Atto680

We isolated splenocytes from the spleen of C57Bl/6 mice and prepared, similarly to the HEK cells, two types of isolated splenocyte samples: single color samples to acquire the spectral signatures of the chromophores and mixed samples. For the cell isolation, the spleen was cut into small pieces, pressed through a strainer and suspended in RPMI medium containing 10% FCS. Erythrocyte lysis buffer was added to the cell suspension to remove erythrocytes. The suspension was centrifuged and the pellet was resuspended in PBS. The labeling of the splenocytes was performed following existing protocols, using Hoechst, CMTPX Red, Alexa647 (ThermoFischer Scientific, Waltham, MA) and Atto680 (AttoTEC, Siegen, Germany). Splenocytes from B1-8^+/+ Jκ^−/− Kusabira Orange mice as well as of mice ubiquitously expressing eGFP were isolated from the spleen in a similar manner as described for C57/Bl6 mice.

The eight fluorophores utilized in this study were obtained commercially in their succinimidyl ester form including Fluorescein, BODIPY FL, AlexaFluor568, AlexaFluor647 (Thermo Fisher Scientific), ATTO488, ATTO680 (ATTO-TEC), Cy3B and Cy5 (GE Healthcare Life Sciences).

The phantom consisted of polymer tubes (600 μm inner dia., MORCAP, Paradigm Optics Inc., USA) filled with either a solution of a near-infrared fluorophore (Atto680, Atto-tec GmbH, Germany) in methanol or an aqueous solution of copper chloride (CuCl₂), which mimicked endogenous absorbers, such as haemoglobin. The Atto680 solution had a concentration of 80 μM, which corresponds to an absorption coefficient of μ_a = 2.3 mm⁻¹ at 680 nm. The fluorescence lifetime of Atto680 in methanol is 2.7 ns. The CuCl₂ solution had a concentration of 0.9 M, i.e. μ_a = 2.5 mm⁻¹ at 742 nm. The tubes were immersed in a scattering lipid suspension with a reduced scattering coefficient of μ_s’ ~ 1 mm⁻¹. The pump fluence was set to 2 mJ/cm² and the probe fluence was set to 4.3 mJ/cm². The phantom was placed onto the Fabry-Perot-based PA scanner. PA image data sets were acquired over a scan area of 5 mm × 16 mm using sequential and interleaved image acquisition. To mimic tissue motion, the phantom was translated by approximately 1 mm halfway during each image acquisition. To compensate for minor fluence changes, the intensity of the images was normalized to that measured in the tubes filled with Atto680 acquired using time-delayed pump and probe pulses. Subsequently, difference images were calculated by subtracting the images obtained using simultaneous and time-delayed pump-probe excitation.