Spectral analysis and sorting of microbial organisms using a spectral sorter.

This chapter discusses the problems related to the application of conventional flow cytometers to microbiology. To address some of those limitations, the concept of spectral flow cytometry is introduced and the advantages over conventional flow cytometry for bacterial sorting are presented. We demonstrate by using ThermoFisher's Bigfoot spectral sorter where the spectral signatures of different stains for staining bacteria are demonstrated with an example of performing unmixing on spectral datasets. In addition to the Bigfoot's spectral analysis, the special biosafety features of this instrument are discussed. Utilizing these biosafety features, the sorting and patterning at the single cell level is optimized using non-pathogenic bacteria. Finally, the chapter is concluded by presenting a novel, label free, non-destructive, and rapid phenotypic method called Elastic Light Scattering (ELS) technology for identification of the patterned bacterial cells based on their unique colony scatter patterns.

Flow Cytometry: The Next Revolution.

Unmasking the subtleties of the immune system requires both a comprehensive knowledge base and the ability to interrogate that system with intimate sensitivity. That task, to a considerable extent, has been handled by an iterative expansion in flow cytometry methods, both in technological capability and also in accompanying advances in informatics. As the field of fluorescence-based cytomics matured, it reached a technological barrier at around 30 parameter analyses, which stalled the field until spectral flow cytometry created a fundamental transformation that will likely lead to the potential of 100 simultaneous parameter analyses within a few years. The simultaneous advance in informatics has now become a watershed moment for the field as it competes with mature systematic approaches such as genomics and proteomics, allowing cytomics to take a seat at the multi-omics table. In addition, recent technological advances try to combine the speed of flow systems with other detection methods, in addition to fluorescence alone, which will make flow-based instruments even more indispensable in any biological laboratory. This paper outlines current approaches in cell analysis and detection methods, discusses traditional and microfluidic sorting approaches as well as next-generation instruments, and provides an early look at future opportunities that are likely to arise.

Bacteria Detection at a Single-Cell Level through a Cyanotype-Based Photochemical Reaction.

The detection of living organisms at very low concentrations is necessary for the early diagnosis of bacterial infections, but it is still challenging as there is a need for signal amplification. Cell culture, nucleic acid amplification, or nanostructure-based signal enhancement are the most common amplification methods, relying on long, tedious, complex, or expensive procedures. Here, we present a cyanotype-based photochemical amplification reaction enabling the detection of low bacterial concentrations up to a single-cell level. Photocatalysis is induced with visible light and requires bacterial metabolism of iron-based compounds to produce Prussian Blue. Bacterial activity is thus detected through the formation of an observable blue precipitate within 3 h of the reaction, which corresponds to the concentration of living organisms. The short time-to-result and simplicity of the reaction are expected to strongly impact the clinical diagnosis of infectious diseases.

High throughput viscoelastic particle focusing and separation in spiral microchannels.

Passive particle manipulation using inertial and elasto-inertial microfluidics have received substantial interest in recent years and have found various applications in high throughput particle sorting and separation. For separation applications, elasto-inertial microfluidics has thus far been applied at substantial lower flow rates as compared to inertial microfluidics. In this work, we explore viscoelastic particle focusing and separation in spiral channels at two orders of magnitude higher Reynolds numbers than previously reported. We show that the balance between dominant inertial lift force, dean drag force and elastic force enables stable 3D particle focusing at dynamically high Reynolds numbers. Using a two-turn spiral, we show that particles, initially pinched towards the inner wall using an elasticity enhancer, PEO (polyethylene oxide), as sheath migrate towards the outer wall strictly based on size and can be effectively separated with high precision. As a proof of principle for high resolution particle separation, 15 µm particles were effectively separated from 10 µm particles. A separation efficiency of 98% for the 10 µm and 97% for the 15 µm particles was achieved. Furthermore, we demonstrate sheath-less, high throughput, separation using a novel integrated two-spiral device and achieved a separation efficiency of 89% for the 10 µm and 99% for the 15 µm particles at a sample flow rate of 1 mL/min-a throughput previously only reported for inertial microfluidics. We anticipate the ability to precisely control particles in 3D at extremely high flow rates will open up several applications, including the development of ultra-high throughput microflow cytometers and high-resolution separation of rare cells for point of care diagnostics.