In-vitro SNFUH imaging of acoustically active liposomes

 

The efficacy of SNFUH for in-vitro biological imaging and indentifying the buried structures is demonstrated on acoustically active liposomes (AAL) which contains small amount of air. AAL have oil layer in which therapeutics can be dissolved, the lipid shell and the air-bubbles interior. These liposomes have potential to carry drugs, ease of conjugation with antibodies, peptides and their acoustic activity could enable them to respond to ultrasound stimulation by releasing their contents. Figure XX depicts a high resolution and remarkably high contrast from AAL, and shows embedded air-bubbles embedded in them.

 

Figure XXB and XXC show AFM topography images and SNFUH phase images of cells, respectively. As expected, AFM topography image shows the typical surface morphology of uniform distribution of liposome’s across the surface with cell size of approximately 500 nm, while SNFUH phase shows remarkably high contrast from the air-bubbles residing well inside the cells. In addition to several other features reminiscent of cell membrane proteins and cellular content are clearly evident. The morphology, spatial scale and distribution of air-bubbles are consistent with prior account of such defects. The buried scattering features (air-bubbles) perturb the specimen acoustic wave, resulting local change in the phase and amplitude of the standing acoustic wave, which was detected by the SPM cantilever “antenna”.

Direct and real-space in-vitro imaging of the presence of air-bubbles and proteins inside liposome’s without any labels or sectioning of cells and shows potential of SNFUH in imaging biological samples under under physiologically viable conditions.

 

In-vitro SNFUH imaging of Malaria Parasites

 

The efficacy of SNFUH in real-time in-vitro imaging of embedded or buried substructures in biology is demonstrated in Fig. XX. It depicts high resolution and remarkably high contrast arising from malaria parasites inside infected red blood cells (RBCs). An early stage direct and real-space in-vitro imaging of the presence of parasites inside RBCs without any labels or sectioning of cells, and under physiologically viable conditions is demonstrated. Plasmodium falciparum strain 3D7 was cultured in-vitro. Parasites were synchronized to within 4 hours using a combination of Percoll purification and sorbitol treatments, cultured to 10% parasitemia, and harvested at the indicated times.

 

SNFUH imaging was performed using the near- contact mode method for imaging soft structures. Fig. XXA shows AFM topography image of the infected RBCs whereas Fig. XXB shows corresponding SNFUH phase image.  Unlike AFM topography image SNFUH phase image shows remarkably high contrast from the parasite residing well inside the RBC. In addition to several other features reminiscent of membrane proteins and sub- cellular contents, multiple parasites are clearly evident. Fig. XXC and D show SNFUH is sensitive to early stage of parasite infection in RBC which is difficult to validate even with commonly used non- invasive techniques like fluorescent tagging.

 

Nanomechanical Hologography for Imaging Nanoparticles in Cells

 

Imaging nanoparticles inside the cells, as demonstrated herein, is an interesting from the view point of studying nanoparticle toxicology studies, and for pharmacokinetics. 

Figure XX shows the SNFUH imaging of cells obtained from mice exposed to single-walled carbon nanohorns (SWCNH)3. Optical images of macrophages collected from both untreated (control) and SWCNH-treated mice sacrificed 7 days after the exposure reveal the presence of variously sized carbonaceous aggregates within the cytoplasm of macrophages from SWCNH- exposed mice (Fig. 4(e)) that are not present in controls (Fig. 4(a)) or in erythrocytes. The region occupied by the nucleus is readily detected and the characteristic macrophage morphology is present in both samples (Fig. 4(b, f) ). The topographic image of an alveolar macrophage from an SWCNH exposed mouse reveals nanoparticles on the surface of the cell (Fig. 4(g)). A striking visualization of the nanohorns was obtained from the SNFUH image of the same macrophage (Fig. 4(h)), revealing several SWCNHs within the cell (marked with arrows in Fig. 4(h)), which are not visible in Fig. 4(d, g). The sizes of these particles are statistically consistent with the size distribution (70–110 nm) established from analyzing several AFM images of the SWCNH solution, indicating that nanoparticles, rather than larger aggregates, were taken up by the macrophages. The contrast measures the phase of the local tip–cell surface coupling, and originates from the difference in elasticity and density between the SWCNH and the cell.