In laboratories around the world, lipids are today observed as dynamic actors in cellular life: from building and maintaining membranes, through storing and mobilizing energy, to managing signaling pathways and the selective passage of molecules. The largest portion of new lipid molecules is formed in the endoplasmic reticulum, but different classes of lipids are purposefully moved to specific destinations – mitochondria, the Golgi apparatus, lysosomes, or the plasma membrane – depending on the function they need to perform. Each of these "addresses" in the cell carries its own recognizable lipid composition, its own lipidome, which participates in defining the organelle's identity and its biochemical niche.

Why it was so difficult to "see" lipids until recently

Light microscopy techniques have been the foundation of cell biology for decades, but they have a resolution limit of around 200–250 nanometers. In living cells, organelles are often only a few tens of nanometers apart, while the membrane bilayer itself – the structure of all cellular membranes – measures approximately 3–4 nanometers in thickness. In such a nanometer-scale world, events at the contacts between organelles, rapid transitions of lipids from one bilayer "leaflet" to another, or asymmetric redistributions within the same membrane often remain below the radar of conventional methods. Classical labeling of proteins with fluorescent reporters describes the "vehicles" (transport proteins) but often misses the "cargo" itself – small, fast, chemically diverse lipid molecules that decisively influence membrane organization and signaling.

New insight at the nanoscale: FACES changes the rules of the game

Researchers from the University of California, San Diego have introduced a chemogenetic approach that enables an unprecedented level of specificity in tracking lipids in living cells. This is the fluorogen-activating coincidence encounter sensing technology – FACES for short – which combines two components: (1) small chemical dyes called fluorogens, which are dark by themselves, and (2) genetically encoded fluorogen-activating proteins (FAPs), which serve as "light switches". Only when the fluorogen and FAP find themselves in the same place at the same time does a fluorescent complex form and the signal "lights up". This turns the light on exactly where a biologically relevant encounter is happening, while everything else remains in the dark – which dramatically reduces background fluorescence and increases contrast.

The key idea is to reverse the usual logic: instead of genetically tagging a protein that indirectly indicates the presence of a lipid, FACES directly "illuminates" the lipid that is chemically modified to carry the fluorogen. As the FAP can be precisely placed in the desired part of the cell (e.g., the outer mitochondrial membrane, a specific cisterna of the Golgi apparatus, or a specific domain of the plasma membrane), the signal appears only when the labeled lipid enters the immediate vicinity of that protein light switch. The result is a high signal-to-noise ratio, location selectivity, and the ability to quantitatively track lipid flows over time.

"Leaflet" by "leaflet": how to distinguish the two sides of the same bilayer

A membrane bilayer consists of two surfaces – leaflets – which do not necessarily have the same composition. The outer leaflet of the plasma membrane is often rich in phosphatidylcholine and sphingomyelin, while the inner leaflet is enriched with phosphatidylethanolamine and negatively charged phosphatidylserine and phosphatidylinositols. This asymmetry affects membrane curvature, the recruitment of specific proteins, and even events like endocytosis, exocytosis, or apoptosis. FACES brings a true breakthrough here: using transmembranally oriented FAP variants, it is possible to separately "illuminate" individual leaflets and track how lipids cross from one side of the bilayer to the other. This provides, for the first time in living cells, a leaflet-specific picture – who is where, at what moment, and at what pace.

From idea to first experiments: how the skeptical gap was bridged

Taming chemistry and biology into a single tool is always a challenge. The questions were numerous: will conjugating the fluorogen to the lipid change its behavior? Can a sufficiently strong and specific signal be achieved without flooding the cell with background glow? Will the FAP, placed on the selected membrane, remain functional and not disrupt the local organization? Through systematic engineering of FAP variants, thoughtful selection of fluorogens (including those with a near-infrared emission maximum, suitable for reducing autofluorescence), and optimization of labeling protocols, a robust, repeatable, and quantitative signal was achieved. This "coincidence" logic ensured that the fluorescence is directly proportional to the actual encounters of lipids and FAPs in space and time.

What FACES is already showing: mitochondria, ER, and trans-Golgi under the magnifying glass

Early work focused on phosphatidylcholine (PC) – the most abundant phospholipid in eukaryotic membranes – and its delivery to mitochondria. The supply of phospholipids to mitochondria is critical for maintaining the functionality of their membranes, energy efficiency, and the organization of cristae. Using FACES, it was possible to distinguish the role of specific lipid transporters and contact sites between the endoplasmic reticulum (ER) and mitochondria, and to quantify the contribution of these "bridges" compared to classical vesicular transport. In the trans-Golgi, meanwhile, transmembrane FAP constructs revealed how the bilayer asymmetry for multiple lipid classes is established and maintained – knowledge that is directly applicable to understanding the sorting of membrane proteins, buffers for signaling lipids, and the mechanisms that determine the physical properties of the membrane.

Why quantitativeness matters

Visualization itself provides a map of events, but numbers allow for hypothesis testing. Since the formation of the fluorescent complex depends on actual encounters between the labeled lipid and the FAP, after calibration, it is possible to derive relative transfer rates from the signal, compare intensities between different subcellular locations, and model fluctuations over time. This yields metric estimates of lipid flow through individual contact sites, and it is particularly valuable that this data can be correlated with variations in metabolism, energy status, or pharmacological interventions.

How FACES complements existing microscopy techniques

Super-resolution methods (such as STED, PALM, or STORM) have pushed the boundaries of what can be seen, but they often require special setups, expensive accessories, or have limitations in the speed of imaging living cells. FACES is designed to work with standard fluorescence microscopy systems: it is sufficient to match the filters and excitation to the selected fluorogen, and to correctly direct the FAP to the desired microdomain. Additionally, the fact that the rest of the cell remains a "dark background" simplifies interpretation and reduces phototoxicity, as it is not necessary to "boost" the general signal to isolate the desired event.

The cell's "traffic hubs": contact sites as highways for lipids

The cell is not a collection of isolated islands, but a network of interconnected compartments. Contact sites between organelles (e.g., ER–mitochondria or ER–plasma membrane) allow for the direct exchange of lipids and ions without membrane fusion. With FACES, such hubs can be imaged live with local selectivity, revealing differences between fast flows (e.g., supplying mitochondria with phosphatidylcholine) and slower, regulatory movements (e.g., remodeling of domains in the trans-Golgi). Such data helps answer the question of where the bottleneck arises, what defines traffic priorities, and how the network reorganizes under stress.

Leaflet-specific changes and the enzymes that maintain them

Leaflet asymmetry is maintained by enzymes called flippases, floppases, and scramblases, which regulate the crossing of lipids from one leaflet to another. Imbalances in their activity disrupt signaling pathways, change adhesion, and can activate immune responses. FACES, by providing the ability to independently "turn on" the signal on a single leaflet, allows for precise mapping of these process flows. This makes it possible, for example, to quantify how quickly and in which direction a phospholipid crosses during receptor activation or under the influence of inhibitors of specific remodeling enzymes.

Methodological architecture: from fluorogen to FAP

The choice of fluorogen is not just a matter of brightness; photophysics, stability in a biological environment, and minimal disruption of membrane dynamics are key. Fluorogens with emission in the near-infrared region reduce autofluorescence and allow for deeper imaging, while FAP variants with high affinity and rapid association increase the fidelity of the coincidence reading. The genetically encoded localization "address" (e.g., signal peptides for the outer mitochondrial membrane or retention in the Golgi) turns the FAP into a precise light marker for the microenvironment where we are interested in the fate of a specific lipid.

Examples of research questions that FACES makes reachable

• How much do individual lipid transfer proteins contribute to the supply of phosphatidylcholine to mitochondria, and how does their role change under metabolic stress?


• How is the asymmetry of the trans-Golgi for different classes of phospholipids established and maintained, and how does it affect the sorting of membrane proteins?


• What is the relative contribution of ER–mitochondria and ER–plasma membrane contacts in the supply of key lipids compared to vesicular transport?


• How does pharmacological inhibition of remodeling enzymes change leaflet-specific distributions and the dynamics of local domains?

From basic science to medicine

Changes in the trafficking of sphingolipids and sterols are linked to neurodegeneration; disorders in phospholipid remodeling are involved in the development of metabolic syndrome and non-alcoholic fatty liver disease; in oncology, the lipid composition of membranes affects the sensitivity of tumor cells to therapy and their invasiveness. FACES offers a way to monitor these changes directly at the site of the event – in a leaflet-specific, organelle-specific, and time-resolved format. This opens the door to diagnostic and therapeutic strategies that target functional flows, not just static quantities.

Collaborations, platforms, and open access

The development of FACES relied on interdisciplinary partnerships between biochemistry, molecular biophysics, pharmacology, and advanced microscopy. Alongside academic programs, philanthropic initiatives focused on organelle communication and membrane biophysics played an important role, encouraging teams to jointly develop tools and compare them with existing methods. The emphasis is on accessibility: the authors want to make the protein constructs and chemical reagents widely available, along with reference protocols and manuals that allow laboratories for rapid implementation without expensive hardware upgrades.

Glossary of terms (quick orientation)

• Lipids – fatty, waxy, or oily molecules that build membranes, store energy, and participate in signaling.


• Organelles – specialized compartments within the cell (e.g., nucleus, mitochondria, Golgi apparatus) with their own lipid composition and function.


• Fluorogens – chemical dyes that do not fluoresce until they form a complex with the appropriate activator protein.


• FAP – fluorogen-activating protein; a genetically encoded "light switch" that "turns on" the signal upon encountering a fluorogen.


• Leaflets – the two sides of the same lipid bilayer that differ in composition and properties.


• Contact sites – membranes of organelles that come close without fusing and allow for the rapid transfer of lipids and ions.

Compatibility with existing instruments

One of the practical advantages of FACES is that it works on standard fluorescence microscopy setups. Specialized optics are not necessary; what is needed is a good imaging plan, an appropriate filter configuration, and carefully modulated FAP expression to avoid overloading the membrane domains. In combination with stable fluorogens and optimized incubation, it is possible to achieve high signal-to-noise ratios and minimal phototoxicity, which is crucial for studying dynamic processes in living cells.

Plans and horizons: mapping "conversations" between organelles in real time

The next step is the systematic mapping of the flows of major lipid classes through the network of contact sites – ER–mitochondria, ER–Golgi apparatus, and all the way to specialized domains of the plasma membrane. A comparison of healthy and pathological states should show whether the flow rates, the geometry and stability of the contact sites, or the leaflet-specific asymmetry that supports signaling platforms change first. A particularly intriguing question is how much rapid rearrangements in microdomains precede larger functional changes, for example, in mitochondrial respiration or in phosphoinositide signaling at the membrane.

Technical nuances that make the difference

Three things are crucial for the success of FACES: chemistry (stable, bioorthogonal ways of conjugating fluorogens to lipids), genetics (precise targeting of FAP to the target microdomain), and physics (sensitive cameras and properly selected filters). When these three come together, the result is a signal that reflects real, local biological interactions – and in a timeframe that is fast enough to capture the short-lived episodes of lipid exchange at contact sites.

Checklist for laboratories introducing FACES

• Define the biological question (e.g., phosphatidylcholine flow to the mitochondrion) and select the appropriate fluorogen conjugate.


• Design the FAP construct with a reliable localization tag (mitochondrial outer membrane, trans-Golgi, plasma membrane).


• Optimize FAP expression to avoid saturation and alteration of the local membrane.


• Calibrate imaging conditions (incubation time, fluorogen concentration, excitation power) according to the desired temporal resolution.


• Establish controls (negative and positive) to assess the specificity and quantitativeness of the signal.

Open potential beyond lipids

Although FACES was developed with a focus on lipids, the same coincidence logic can be extended to other biomolecules that can be chemically labeled with bioorthogonal reactions – for example, certain sugars in glycoproteins. This makes FACES a platform: by modulating the chemistry and the choice of FAP location, it is possible to design sensors tailored for a specific biological question, from metabolism to intracellular signaling.

Names, dates, and context

The announcement about FACES attracted the attention of the scientific community in mid-October 2025, just before today's date (October 22, 2025), with detailed descriptions of the concept, authors, and applications. The authors listed include William M. Moore as the first author, Itay Budin as the corresponding author, along with collaborators Robert J. Breu, Caroline H. Knittel, Ellen Wrightsman, Brandon Hui, Christopher J. Obara, and Neal K. Devaraj. The work fits into the broader framework of research on communication between organelles and into programs supporting advanced imaging technologies, and the intention to make the key components – the protein "switch" and the associated fluorogens – available to the wider research community is also emphasized.

For further orientation

Readers who wish to deepen their introduction to the topic can recall the fundamental structure of membranes and the role of phospholipids through review textbook materials on membrane biology, the basics of bioorthogonal chemistry, and the concept of membrane contact sites between organelles. For the context of current research in the field of organelle communication, it is also useful to follow initiatives focused on developing methods that quantify lipids with high spatio-temporal resolution. Note on the date: the information in the text is aligned with the state of affairs as of October 22, 2025.