Chapter 3 – Making Light Work in Biology  65

whether the protein is in the folded conformational state. Also, chlorophyll, which is a key

molecule in plants as well as many bacteria essential to the process of photosynthesis (see

Chapter 2), has significant fluorescent properties.

Note also that there is sometimes a problematic issue with in vitro fluorimetry known

as the “inner filter effect.” The primary inner filter effect (PIFE) occurs when the absorption

of a fluorophore toward the front of the cuvette nearest the excitation beam entry point

reduces the intensity of the beam experienced by fluorophores toward the back of the cuvette

and so can result in apparent nonlinear dependence of measured fluorescence with sample

concentration. There is also a secondary inner filter effect (SIFE) that occurs when the fluor­

escence intensity decreases due to fluorophore absorption in the emission region. PIFE in

general is a more serious problem than SIFE because of the shorter wavelengths for exci­

tation compared to emission. To properly correct these effects requires a controlled titra­

tion at different fluorophore concentrations to fully characterize the fluorescence response.

Alternatively, a mathematical model can be approximated to characterize the effect. In prac­

tice, many researchers ensure that they operate in a concentration regime that is sufficiently

low to ignore the effect.

3.2.3  FLOW CYTOMETRY AND FLUORESCENCE-​ASSISTED CELL SORTING

The detection of scattered light and fluorescence emissions from cell cultures are utilized

in powerful high-​throughput techniques of flow cytometry and fluorescence-​assisted cell

sorting (FACS). In flow cytometry, a culture of cells is flowed past a detector using con­

trolled microfluidics. The diameter of the flow cell close to the detector is ~10⁻⁵ m, which

ensures that only single cells flow past the detector at any one time. In principle, a detector

can be designed to measure a variety of different physical parameters of the cells as they flow

past, for example, electrical impedance and optical absorption. However, by far the most

common detection method is based on focused laser excitation of cells in the vicinity of

a sensitive photodetector, which measures the fluorescence emissions of individual cells as

they flow past.

Modern commercial instruments have several different wavelength laser sources and

associated fluorescence detectors. Typically, cells under investigation will be labeled with a

specific fluorescent dye. The fluorescence readout from flow cytometry can therefore be used

as a metric for purity of subsequent cell populations, that is, what proportion of a subsequent

cell culture contains the original labeled cell. A common adaptation of flow cytometry is to

incorporate the capability to sort cells on the basis of their being fluorescently labeled or not,

using FACS. A typical FACS design involves detection of the fluorescence signature with

a photodetector that is positioned at 90° relative to the incident laser beam, while another

photodetector measures the direct transmission of the light, which is a metric for size of

the particle flow past the detector that is thus often used to determine if just a single cell is

flowing past as opposed to, more rarely, two or more in the line with the incident laser beam

(Figure 3.1c).

Cells are usually sorted into two populations of those that have a fluorescence intensity

above a certain threshold, and those that do not. The sorting typically uses rapid electrical

feedback of the fluorescence signal to electrostatics plates; the flow stream is first interrupted

using piezoelectric transducers to generate nanodroplets, which can be deflected by the

electrostatic plates so as to shunt cells into one of two output reservoirs. Other commer­

cial FACS devices use direct mechanical sorting of the flow, and some bespoke devices have

implemented methods based on optical tweezers (OTs) (Chapter 6).

FACS results in a very rapid sorting of cells. It is especially useful for generating purity in

a heterogeneous cell population. For example, cells may have been genetically modified to

investigate some aspect of their biology; however, the genetic modifications might not have

been efficiently transferred to 100% of the cells in a culture. By placing a suitable fluorescent

marker on only the cells that have been genetically modified, FACS can then sort these effi­

ciently to generate a pure culture output that contains only these cells.