Dynamic measurement of the height and volume of migrating cells by a novel fluorescence microscopy technique

Abstract

We propose a new technique to measure the volume of adherent migrating cells. The method is based on a negative staining where a fluorescent, non cell-permeant dye is added to the extracellular medium. The specimen is observed with a conventional fluorescence microscope in a chamber of uniform height. Given that the fluorescence signal depends on the thickness of the emitting layer, the objects excluding the fluorescent dye (i.e., cells) appear dark, and the decrease of the fluorescent signal with respect to the background is expected to give information about the height and the volume of the object. Using a glass microfabricated pattern with steps of defined heights, we show that the drop in fluorescence intensity is indeed proportional to the height of the step and obtain calibration curves relating fluorescence intensity to height. The technique, termed fluorescence displacement method, is further validated by comparing our measurements with the ones obtained by atomic force microscopy (AFM). We apply our method to measure the real-time volume dynamics of migrating fish epidermal keratocytes subjected to osmotic stress. The fluorescence displacement technique allows fast and precise monitoring of cell height and volume, thus providing a valuable tool for characterizing the three-dimensional behaviour of migrating cells.

Integrated automated nanomanipulation and real-time cellular surface imaging for mechanical properties characterization

“…These results are, however, in good agreement with the fluorescent microscopy technique and AFM results accomplished in Ref. 16 for the cells.…”

“…In Figs. 16(g) and 16(h), the microcantilever's tip is brought up a few nm to avoid the probe's tip to be in contact with the glass substrate before touching the corneal cell. The time increment to send command signals is now reduced to 1 s expecting a faster speed.…”

“…They have compared their measurements with the AFM results. 16,17 Some methods are concentrated on precisely measuring the density and spatial distribution of the keratocytes as the fibroblasts-like cells by using the ultra-high resolution optical coherence tomography (OCT). 18 An algorithm to distinguish the joint outlines of the overlapped cells is developed through the image processing algorithm and presented in Ref.…”

Effect of an osmotic stress on multicellular aggregates

“…To measure the evolution of the cellular volume in real time, we use the fluorescence exclusion principle recently re-adapted by Bottier et al [18]. Cells are cultured in a microfluidic device in Polydimethylsiloxane (PDMS), with a constant height of 20.9 lm.…”

Nanoscale colocalization of NK cell activating and inhibitory receptors controls signal integration

“…The use of a monolayer of NIH3T3 cells is a novel experimental approach to study the NK IS. NIH3T3 present a small height and thickness revealed by atomic force microscopy studies [44]. This fact makes NIH3T3 a promising cell line to use in NK IS studies.…”