Fluorescence imaging of stained red blood cells with simultaneous resonance Raman photostability analysis.

Optical spectroscopic imaging of biological systems has important applications in medical diagnosis, biochemistry, and image-guided surgery. Vibrational spectroscopy, such as Raman scattering, provides high chemical selectivity but is limited by weak signals and a large fluorescence background. Fluorescence imaging is often used by introducing specific dyes in biological systems to label different system parts and to increase the image contrast. However, the extrinsic fluorescence of the staining molecules often masks the intrinsic vibrational signals of biomolecules, which could also be simultaneously detected using the same excitation laser source. Therefore, fluorescence staining is often accompanied by the loss of other important complimentary information. For example, the high laser power often used for the rapid, high-quality imaging could lead to photo-induced suppression or bleaching of the fluorescence and Raman signals resulting in sample photodamage. Therefore, simultaneous imaging and photodamage analysis need to be performed in a controlled bioimaging experiment. Here we perform simultaneous spectroscopic bioimaging and photostability analysis of rhodamine 6G (R6G) stained red blood cells (RBCs) using both fluorescence and resonance Raman imaging in a single 532 nm laser excitation experiment. We develop a corresponding data processing algorithm which allows separation of the two spectroscopic signals. We control the relative intensity of the R6G and RBC signals by varying the excitation laser power and simultaneously monitor the photostability of RBCs. We observe no significant photodamage of RBCs through the absence of changes in the relative Raman peak intensities. Conversely, the R6G molecules show bleaching with the suppression of both the fluorescence and resonance Raman signals. Our approach may be generalized to other types of stained cells with the appropriate selection of fluorescent dyes and excitation sources.

Characterization of carrier erythrocytes for biosensing applications.

Erythrocyte abundance, mobility, and carrying capacity make them attractive as a platform for blood analyte sensing as well as for drug delivery. Sensor-loaded erythrocytes, dubbed erythrosensors, could be reinfused into the bloodstream, excited noninvasively through the skin, and used to provide measurement of analyte levels in the bloodstream. Several techniques to load erythrocytes, thus creating carrier erythrocytes, exist. However, their cellular characteristics remain largely unstudied. Changes in cellular characteristics lead to removal from the bloodstream. We hypothesize that erythrosensors need to maintain native erythrocytes' (NEs) characteristics to serve as a long-term sensing platform. Here, we investigate two loading techniques and the properties of the resulting erythrosensors. For loading, hypotonic dilution requires a hypotonic solution while electroporation relies on electrical pulses to perforate the erythrocyte membrane. We analyze the resulting erythrosensor signal, size, morphology, and hemoglobin content. Although the resulting erythrosensors exhibit morphological changes, their size was comparable with NEs. The hypotonic dilution technique was found to load erythrosensors much more efficiently than electroporation, and the sensors were loaded throughout the volume of the erythrosensors. Finally, both techniques resulted in significant loss of hemoglobin. This study points to the need for continued development of loading techniques that better preserve NE characteristics.

Subcellular measurements of mechanical and chemical properties using dual Raman-Brillouin microspectroscopy.

Brillouin microspectroscopy is a powerful technique for noninvasive optical imaging. In particular, Brillouin microspectroscopy uniquely allows assessing a sample's mechanical properties with microscopic spatial resolution. Recent advances in background-free Brillouin microspectroscopy make it possible to image scattering samples without substantial degradation of the data quality. However, measurements at the cellular- and subcellular-level have never been performed to date due to the limited signal strength. In this report, by adopting our recently optimized VIPA-based Brillouin spectrometer, we probed the microscopic viscoelasticity of individual red blood cells. These measurements were supplemented by chemically specific measurements using Raman microspectroscopy.

Active targeting of chemotherapy to disseminated tumors using nanoparticle-carrying T cells.

Tumor cells disseminate into compartments that are poorly accessible from circulation, which necessitates high doses of systemic chemotherapy. However, the effectiveness of many drugs, such as the potent topoisomerase I poison SN-38, is hampered by poor pharmacokinetics. To deliver SN-38 to lymphoma tumors in vivo, we took advantage of the fact that healthy lymphocytes can be programmed to phenocopy the biodistribution of the tumor cells. In a murine model of disseminated lymphoma, we expanded autologous polyclonal T cells ex vivo under conditions that retained homing receptors mirroring lymphoma cells, and functionalized these T cells to carry SN-38-loaded nanocapsules on their surfaces. Nanocapsule-functionalized T cells were resistant to SN-38 but mediated efficient killing of lymphoma cells in vitro. Upon adoptive transfer into tumor-bearing mice, these T cells served as active vectors to deliver the chemotherapeutic into tumor-bearing lymphoid organs. Cell-mediated delivery concentrated SN-38 in lymph nodes at levels 90-fold greater than free drug systemically administered at 10-fold higher doses. The live T cell delivery approach reduced tumor burden significantly after 2 weeks of treatment and enhanced survival under conditions where free SN-38 and SN-38-loaded nanocapsules alone were ineffective. These results suggest that tissue-homing lymphocytes can serve as specific targeting agents to deliver nanoparticles into sites difficult to access from the circulation, and thus improve the therapeutic index of chemotherapeutic drugs with unfavorable pharmacokinetics.