These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

Ultrafast real-time optical imaging is an indispensable tool for studying dynamical events such as shock waves, chemical dynamics in living cells, neural activity, laser surgery and microfluidics. However, conventional CCDs (charge-coupled devices) and their complementary metal-oxide-semiconductor (CMOS) counterparts are incapable of capturing fast dynamical processes with high sensitivity and resolution. This is due in part to a technological limitation-it takes time to read out the data from sensor arrays. Also, there is the fundamental compromise between sensitivity and frame rate; at high frame rates, fewer photons are collected during each frame-a problem that affects nearly all optical imaging systems. Here we report an imaging method that overcomes these limitations and offers frame rates that are at least 1,000 times faster than those of conventional CCDs. Our technique maps a two-dimensional (2D) image into a serial time-domain data stream and simultaneously amplifies the image in the optical domain. We capture an entire 2D image using a single-pixel photodetector and achieve a net image amplification of 25 dB (a factor of 316). This overcomes the compromise between sensitivity and frame rate without resorting to cooling and high-intensity illumination. As a proof of concept, we perform continuous real-time imaging at a frame speed of 163 ns (a frame rate of 6.1 MHz) and a shutter speed of 440 ps. We also demonstrate real-time imaging of microfluidic flow and phase-explosion effects that occur during laser ablation.

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A fundamental challenge of biology is to understand the vast heterogeneity of cells, particularly how cellular composition, structure, and morphology are linked to cellular physiology. Unfortunately, conventional technologies are limited in uncovering these relations. We present a machine-intelligence technology based on a radically different architecture that realizes real-time image-based intelligent cell sorting at an unprecedented rate. This technology, which we refer to as intelligent image-activated cell sorting, integrates high-throughput cell microscopy, focusing, and sorting on a hybrid software-hardware data-management infrastructure, enabling real-time automated operation for data acquisition, data processing, decision-making, and actuation. We use it to demonstrate real-time sorting of microalgal and blood cells based on intracellular protein localization and cell-cell interaction from large heterogeneous populations for studying photosynthesis and atherothrombosis, respectively. The technology is highly versatile and expected to enable machine-based scientific discovery in biological, pharmaceutical, and medical sciences.

The quantum nature of the electromagnetic field imposes a fundamental limit on the sensitivity of optical precision measurements such as spectroscopy, microscopy, and interferometry [1]. The so-called quantum limit is set by the zero-point fluctuations of the electromagnetic field, which constrain the precision with which optical signals can be measured [2,3,4]. In the world of precision measurement, laser-interferometric gravitational wave (GW) detectors [4,5, 7] are the most sensitive position meters ever operated, capable of measuring distance changes on the order of 10 −18 m RMS over kilometer separations caused by GWs from astronomical sources [8]. The sensitivity of currently operational and future GW detectors is limited by quantum optical noise [7]. Here we demonstrate a 44% improvement in displacement sensitivity of a prototype GW detector with suspended quasi-free mirrors at frequencies where the sensitivity is shot-noise-limited, by injection of a squeezed state of light [1,2,3]. This demonstration is a critical step toward implementation of squeezing-enhancement in large-scale GW detectors.

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Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system’s utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million.

We report on an all-sky search with the LIGO detectors for periodic gravitational waves in the frequency range 50 -1000 Hz and with the frequency's time derivative in the range ÿ1 10 ÿ8 Hz s ÿ1 to zero. Data from the fourth LIGO science run (S4) have been used in this search. Three different semicoherent methods of transforming and summing strain power from short Fourier transforms (SFTs) of the calibrated data have been used. The first, known as StackSlide, averages normalized power from each SFT. A ''weighted Hough'' scheme is also developed and used, which also allows for a multiinterferometer search. The third method, known as PowerFlux, is a variant of the StackSlide method in which the power is weighted before summing. In both the weighted Hough and PowerFlux methods, the weights are chosen according to the noise and detector antenna-pattern to maximize the signal-to-noise ratio. The respective advantages and disadvantages of these methods are discussed. Observing no evidence of periodic gravitational radiation, we report upper limits; we interpret these as limits on this radiation from isolated rotating neutron stars. The best population-based upper limit with 95% confidence on the gravitational-wave strain amplitude, found for simulated sources distributed isotropically across the sky and with isotropically distributed spin axes, is 4:28 10 ÿ24 (near 140 Hz). Strict upper limits are also obtained for small patches on the sky for best-case and worst-case inclinations of the spin axes.