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.

Endometriosis Mimicking a Cecum Mass with Complete Bowel Obstruction: An Infrequent Cause of Acute Abdomen.

Endometriosis is a common entity among fertile women which unfortunately manifests through variable symptomatology. Intestinal involvement in endometriosis is quite common and can simulate several diseases such as Crohn's disease, appendicitis, tubo-ovarian abscess, or malignant tumors. Intestinal obstruction due to endometriosis is rare, and preoperative diagnosis is difficult because the signs and symptoms are nonspecific and can be easily confused. In the case of patients without a history of endometriosis, diagnosis is further complicated. We present a case of a 41-year-old female patient. She presented to the emergency room with complete bowel obstruction and a mass in the cecum. Surgery was decided, and the patient underwent full recovery. Endometriosis was the final diagnosis for the observed condition.

A nail in the appendix, accidental discovery on an asymptomatic patient.

Inadvertent ingestion of foreign bodies is a common condition within clinical practice. It rarely produces any symptoms. The diagnosis is difficult since most patients do not recall having swallowed any object. Needles, pins, keys, nails and bones are among the most commonly ingested foreign bodies. Severe complications are uncommon, but if present they can put patients' lives at risk. Although extremely rare, the ingested foreign body may end lodging in the appendix, posing a challenge for the clinical team. Once the exact location of the object is confirmed, the extraction of the foreign object must be performed to avoid complications. The present report describes a case of a young adult patient, who presented to the emergency room after a routine medical examination. A 30 mm metallic nail was discovered in the tip of the appendix. After a failed endoscopic approach an appendectomy was performed, and the patient underwent a complete recovery.

Colonic perforation due to amebiasis, a rare and lethal complication.

Amebiasis is still a major healthcare concern, especially in developing countries like Ecuador. The lack of sanitary control and hygiene measures make parasites infections still a burden for patients and physicians. Despite infections due to this parasites are usually mild, severe infections and fatal outcomes although rare still occur. Bowel perforation is a rare complication of amebiasis, and unfortunately, it continues to be almost fatal. We present a case of an Ecuadorian patient who presented to the emergency room with an acute abdomen, despite adequate surgery and critical care, the patient regrettably died. Bowel perforation due to Entamoeba histolytica was the final diagnosis.

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.

Multicomponent One-Pot Synthesis of 3-Tetrazolyl and 3-Imidazo[1,2-a]pyridin Tetrazolo[1,5-a]quinolines.

A series of 18 3-tetrazolyl-tetrazolo[1,5-a]quinolines were synthesized in 21-90% yields via a novel one-pot Ugi-azide/SNAr/ring-chain azido-tautomerization process. We report also the synthesis of 10 3-imidazo[1,2-a]pyridin-tetrazolo[1,5-a]quinolines in 28-94% yields via a novel one-pot Groebke-Blackburn-Bienaymé/SNAr/ring-chain azido-tautomerization process. Both synthetic strategies involve the use of microwaves or ultrasound, and catalyst-free conditions. Finally, we show the synthesis of the tetrazolo[1,5-a]quinoline-3-carbaldehyde and tetrazolo[1,5-a]quinoline-3-dimethyl acetal at room temperature in methanol as solvent.

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.

In situ engineering of the lymph node microenvironment via intranodal injection of adjuvant-releasing polymer particles.

Recent studies have demonstrated a simple, potentially universal strategy to enhance vaccine potency, via intralymph node (i.LN) injection. To date, intranodal immunization studies have focused on the delivery of unadjuvanted vaccines (e.g., naked DNA, peptide, or protein). We hypothesized that combining i.LN vaccination with controlled release biomaterials permitting sustained dosing of molecular adjuvants to the local tissue microenvironment would further enhance this promising vaccination strategy. To test this idea, we encapsulated the Toll-like receptor-3 ligand poly(inosinic:cytidylic acid) (polyIC) in biodegradable poly(lactide-co-glycolide) microparticles (MPs) designed to remain extracellular and release polyIC in the LN over several days. Intranodal injection of MPs increased persistence of polyIC in LNs compared to the same dose of soluble polyIC or polyIC formulated in nanoparticles, leading to increased accumulation of Toll-like receptor agonist in LN-resident antigen presenting cells and more enduring dendritic cell activation. Intralymph node injection of ovalbumin mixed with polyIC-releasing MPs enhanced the humoral response and expanded ovalbumin-specific T cells to frequencies as high as 18% among all CD8(+) cells following a single injection (8.2-fold greater than the same vaccine given i.m.), a response that could not be matched by antigen mixed with polyIC-loaded nanoparticles or a 10-fold greater dose of soluble polyIC. Thus, i.LN immunization with slow release-formulated adjuvants may be a broadly applicable strategy to enhance therapeutic or prophylactic vaccines.