Label free imaging and deep tracking of single biological nanoparticles in free solution by reflection enhanced dark field scattering microscopy

Label-free single-particle imaging approach for ultra-rapid detection of pathogenic bacteria in clinical samples

“…3.5 × 10 −15 M (2.1 × 10 6 counts/mL) without convection, which is much better than the SPRM of 1.2 × 10 −13 M (7.2 × 10 7 counts/mL) at the same field of view for 5-min counting of particles. We attribute this to the significantly different depth of field ( 24 ). With the increasing flow rate, as well as the screening time, the sensitivity can be further improved as shown in Fig.…”

“…1 A demonstrates the detection principle of bacteria by a single-particle imaging approach. The sample is placed on a REDFSM with a long focus objective to enhance the depth of field ( 24 ), so that more particles can be observed in free solution. Compared with other label-free single-particle imaging methods, such as SPRM ( 29 ) or iSCAT ( 21 ), which require the binding (or approaching within a few hundred nanometers) of particles to the solid–liquid interface, the direct imaging of particles in free solution with large depth of field by dark-field scattering microscopy is much more efficient than the above interfacial imaging approaches.…”

“…100 × 100 μm) is quite a low probability event at low concentration. A polished Si chip is used to enhance the sensitivity and the depth of field by a reflection-enhanced mode as shown in our recent work ( 24 ). As the scattering intensity depends on the scattering cross-section of the particle, the fluctuation of scattering intensity in free solution reflects the morphological heterogeneity of the particle as indicated in Fig.…”

“…Besides morphological identification, the size of particles is another parameter that can be used for further GBS detection. However, particle size can be directly determined by Brownian motion in free solution based on the Stokes–Einstein equation ( 24 ). The manipulation of convection for particle screening makes the size determination by pure Brownian motion difficult.…”

“…In response to these challenges, we developed reflection-enhanced dark-field scattering microscopy (REDFSM) ( 24 ) that is able to directly identify single bacteria in free solution in an incubation-free manner without using any expensive biological reagents. By tracking the scattering intensity variation of single particles, REDFSM can directly identify the morphological heterogeneity of single particles with sizes smaller than the diffraction limit, facilitating the direct morphological identification of bacteria in a complex matrix in a label-free manner.…”