= Flow cytometer
A \b[<flow cytometer>] is an instrument that measures optical signals as suspended <particles> pass through an illuminated observation region. Its fluidics positions the sample stream, its optical system excites and collects light, and its electronics converts detector pulses into recorded events. <Flow cytometry> is the measurement technique; a <flow cytometer> is the apparatus implementing it.
In a common design, <hydrodynamic focusing> narrows the sample stream within surrounding fluid so particles encounter a more reproducible region of a <laser> beam. Collected <fluorescence> and scattered light reach <photodetectors> through optical selection elements. A pulse can be summarized by its height, integrated area and width. The FACSCelesta manual distinguishes the maximum digitized intensity from the integral over the electronic acquisition window.<BD FACSCelesta Flow Cytometer User’s Guide by BD Biosciences>[Chapter 7, Technical overview, printed p. 115]{ref} Signal area and peak height are different measurements even when both increase with the brightness of a selected particle population.
For an original ideal pulse model,
$$
p(t)=H\exp\left(-\frac{(t-t_0)^2}{2\sigma^2}\right),\qquad
\int_{-\infty}^{\infty}p(t)\,dt=H\sigma\sqrt{2\pi}.
$$
Two pulses with the same height $H$ but widths $\sigma$ and $2\sigma$ have areas in ratio 1:2. Conversely, equal areas can arise from different heights and durations.
\Image[/flow-cytometer-pulse-width.png]
{title=Original equal-height Gaussian pulses: doubling the width doubles the ideal integrated signal}
Real acquisition windows and thresholding modify the infinite-window model. Pulse-shape comparisons can help identify coincident particles, but no one pulse parameter guarantees a single intact <biological cell>.
A primary experimental example measured <green fluorescent protein> expression in a <genetic toggle switch> with a FACSCalibur instrument. It used 488 nm excitation, a 515–545 nm emission filter and separately acquired cultures; single-cell distributions distinguished alternative expression populations.<Construction of a genetic toggle switch in Escherichia coli by Timothy S. Gardner and others>[p. 342, Methods, Assay of gene expression; Figure 5]{ref} That experiment illustrates the chain from biological reporter to photons, detector signal and population interpretation.
A <flow cytometric DNA-content assay> similarly requires controlled staining, a suitable event-selection rule and detector operation within its quantitative range. Adjusting detector gain changes recorded signal without changing cellular DNA. Instrument response, sample preparation and biological interpretation therefore require distinct controls.
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