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| Term | Abbreviation | Description |
|---|---|---|
| Signal-to-noise ratio | S/N | The signal to noise ratio is the ratio of the power of the signal to that of the noise. For example, in fluorimetry it would be the ratio of the square of the fluorescence intensity to the square of the intensity of the background noise. |
| Slit width | The slit width determines the amount of light entering the spectrofluorometer or spectrophotometer. A larger slit reduces the signal-to-noise ratio but reduces the wavelength resolution. | |
| Smoothing | Various methods of smoothing can be applied to improve the signal-to-noise ratio. For instance, data points recorded over time [s] or over a range of wavelengths [nm] can be smoothed by averaging n data points per interval. Then the average of the n points per smoothing interval can be taken for each successively recorded data point across the time range or range of the spectrum to give a n-point moving average smoothing. This method decreases the noise of the signal, but clearly reduces the time or wavelength resolution. More advanced methods of smoothing are applied to retain a higher time resolution or wavelength resolution. | |
| Spectrophotometer | A spectrophotometer is an instrument that consists of an entrance slit, a dispersion device (see dispersion devices and a detector for the purpose of measuring the intensity of light emerging from a sample across a given wavelength range. A light source is also necessary in order for the instrument to function, and this may be located within the instrument or from an external source using lightguides or other optics. | |
| Spectrophotometry | Spectrophotometry is the use of a spectrophotometer to measure the transmittance, reflectance or remittance of a material as a function of wavelength. See transmission spectrophotometry, reflectance spectrophotometry and remission spectrophotometry. | |
| Spectroscopy | Spectroscopy is a broader term than spectrophotometry in that it is concerned with the investigation and measurement of spectra produced when matter interacts with or emits any form electromagnetic radiation. | |
| Spline | Some spectrofluorometer or spectrophotometer software offers the possibility of spline interpolation of the spectral data points. This makes use of a polynomial (the number of spline points is entered by the user) to interpolate the curve between the data points. | |
| Stability | Stability determines the accuracy of intensity and absorbance measurements as a function of time. Instability (see drift introduces systematic errors in the accuracy of fluorescence and absorbance measurements. | |
| Steady state | A system is in a steady state if the state variables of a dynamic system do not change over time due to exchange processes with the environment, which compensate for internal dissipative transformations — such as chemical reactions or diffusion — and thus prevent any changes of the system and externalize dissipative changes to the environment. The dynamic nature of the steady state differentiates it from the thermodynamic equilibrium state. {Quote} Steady states can be obtained only in open systems, in which changes by internal transformations, e.g., O2 consumption, are instantaneously compensated for by external fluxes across the system boundary, e.g., O2 supply, thus preventing a change of O2 concentration in the system (Gnaiger 1993). Mitochondrial respiratory states monitored in closed systems satisfy the criteria of pseudo-steady states for limited periods of time, when changes in the system (concentrations of O2, fuel substrates, ADP, Pi, H+) do not exert significant effects on metabolic fluxes (respiration, phosphorylation). Such pseudo-steady states require respiratory media with sufficient buffering capacity and substrates maintained at kinetically-saturating concentrations, and thus depend on the kinetics of the processes under investigation. {end of Quote: BEC 2020.1}. Whereas fluxes may change at a steady state over time, concentrations are maintained constant. The 'respiratory steady state' (Chance and Williams 1955) is characterized by constant fluxes (O2 flux, H2O2 flux) and measured variables of state (cytochrome redox states, Q redox state, NADH redox state, mitochondrial membrane potential). High-resolution respirometry allows for the measurement of several parameters (e.g. O2 flux, H2O2 flux, mitochondrial membrane potential) at pseudo-steady states, when changes of concentrations in the closed system do not exert any control on fluxes. Combination with the Titration-Injection microPump (TIP2k) allows operation with programmable titration regimes at steady-state ADP concentration (Gnaiger 2001), oxygen concentration (oxystat mode; Gnaiger et al 2000, Harrison et al 2015) or steady-state pH (pH-stat more), yielding an expanded flexibility in experimental design by combining the technical advantages of closed and open systems approaches. | |
| Stray light | Stray light is defined as the detected light of any wavelength that lies outside the bandwidth of the selected wavelength. In the presence of stray light of intensity Is, the equation for transmittance (T) becomes T = (I + Is)/(I0 + Is) where I0 is the incident light intensity and I is the transmitted light intensity. Clearly, the lower the value of I, the more dominant becomes the stray light term and so can cause errors in the quantification of low fluorescence signals or at high levels of absorbance. | |
| Transmission spectrophotometry | In the transmission mode, the incident light passes through the sample cuvettes and the emergent light reaches the detector directly. Before absorbance measurements can be made, a balance is carried out. | |
| Transmittance | T | When light enter a sample, transmittance (T) is the fraction of the intensity (I) of the light emerging from the sample compared with the incident light intensity (I0): T = I/I0. |
| Wavelength averaging | Wavelength averaging is the averaging of several adjacent data points across the recorded spectrum (spectral smoothing), to improve the signal-to-noise ratio. For example, if the instrument recorded 5 data points per nm, the average of the 5 points can be taken for each successive nm across the range of the spectrum to give a 5-point smoothing. This method clearly reduces the wavelength resolution. | |
| Wavelength range | The minimum and the maximum wavelengths over which an absorbance spectrum is measured are described in terms of the wavelength range. It is determined mainly by the specifications of the spectrophotometer and the type of light source used, and the characteristic absorbance spectrum of the sample being investigated. | |
| White balance | In reflectance spectrophotometry and remission spectrophotometry a white balance is carried out to determine the intensity of the incident light (I0) for the purpose of quantitative absorbance measurements. In reflectance spectrophotometry, a mirror can be used whereas in remission spectrophotometry a standard white tile is more appropriate. |
