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21

Single Cell Analysis and Multiomics

21.1

Experimental Methods

Early work on the phenotypic heterogeneity of the descendants of a single bacterial

cell relied on the microfluidic isolation of the individual descendants. ³ The expo-

nential growth in numbers means that a complete examination of all the descendants

rapidly becomes impracticable. In eukaryotic work, individual cells are isolated from

tissue. Continual advances in the technologies of DNA, RNA, protein, metabolite,

etc., characterization mean that many are now sensitive enough to work with material

from a single cell. Nevertheless, challenges remain. Technical variability in the omics

technique may artefactually exaggerate the heterogeneity of a collection of single

cells. It can be empirically estimated by diluting a sample of known composition

and running it through the experimental protocol, but even dilution down to the scale

required is challenging.

Single cell analysis also encompasses the sorting of cells to produce phenotyp-

ically pure collections. If the goal of the investigation is to correlate phenotype

with genome, proteome, etc. in order to understand the underlying mechanisms of

phenotype production, genomic, etc. data can be collected from multiple cells simul-

taneously; here it is assumed that they are genetically, etc. homogeneous, and the

investigator will likely compare genome, proteome, etc. with those of cells differing

phenotypically.

The sorting has been largely automated; cells are labelled according to pheno-

type, for example with a fluorescent marker or magnetic bead functionalized to bind

specific molecules characteristically expressed on the surface of the cells. As they

pass through a microfluidic channel, the labelled cells are separated from the rest. ⁴

If single cells need to be isolated, manual micromanipulation is required. It can

be accomplished by using micropipettes to move cells around under a microscope.

More sophisticated is the use of laser tweezers to capture and move cells. In laser

capture microdissection, a focused laser melts a synthetic polymer film placed over a

piece of tissue, which then adheres to the selected cells, which can be then removed

by lifting up the film.

Quantitative dynamical phenotypic characterization can be achieved by observing

cells placed on a planar optical waveguide. ⁵ The waveguide can be precoated with

practically any substrate of interest, appropriate to the investigation. Multiple param-

eters pertaining to the shape of the cell can be measured using optical waveguide

lightmode spectroscopy (OWLS), and the high time resolution allows phase portraits

of individual cells to be readily obtained. ⁶

3 Wakamoto et al. (2005).

4 These techniques various acronyms such as FACS (fluorescence-activated cell sorting) or MACS

(magnetic-activated cell sorting). See Hu et al. (2016) for many references.

5 Ramsden et al. (1995), Horvath et al. (2008).

6 Aref et al. (2009, 2010); see Fig. 16.1.