Scanning cytometry with a LEAP: Laser‐enabled analysis and processing of live cells in situ

Abstract

Background: Scanning cytometry now has many of the features (and power) of multiparameter flow cytometry while keeping its own advantages as an imaging technology. Modern instruments combine capabilities of scanning cytometry with the ability to manipulate cells. A new technology, called LEAPä (laser-enabled analysis and processing), offers a unique combination of capabilities in cell purification and selective macromolecule delivery (optoinjection). Methods: LEAP-mediated cell purification and optoinjection effects were assessed in model experiments using adherent and suspension cell types and cell mixtures plated and processed at different densities. Optoinjection effects were visualized by delivering fluorescent dextrans into cells. Results were analyzed using the LEAP instrument's own imaging system as well as by fluorescence and confocal microscopy. Results: Live cell samples (adherent and suspension) could be purified to 90-100% purity with 50-90% yield,

A computational platform for robotized fluorescence microscopy (I): High‐content image‐based cell‐cycle analysis

“…A valuable feature of the developed approach is, indeed, the possibility to perform multiple acquisitions on the same sample. The ability to relocate events based on a first analysis has been already validated in both slide-based LSC and high-resolution microscopy (1,3,14,(50)(51)(52)(53)(54)(55)(56). We demonstrated that, in our approach, a first acquisition could be exploited to target in-silico identified subpopulations.…”

Application of Theranostics to Measure and Treat Cell Heterogeneity in Cancer

“…Laser capture microdissection (LCM) is routinely used to retrieve groups of cells [79] or even single cells [80] from fixed and frozen tissues, and LCM instrumentation for live cells is being developed [81,82].…”

Methods of Cell Purification: A Critical Juncture for Laboratory Research and Translational Science

“…Microinjection can be accomplished using the same laser that will subsequently be used to perform ablation. It has been reported that cell purities in excess of 99% and cell yields of 90% can be obtained from a wide range of sample sizes (from 10 1 to 10 8 cells) using in situ laser purification [Koller et al, 2004;Szaniszlo et al, 2006]. It has been shown that laser microdissection can be used to preserve viability in neurons during purification, whereas more widely used techniques like FACS might damage such a fragile cell type [Hempel et al, 2007].…”

“…In laser dissection, computer software is used to guide a medical laser as it ablates nonfluorescent cells in a heterogeneous mixture [Koller et al, 2004;Szaniszlo et al, 2006;Hempel et al, 2007]. When tissue is explanted from transgenic animals harboring fluorescent reporter gene(s), cells expressing the fluorescent reporter gene can be isolated efficiently using laser dissection techniques.…”

“…Low purity Reagents can be expensive Mummery, 2003;Nemir, 2006;Yuasa, 2005;Laflamme, 2007;Ieda, 2010 Laser dissection Accurate and efficient Gentle to cells of interest Can be expensive Requires expertise Koller, 2004;Szaniszlo, 2006;Hempel, 2007 T he strengths and weaknesses of each purification method must be considered when designing experiments for cell biology and cell-based therapeutics. For example, cell sorting based on surface protein expression is more amenable to translational projects since it does not require permanent manipulation of the cell, but expression of these proteins may not be as precise as the expression of genes in the nucleus.…”