comes into contact with an adhering surface. Therefore, the shape that is observed is the true shape of the hydrated molecule in solution and has not been distorted by attaching itself and flattening against the supporting film. There are no stains or chemical fixatives to distort the sample. When stained, the sample can be damaged in many ways, such as flattening and twisting. Enables one to control the chemical environment so that examination of different functional states of molecules is possible. DISADVANTAGE When the sample adheres to the carbon grid, it could stick in a preferential orientation. If this happens, then information will be missing from the final image set (a missing cone), and the resolution of the calculated model in that direction will be absent. Very low signal to noise ratio. Biological macromolecules are normally made up of carbon, hydrogen, oxygen, and nitrogen. The electron absorption of such molecules is very low. Sample must be maintained at less than 135 degrees Celsius. ***