The propagation of HCV requires host–virus interactions that support infection, replication, and viral particle assembly. 2] Genotypes 1a and 1b of HCV induce changes in lipid metabolism and cause the formation of endoplasmic reticulum (ER)-derived membranous webs on which HCV replicates. HCV also induces the accumulation of lipid droplets (LDs), known as steatosis, on which certain HCV proteins are known to reside. Currently, there is no method that permits the observation of spatiotemporal relationships between HCV RNA and alterations in host-cell lipids. Coherent anti-Stokes Raman scattering (CARS) microscopy is a powerful, multiphoton, vibrational imaging modality that is ideal for imaging lipids in live, unstained cells and tissues. Selective imaging of lipids is easily achieved by tuning the frequency difference between two pulsed excitation lasers to match the vibrational frequency of the C H bonds that are abundant in lipids. The use of pulsed, near-IR excitation sources enables the easy combination of CARS with other nonlinear imaging techniques, such as two-photon fluorescence (TPF) microscopy. Herein we establish methods that combine CARS and TPF microscopies to simultaneously examine the subcellular localization of HCV replicon RNA (Figure 1), a noninfectious cell model for HCV replication, and changes in lipid phenotype in live Huh-7 hepatoma cells. The approach is also applicable to cell culture models for HCV infection. First we investigated the localization of LDs in CARS mi ACHTUNGTRENNUNGcrosACHTUNGTRENNUNGcopy images of Huh-7 cells that were treated with only the DMRIE-C transfection reagent (mock-transfected). These cells contained LDs that dominated the CH2 vibrational resonance and corresponded to a size range of 0.3–2 mm (see Figure S1 in the Supporting Information). However, when Huh-7 cells were transfected with lipoplexes comprising transfection reagent containing HCV RNA from the pFK-I389luc/NS3-3’/5.1 subACHTUNGTRENNUNGgenomic replicon (Figure 1B), 14] we observed a trend for increased lipid density in the living cells exposed to HCV RNA as compared to mock-transfected cells (0.35 0.12 vs. 0.25 0.10 a.u. , respectively) that was consistent with the initiation of changes in lipid metabolism by the HCV replicon RNA (Figure S1). To image HCV RNA by TPF, the replicon RNAs were labeled with a two-photon fluorophore (fluorescein) at either the 5’ end of the positive strands, according to Figure 1C, or along the length of the RNA (see the Supporting Information). For simultaneous imaging with combined CARS and TPF microscopies, we used a 711 nm (2 ps) laser beam as both the pump beam for CARS and the excitation beam for TPF. The fluorescein molecules attached to fully labeled and 5’-labeled HCV RNA in lipoplexes were easily probed by TPF, and the fluorescence was stable over a continuous scan of more than five minutes (Figure S2), perhaps due to solid stacking among lipid and RNA molecules in the lipoplexes. We utilized 5’-labeled RNA to study the localization of HCV RNA in Huh-7 cells because activity studies measuring the luciferase genetic reporter demonstrated that 5’-labeled RNA was replication competent (45 17% activity compared to unlabeled RNA), whereas fully labeled RNA was not. We observed that the HCV RNA–liposome lipoplexes were condensed into tightly packed structures that gave strong TPF signals up to 8 h post-transfection (Figure 2C and D). As previously demonstrated, we observed that the HCV RNA localized to the perinuclear region and on or near to LDs/lipoplexes (Figure 2D). The cells showed a progressive increase in LDs during the first 16 h after transfection, as shown in Figure 2B and D and as quantified in Figure 2E (see the Supporting Information). There was a positive correlation between the density of LDs and the levels of HCV RNA, that is, the cells with the highest density of LDs were also transfected with the highest amount of RNA (Figure 2E). To our knowledge, this is the first detailed, live-cell quantification of total LDs over time in cells expressing HCV RNA and proteins. At 16 h post-transfection, the fluorescence signals were significantly reduced and more diffuse, and, by 24 h, the 5’-labeled RNA was no longer visible by TPF (Figure 2C). These observations are consistent with the half-life of the labeled RNA. Since replication of the labeled RNA did not involve the incorporation of new fluorophores, our ability to image viral RNA was limited to the lifetime of the fluorescently labeled RNA that was initially delivered to the cells. According to luciferase assays of cellular lysates from Huh-7 cells transfected with unlabeled RNA, the luciferase signal was 100-fold higher in cells transfected with viral RNA than in mock-transfected cells at 2–6 h post-transfection (data not shown). This implies that transfected HCV RNA enters the cells and begins dissociating from liposomes within 2–6 h, followed by translation of encoded viral proteins and replication of the viral RNA. Thus, we believe that the significant increase in LDs observed after 16 h can be attributed directly to the effects of fluorescently labeled RNA [a] X. Nan, Prof. X. S. Xie Department of Chemistry and Chemical Biology, Harvard University 12 Oxford Street, Cambridge, MA 02138 (USA) [b] Dr. A. M. Tonary, Prof. A. Stolow, Prof. J. P. Pezacki The Steacie Institute for Molecular Sciences National Research Council of Canada 100 Sussex Drive, Ottawa, K1A 0R6 (Canada) Fax: (+1)613-952-0068 E-mail : John.Pezacki@nrc-cnrc.gc.ca [] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://www.chembiochem.org or from the author.