6 from a single gene inside the liposomes was also detected and quantitatively evaluated using a ﬂuorogenic substrate and FACS [34]. 5. High-Throughput Analysis of Liposomes Using an FACS 5.1. Application of an FACS to Liposome Measurement. In the liposome-based IVC, an extremely large number (more than 108) of liposomes are created for in vitro protein synthesis beginning with a single DNA. Liposomes that encapsulate a gene of interest are screened from the large population of liposomes for the desired protein function. The protein function expressed inside an individual liposome should be detected and quantitatively analyzed to identify the liposomes for sorting. Protein production and function inside the liposome are often measured and quantiﬁed using analytical tools such as a ﬂuorescence spectrometer and ﬂuorescence microscope [31]. A ﬂuorescence spectrometer detects an averaged ﬂuorescence signal from an ensemble of liposomes. A ﬂuorescence microscope measures a ﬂuo- rescence signal from an individual liposome where proteins are synthesized from genes. Microscopy measurements for liposomes provide data on the morphology (shape and size) and ﬂuorescence intensity of a liposome (internal reaction). However, this technique is only eﬀective for a much smaller liposome population than the population required for statistical analysis and gene screening in IVC- based directed evolution of proteins. FACS is a promising technique for observing a large population of liposomes because of its capacity for high-throughput analysis and simultaneous measurement of multiple characteristics. An FACS is a powerful experimental apparatus for ana- lyzing and sorting live cells simultaneously. The apparatus comprises a ﬂuidics system for transporting one cell at a time, an interrogation system for detecting the cell by laser illumination, and a sorting system for collecting the cells of interest from one to millions of cells. Using this technique, cells exhibiting a speciﬁc biological characteristic are separated from a heterogeneous population of cells using ﬂuorescence and light scattering from individual cells in the population. The FACS was invented in the late 1960s, commercialized in the early 1970s, and has been utilized since then for basic studies in cell biology as well as clinical applications such as diagnosis, disease classiﬁcation, and in vivo therapies [38]. Recently, FACS measurements have not only been used for cell-oriented applications but also for molecular screening in directed evolution of proteins [15, 39]. In addition, the FACS has been utilized for measuring nonbiological particles such as submicron-size liposomes [40] and double emulsion droplets [41] for particle size and ﬂuorescent marker entrapment. We have used FACS to characterize liposomes for structure and biochemical reactions. We ﬁrst successfully detected a GFP synthesis in lipo- somes using FACS based on ﬂuorescence signals from the synthesized GFP [22]. For liposome structure, the internal aqueous volume and membrane volume of indi- vidual liposomes were quantitatively evaluated using light Journal of Nucleic Acids scattering intensity data from an FACS measurement [42]. The liposome population selected using these structural parameters was sorted using an FACS and observed by opti- cal microscopy. The structural parameters generated using the FACS correlated with liposome structural heterogeneity, as demonstrated by microscopy observations. Population analysis of giant liposomes with an FACS was used to identify the subpopulation of unilamellar liposomes in a 2D contour map of surface area and internal aqueous volume generated for giant liposomes [43] (Figure 5). Furthermore, substructure of the multilamellar giant liposomes has been identiﬁed by encapsulating β-glucuronidase synthesis in liposome, and analyzed by an FACS [34]. 5.2. Evaluation of an In-Liposome Reaction Using FACS. Analysis of biochemical reactions in liposomes using an FACS is based on a quantitative evaluation of liposome size and reaction products in liposomes. Liposome size is evaluated by measuring the ﬂuorescence intensity of a ﬂuorescent protein as a volume marker molecule, which is encapsulated in a liposome at a high concentration [24, 34]. The ﬂuorescence intensity of the marker protein is converted to the number of marker molecules in a liposome and then to volume of the internal aqueous phase in the liposome. The reaction product is quantiﬁed by measuring the ﬂuorescence intensity of new synthesized proteins or the ﬂuorescent product of expression of a protein function [20, 25]. Through this analytical method using an FACS, a large population of liposomes can be measured for size and reaction as well as analyzed throughout a population or subpopulation that is deﬁned by reactivity and a speciﬁc size [33]. For liposome- based IVC enzyme screening, a ﬂuorescent volume marker and ﬂuorogenic substrate are encapsulated in liposomes for a screening assay using an FACS (Figure 5, left). Details on this screening system are described in Section 6. Using our system, GFP synthesis inside the liposomes was quantitatively evaluated, and the inﬂuence of lipid membrane composition on protein synthesis was discussed [44]. The study suggested that phospholipids and other liposomal membrane components for liposome preparation should neither inhibit nor impair the protein synthesis reaction steps. Furthermore, GFP synthesis inside the lipo- somes proceeds similarly to that in the test tube in spite that liposomes have very high surface-to-volume ratio in comparison to a test tube. This indicates that phospholipids and other liposomal membrane components for liposome preparation neither inhibit nor impair the protein synthesis reaction steps [20]. Consequently, liposome provides a reaction environment that is equally good as a test tube and provides an extremely large number (more than 1010/100 μL reaction volume) of microcompartments. 6. Liposome-Based IVC for Directed Evolution of Proteins 6.1. Liposome-Based IVC. We constructed a novel gene screening system using a liposome-based IVC for directed evolution of proteins [24, 25]. Liposome-based IVC is a technique used to link genotype and phenotype. The idea