Micro fluorescence in situ hybridization (μFISH) for spatially multiplexed analysis of a cell monolayer

Abstract

We here present a micrometer-scale implementation of fluorescence in situ hybridization that we term μFISH. This μFISH implementation makes use of a non-contact scanning probe technology, namely, a microfluidic probe (MFP) that hydrodynamically shapes nanoliter volumes of liquid on a surface with micrometer resolution. By confining FISH probes at the tip of this microfabricated scanning probe, we locally exposed approximately 1000 selected MCF-7 cells of a monolayer to perform incubation of probes — the rate-limiting step in conventional FISH. This method is compatible with the standard workflow of conventional FISH, allows re-budgeting of the sample for various tests, and results in a ~ 15-fold reduction in probe consumption. The continuous flow of probes and shaping liquid on these selected cells resulted in a 120-fold reduction of the hybridization time compared with the standard protocol (3 min vs. 6 h) and efficient rinsing, thereby shortening the total FISH assay time for centromeric probes. We further demonstrated spatially multiplexed μFISH, enabling the use of spectrally equivalent probes for detailed and real-time analysis of a cell monolayer, which paves the way towards rapid and automated multiplexed FISH on standard cytological supports.Electronic supplementary materialThe online version of this article (doi:10.1007/s10544-016-0064-0) contains supplementary material, which is available to authorized users.

Rapid micro fluorescence in situ hybridization in tissue sections

“…We aimed to enumerate the copy numbers of chromosome 17 in FFPE tissue sections, and for this, we adapted the MFP-based FISH method 29 . In contrast to the “fresh” cells we used for our previous study, FFPE samples undergo a series of preparation steps affecting the sample integrity 32–38 .…”

“…In contrast to the traditional microfluidic devices, the MFP is a scanning, non-contact technology, which allows reagents to be shaped in the “open space.” This allows microfluidic-based FISH to be performed on cells in selected regions of the tissue section without physical contact between the device and the cytological sample. In previous work, 29 we introduced the concept of microfluidic-based FISH (μFISH) with an MFP where we demonstrated spatially multiplex micro-scale FISH on cells. To demonstrate the diagnostic use of μFISH with an MFP, here we present methods and protocols for rapid HER2 status assessment of FFPE cell blocks and tissue sections while also using small volumes of FISH probes.…”

FISH and chips: a review of microfluidic platforms for FISH analysis

“…The device can essentially scan over a cell layer or tissue slice, measuring in zones of interest in a fast manner. The probe was first applied to FISH on breast cancer cell lines with about 1000 cells interrogated at a time [75]. Signals were obtained with hybridization times as short as 3 min with 0.6 µL of probe, followed by a 2-min wash, faster than any other method reported for FISH.…”

“…Reproduced with permission from Ref. [77] investigation varied, some using only 50-100 cells [41,52,58] or on the order of 1000 cells [57,75], others at tens of thousands [56,59].…”

Microfluidics-assisted fluorescence in situ hybridization for advantageous human epidermal growth factor receptor 2 assessment in breast cancer

“…8 Recently, for implementing rapid, economical, and multiplexed FISH for cell analysis, Huber et al proposed scanning a vertically oriented capillary that creates a hydrodynamic flow of FISH probe on a small area (~0.096 mm 2 ) of the cell slide surface. 9 However, this technique can work only with fast hybridization centromere probes and has a small footprint due to the limited size of the capillary.…”