Partitioning and subsampling statistics in compartment-based quantification methods.

The precision of compartment-based quantification methods is subject to multiple effects, of which partitioning and subsampling play a major role. Partitioning is the process of aliquoting the sample liquid and consequently the contained target molecules, whereas subsampling denotes the fact that usually only a portion of a sample is analyzed. In this work, we present a detailed statistical description comprising the effects of partitioning and subsampling on the relative uncertainty of the test result. We show that the state-of-the-art binomial model does not provide accurate results for the level of subsampling present when analyzing the nucleic acid content of single specific cells. Hence, in this work we address partitioning and subsampling effects separately and subsequently combine them to derive the relative uncertainty of a test system and compare it for single cell content analysis and body fluid analysis. In point-of-care test systems the area for partitioning and detection is usually limited, which means that a trade-off between the number of partitions (related to a partitioning uncertainty) and the amount of analyzed volume (related to a subsampling uncertainty) might be inevitable. In case of low target concentration, the subsampling uncertainty is dominant whereas for high target concentration, the partitioning uncertainty increases, and a larger number of partitions is beneficial to minimize the combined uncertainty. We show, that by minimizing the subsampling uncertainty in the test system, the quantification uncertainty of low target concentrations in single cell content analysis is much smaller than in body fluid analysis. In summary, the work provides the methodological basis for a profound statistical evaluation of partitioning and subsampling effects in compartment-based quantification methods and paves the way towards an improved design of future digital quantification devices for highly accurate molecular diagnostic analysis at the point-of-care.

Fusing MEMS technology with lab-on-chip: nanoliter-scale silicon microcavity arrays for digital DNA quantification and multiplex testing.

We report on the development of a microfluidic multiplexing technology for highly parallelized sample analysis via quantitative polymerase chain reaction (PCR) in an array of 96 nanoliter-scale microcavities made from silicon. This PCR array technology features fully automatable aliquoting microfluidics, a robust sample compartmentalization up to temperatures of 95 °C, and an application-specific prestorage of reagents within the 25 nl microcavities. The here presented hybrid silicon-polymer microfluidic chip allows both a rapid thermal cycling of the liquid compartments and a real-time fluorescence read-out for a tracking of the individual amplification reactions taking place inside the microcavities. We demonstrate that the technology provides very low reagent carryover of prestored reagents < 6 × 10-2 and a cross talk rate < 1 × 10-3 per PCR cycle, which facilitate a multi-targeted sample analysis via geometric multiplexing. Furthermore, we apply this PCR array technology to introduce a novel digital PCR-based DNA quantification method: by taking the assay-specific amplification characteristics like the limit of detection into account, the method allows for an absolute gene target quantification by means of a statistical analysis of the amplification results.

Imaging the Nonlinear Plasmoemission Dynamics of Electrons from Strong Plasmonic Fields.

We use subcycle time-resolved photoemission microscopy to unambiguously distinguish optically triggered electron emission (photoemission) from effects caused purely by the plasmonic field (termed "plasmoemission"). We find from time-resolved imaging that nonlinear plasmoemission is dominated by the transverse plasmon field component by utilizing a transient standing wave from two counter-propagating plasmon pulses of opposite transverse spin. From plasmonic foci on flat metal surfaces, we observe highly nonlinear plasmoemission up to the fifth power of intensity and quantized energy transfer, which reflects the quantum-mechanical nature of surface plasmons. Our work constitutes the basis for novel plasmonic devices such as nanometer-confined ultrafast electron sources as well as applications in time-resolved electron microscopy.

Short-range surface plasmonics: Localized electron emission dynamics from a 60-nm spot on an atomically flat single-crystalline gold surface.

We experimentally and theoretically visualize the propagation of short-range surface plasmon polaritons using atomically flat single-crystalline gold platelets on silicon substrates. We study their excitation and subfemtosecond dynamics via normal-incidence two-photon photoemission electron microscopy. By milling a plasmonic disk and grating structure into a single-crystalline gold platelet, we observe nanofocusing of the short-range surface plasmon polariton. Localized two-photon ultrafast electron emission from a spot with a smallest dimension of 60 nm is observed. Our novel approach opens the door toward reproducible plasmonic nanofocusing devices, which do not degrade upon high light intensity or heating due to the atomically flat surface without any tips, protrusions, or holes. Our nanofoci could also be used as local emitters for ultrafast electron bunches in time-resolved electron microscopes.