Biophysical Techniques and Instrumentation: Key Terms and Glossary

A purity indicator from the different shapes of nucleic-acid and protein spectra. It is not a concentration, and it is blind to salt, ethanol, and many carbohydrates.

The lateral diffraction limit, about λ divided by twice the numerical aperture. For green light and a high-numerical-aperture oil lens it is about 0.2 µm, and axial resolution is poorer.

log₁₀ of incident intensity over transmitted intensity. Under the Beer–Lambert law it equals εcl for a clear solution of one chromophore whose molar absorption coefficient is constant.

Removal of photons whose energy matches an allowed gap, so the transmitted beam is weaker. A detector cannot treat absorption, emission, and scattering as the same event.

The peptide carbonyl stretch, near 1600–1700 cm⁻¹, the main infrared reporter of backbone hydrogen bonding. It sits under water's bending mode, and a secondary-structure fraction fitted to it is not unique.

Infrared sampling by an evanescent wave at a high-refractive-index crystal, penetrating on the order of a micrometre, one way around water's opaque bend. It is a geometry, not a recipe for a particular protein.

The extra staying power of a multivalent binder that remains down after one contact lets go. A one-to-one fit then does not return the solution dissociation constant of a single site.

A fragment that keeps the N-terminus when a peptide breaks at the peptide bond. A ladder of such ions differs by residue masses, except where two residues share a mass.

The MRI contrast that arises because deoxyhaemoglobin is paramagnetic and alters the field experienced by nearby water. It is a vascular proxy, not a spectrum of haemoglobin and not a record of spikes.

Constructive interference from a crystal only when nλ = 2d sinθ, so spot position reports plane spacing and the beam is deflected by 2θ. Intensity is recorded; phase is not.

The area of the excess heat-capacity peak on unfolding, the heat measured directly. It exceeds the van’t Hoff enthalpy when intermediates or independent domains broaden the transition.

A patch left on an intact cell. The bath cannot change the cytoplasmic face, and the cell's resting potential sits in series with the pipette, so the absolute membrane potential is usually unknown.

The ladder of m/z peaks from one protein carrying different numbers of charges. Adjacent peaks give the charge and therefore the mass, and the same composition still means the same mass.

The shielding of a nucleus by nearby electrons, expressed in parts per million relative to a reference so that the number does not depend on the field. Dispersion of shifts reports that a fold exists; it is not a structure.

Subtraction of spectral overlap so that a bright fluorescence channel does not paint a false signal into the next. Without it a single fluorophore can look double-positive.

Current divided by the driving force away from the reversal potential, for a pathway that behaves ohmically. Many channels rectify, so the current is not a straight line.

An aperture in a plane conjugate to the focus, rejecting light from above and below that plane. The improvement is an optical section, while lateral resolution stays at the diffraction limit.

The oscillating transfer of spatial frequencies in a defocused electron image, including zeros. A raw cryo image looks modulated for that reason, and several defocus values are used so missing frequencies can be filled.

A two-dimensional spectrum whose cross-peaks connect nuclei that share a scalar coupling, typically across two or three bonds: neighbours in the covalent structure.

The wavelength of an accelerated electron, a few picometres at the voltages used for biological microscopes. Dose, contrast, and preparation, not that wavelength, set the resolution that is actually reached.

A set of reversed or shuffled sequences used to estimate how often a random peptide-spectrum match scores as well as a real one.

A pulsed electron-resonance method that measures distances of about 2–8 nm between two spin labels. The distance is between the unpaired electrons on their tethers, not between α-carbons.

Contrast from interference of two closely spaced polarised beams, showing the gradient of optical path. It is not a height map, and birefringent plastic spoils it.

The charge separation whose change during a vibration allows that vibration to absorb infrared light. A symmetrical non-polar stretch is often silent.

How much the refractive index changes with concentration. In a light-scattering mass it is squared, and a glycoprotein, a nucleic acid, or a protein–detergent complex does not share the value of a simple protein in water.

Two cells stuck together and recorded as one event, looking large and double-positive. Pulse width is the usual clue that the event is not one double-labelled cell.

A scalp recording of synaptic currents summed over a wide population and blurred by the skull. Time resolution is fine next to MRI, spatial specificity is poor, and an open probability cannot be read from it.

Bulk solvent dragged towards the cathode by a charged capillary wall, often faster than the analytes' own electrophoresis, so cations, neutrals, and anions can pass one detector.

Soft ionisation in which charged droplets shrink until gas-phase ions remain. Proteins typically carry many charges, and non-covalent complexes can survive.

A photon leaving as a molecule returns to a lower state. The emitted photon is almost always of lower energy than the one absorbed.

A brief extracellular voltage produced by current in the tissue, typically tens to hundreds of microvolts. The electrode does not seal, does not clamp, and does not report a conductance or an open probability.

The expected fraction of an accepted list of peptide identifications that is chance. A stated rate is a property of the list, not a proof that one weak peptide is correct, and absence from the list is not absence from the cell.

Wavelengths below about 250 nm, where the peptide bond absorbs and where water, oxygen, and many buffer ions close the window.

Circular dichroism of the peptide bond, roughly 180–250 nm, reporting backbone conformation as a mixture of helix, sheet, and disorder. It does not assign a residue and it is not an atomic structure.

Light deflected at small angles as a cell passes a laser in a flow cytometer. It depends on size and also on refractive index and shape, and the cytometer does not say where in the cell a label sat.

The distance at which Förster resonance energy transfer is half efficient. Efficiency falls with the sixth power of distance and, for the dye pairs used on proteins, is informative at about 2–10 nm, not across a bond and not across a cell.

The number in the electron-resonance condition that is about 2 for a free electron and is shifted by the local electronic structure. The spectrum is usually plotted against field at fixed microwave frequency.

Separation by hydrodynamic size on a porous bed. Large molecules elute first, and an apparent mass is valid only for the shape of the standards.

The very high resistance formed when a polished pipette sticks to a clean membrane, so current through the patch has nowhere else to go and a picoampere step can be seen. These notes do not describe how to make the pipette.

The resting voltage as a permeability-weighted combination of the ions that can cross. Chloride is written on the opposite side of the cations because its charge is negative.

A plot of the logarithm of small-angle scattering intensity against the square of the scattering vector. The slope gives a radius of gyration only while that product is small, and only for a sample that is not a mixture.

A spectrum that correlates each amide proton with its attached nitrogen, giving about one backbone peak per residue that has an amide proton. Proline is absent, and a shifted peak is a changed environment, not automatically a contact.

The radius of a sphere that would diffuse at the observed rate, from the Stokes–Einstein relation. A rod and a sphere with the same hydrodynamic radius are not the same geometric size.

Uptake of solvent deuterium by backbone amides, slower when they are hydrogen-bonded or buried. The peptide map reports protection and opening, back-exchange makes the uptake an underestimate, and the pattern is not itself a structure.

The lower absorbance of stacked bases in a duplex compared with free bases. A melting curve at 260 nm therefore reports unstacking, not how much DNA was weighed in.

An excised patch with the cytoplasmic face towards the bath, so a cytoplasmic ligand can be applied. Diffusible partners that were in the cell are gone.

A non-radiative return of electronic energy as heat, including the drop from a higher singlet to the first excited singlet before emission.

A spin flip that populates the triplet. Return from the triplet is phosphorescence, slow and usually quenched in aerated solution at room temperature.

Retention by net charge at the working pH, released by salt or by a move towards the isoelectric point. Elution order is not a strict order of isoelectric points, because the pattern of charges matters as well as the sum.

Migration in a pH gradient until the net charge is zero, at the isoelectric point. A phosphate typically moves that point more acidic, a change a size gel may miss.

Centrifugation to equilibrium in a density gradient. Particles band at their buoyant density whatever their size, and further spinning does not move them.

The through-bond splitting of NMR lines by bonding electrons, quoted in hertz and independent of field strength. A three-bond coupling depends on a dihedral angle and does not report two groups that merely sit next to each other.

The diagram of absorption, vibrational relaxation, fluorescence, internal conversion, and intersystem crossing. Emission almost always leaves from the lowest vibrational level of the first excited singlet.

The fraction of the bead's interior volume that a molecule can enter, from its elution volume between the void and the total volume. It is not a dissociation constant.

The mean time in the excited state, the reciprocal of the sum of radiative and non-radiative rate constants. Collisional quenching shortens it; static quenching does not.

The distance between a dye and the epitope it is supposed to mark, often a primary plus a secondary antibody. It is a floor under any biological claim of nanometre resolution.

Uptake or release of protons on binding, which puts the buffer's ionisation enthalpy inside the heat that is observed. Repeating the titration in buffers of different ionisation enthalpy separates the two contributions.

How fast the force rises while a molecule is pulled. An unfolding force is an escape over a barrier and rises approximately with the logarithm of this rate, so a number without a rate cannot be compared with another.

How finely the centre of an isolated diffraction-limited spot can be found, improving roughly with the square root of the number of photons. Precision is not the resolution of a structure if two molecules occupied the same frame.

Mass divided by charge number, the quantity an analyser sorts. A mass is recovered only once the charge state is known.

The nucleus-specific constant γ that sets the resonance frequency in a field, ν = |γ|B₀/2π. A nucleus with zero spin has none and cannot be observed.

Soft ionisation from a matrix crystal that absorbs a laser pulse, usually producing a singly charged ion. It reports a mass and is not a measurement of the fold.

The limit in which molecules bind as fast as flow and diffusion deliver them, so the rising curve is not the molecular on-rate. A molecular on-rate does not change with flow rate; a transport-limited rate does.

Circular-dichroism ellipticity divided by the number of residues, so chains of different length can be compared. An error in concentration or path scales the whole curve.

The share of fluorescence that returns after a region is bleached, the molecules free to move in on that timescale. Recovery in a fixed sample, where diffusion is impossible, means the live-cell curve is not mobility.

The ε in the Beer–Lambert law, with units that make εcl dimensionless when concentration and path are stated. It belongs to one chromophore in one solvent, pH, and temperature.

A state that often keeps a far-ultraviolet circular-dichroism spectrum while losing the near-ultraviolet signal: the backbone is still ordered and the side chains are not packed.

Electrospray of a complex under conditions chosen so that non-covalent assembly can survive. The mass reports stoichiometry of what endured the spray, not the arrangement of subunits.

Circular dichroism of aromatic side chains and disulphides in asymmetric environments, roughly 250–320 nm, reporting tertiary packing rather than secondary structure.

A dense salt dried around an isolated particle so the particle appears light in projection. The image is a map of the stain, not a photograph of unstained density.

The membrane voltage at which one ion is at equilibrium, set by its concentration ratio, its charge, and the temperature. Channels decide whether the membrane actually sits there; they do not change the equilibrium voltage itself.

A two-dimensional spectrum whose cross-peaks connect nuclei through the nuclear Overhauser effect. Intensity falls off as about the sixth power of distance and is useful out to roughly 5–6 Å.

n sin θ, the refractive index of the medium times the sine of the half-angle the lens accepts. Together with wavelength it sets the diffraction-limited resolution; magnification does not.

The fraction of time a channel spends open, taken over many events. One opening is not that fraction, and an empty patch is not proof that the cell lacks the channel.

A tightly focused laser that holds a dielectric bead at the focus. For small displacements it is a spring, and the record is force and displacement, not an image of a fold.

An analyser that reads the frequency of ion oscillation about a central electrode. It gives high resolution without a superconducting magnet.

A patch withdrawn from the whole-cell configuration so that the extracellular face meets the bath, the arrangement used for an extracellular ligand. The patch has already been excised and dialysed.

Conversion of a phase shift, from optical path through the specimen, into intensity. Thick objects are surrounded by a halo, and the image is not a map of absorbance.

The missing phase of each diffracted beam. Without it a Fourier map of the electron density cannot be computed, however carefully the intensities were measured.

The ease with which the electron cloud distorts. A vibration appears in the Raman spectrum if the polarisability changes.

Particles that sit in the ice in only one or two views, leaving a missing wedge and an anisotropic map. The air–water interface can also unfold particles before they freeze.

An analyser that passes one m/z window, the ions that have a stable trajectory in an oscillating field between four rods. Resolution is modest, and the device is the selector in tandem instruments.

The fraction of absorbed photons that are re-emitted. A new non-radiative path lowers it, and brightness in an experiment is the product of yield and the molar absorption coefficient.

The size taken from the Guinier slope. For a mixture it can belong to no species that is actually present.

Inelastic scattering that gains or loses a vibrational quantum. It needs a change in polarisability, which is a different selection rule from infrared absorption.

Centrifugation that separates by sedimentation coefficient while zones are still moving. If it is left until equilibrium, the rate information is gone and only density remains.

Elastic redirection of a wave, with the photon energy unchanged. It is the basis of dynamic light scattering and is not an absorption.

Centrifugal acceleration divided by standard gravity. It depends on angular speed and on distance from the rotor axis, so a speed in revolutions per minute does not state the force.

Capture of a dissociated molecule by a neighbouring site before it escapes, which slows the observed off-rate. A true off-rate does not depend on how much ligand was immobilised.

How much longer a compound is retained than one that does not interact with the stationary phase, (t_R − t₀)/t₀. It is a partition, not an identity.

The voltage at which the current through a pathway changes sign. If only one ion flows, it equals that ion's Nernst potential, which is how a selectivity test is read.

Retention by hydrophobicity, released by an organic-solvent gradient. Peptides behave; many folded proteins are unfolded by the solvent.

Electrophoresis after dodecyl sulphate has denatured the chain and swamped its own charge, so the sieve sorts roughly by length. A band is one mobility, not proof of purity, and not the mass of an oligomer the detergent took apart.

Size-exclusion chromatography followed by multi-angle light scattering and a concentration detector, giving the mass of what is in the peak without a globular calibration. Co-eluting species produce a weight average, not a new oligomer.

A lower-speed analytical run in which sedimentation balances diffusion. For an ideal single species the profile gives molar mass with almost no dependence on shape.

A high-speed analytical run in which a boundary moves. Its speed gives the sedimentation coefficient and its spread contains diffusion, so heterogeneity is seen directly.

The requirement that the radiation couple to the motion being measured. Containing a bond does not mean the spectrum will show it.

Bound mass against time at a surface where a plasmon reports the refractive index. Association mixes the on-rate with concentration; dissociation, in buffer alone, reports the off-rate if artefacts are absent.

The resistance between the pipette and the membrane, which makes the membrane voltage equal the command minus current times that resistance. The error is worst for large currents.

A three-dimensional map built by aligning many noisy copies. One particle is not a structure, and a mixture of conformations, averaged together, blurs or invents a state that never existed.

Control of membrane voltage over the region whose current is being interpreted. It fails in a cell with processes: the pipette holds the soma and does not hold a distant membrane.

A small paramagnetic group, most often a nitroxide, attached at a chosen cysteine. Its lineshape reports motion of the probe, related to but not identical with motion of the backbone.

A confocal scan overlaid with a depletion beam that returns fluorophores to the ground state everywhere except a smaller central spot. The spot shrinks as the depletion beam is made stronger, at a cost in intensity and photodamage.

The factor that makes the ratio of unquenched to quenched intensity rise linearly with quencher concentration in collisional quenching. An upward curve usually means static quenching is present as well.

The gap between the absorption maximum and the emission maximum. Emission is at longer wavelength because vibrational energy and solvent reorganisation have already been lost as heat.

Light that reaches the detector without passing through the sample as the measurement assumes. At high absorbance it is a large fraction of the true signal and the reading flattens.

The relation that removes frictional shape by combining the sedimentation coefficient with the diffusion coefficient, giving molar mass. Svedberg units themselves do not add when subunits assemble.

The midpoint of an unfolding transition watched by heat capacity. A ligand that binds the native state raises it, which shows binding and is not itself a dissociation constant.

Separation by flight time after acceleration, with m/z proportional to the square of that time. The range is wide, which is why it partners singly charged MALDI ions.

The spring constant of an optical trap. Equipartition can calibrate it from the positional variance if the detector is calibrated and filtering has not hidden part of the motion.

A way of keeping a slowly relaxing component of a coupled pair of nuclei, used with isotope enrichment so that a large, slowly tumbling protein can still show resolved peaks.

A clamp that uses one electrode to measure voltage and another to pass current, required when the currents are too large for a single patch pipette to control, as in a full-grown oocyte.

Excitation that requires two infrared photons to arrive together, a rate significant only at the focus. It confines the event and scatters less in tissue, and it is not, by itself, a super-resolution method.

The longitudinal time for magnetisation along the field to regrow, and the transverse time for magnetisation to lose phase. T₂ sets the linewidth, and a statement that a protein is dynamic is incomplete until the timescale is named.

Plate height as a function of linear flow: a term for unequal paths, a term for longitudinal diffusion that dominates when flow is very slow, and a term for slow transfer into the stationary phase that dominates when flow is very fast.

The enthalpy inferred from how sharp a transition is. It equals the calorimetric enthalpy only for a two-state unfolding with no intermediates.

Water frozen faster than it crystallises, so a macromolecule stays hydrated without stain. Crystalline ice would diffract and damage the specimen.

The length λ that sets a photon's energy through E = hc/λ. A shorter wavelength is a larger energy and therefore a different kind of molecular transition.

The reciprocal of wavelength, usually in cm⁻¹, and the usual axis of a vibrational spectrum because it is proportional to energy.

Transfer of a protein gel to a membrane and probing with an antibody. The name follows Southern's; it is not a compass direction, and the evidence is only as good as the antibody.

The configuration after the patch is ruptured, so the pipette solution is continuous with the cytoplasm and the record sums the cell. Soluble regulators wash out, and series resistance drops the membrane off the command when current is large.

The ratio of the concentration of binding sites to the dissociation constant, which decides whether one isothermal titration can return stoichiometry, enthalpy, and affinity together. Outside a middle window the isotherm is still valid and simply lacks one of those numbers.

A fragment that keeps the C-terminus of the same cleavage. Leucine cannot be told from isoleucine by the mass difference alone.

The intensity-weighted mean size that dynamic light scattering quotes first. Because scattered intensity grows steeply with mass, a trace of aggregate can dominate it.

The orientation factor inside the Förster radius. It equals 2/3 only when both dipoles randomise quickly relative to the lifetime, and a change in transfer can be an angle rather than a distance.