IR-Live: fabrication of a low-cost plastic microfluidic device for infrared spectromicroscopy of living cells

Abstract

Water is a strong mid-infrared absorber, which has hindered the full exploitation of label-free and non-invasive infrared (IR) spectromicroscopy techniques for the study of living biological samples. To overcome this barrier, many researchers have built sophisticated fluidic chambers or microfluidic chips wherein the depth of the liquid medium in the sample compartment is limited to 10 μm or less. Here we report an innovative and simple way to fabricate plastic devices with infrared transparent view-ports enabling infrared spectromicroscopy of living biological samples; therefore the device is named "IR-Live". Advantages of this approach include lower production costs, a minimal need to access a micro-fabrication facility, and unlimited mass or waste exchange for the living samples surrounding the view-port area. We demonstrate that the low-cost IR-Live in combination with microfluidic perfusion techniques enables long term (>60 h) cell culture, which broadens the capability of IR spectromicroscopy for studying living biological samples. To illustrate this, we first applied the device to study protein and lipid polarity in migrating REF52 fibroblasts by collecting 2-dimensional spectral chemical maps at a micrometer spatial resolution. Then, we demonstrated the suitability of our approach to study dynamic cellular events by collecting a time series of spectral maps of U937 monocytes during the early stage of cell attachment to a bio-compatible surface.

Subcellular mapping of living cells via synchrotron microFTIR and ZnS hemispheres

“…The use of a thinner spacer will also reduce the contribution from the medium above the attached cell further improve the SNR of the measurement. Previous works have shown that cell remains viable for several hours when sandwiched between windows with a gap of a few μm [ 20 , 36 ] and > 60 h when subjected in a flow device [ 15 ]. Nevertheless, the current procedure enables the demonstration of the principle of subcellular imaging of living cells with the improved spatial resolution.…”

“…When measuring live cells by FTIR, three major challenges to be considered are the strong IR absorbance of water in the cell culture medium and within the cell itself, the biocompatibility of the IR substrate and the limited spatial resolution relative to visible microscopy and in terms of subcellular structures. To experimentally overcome the strong absorbance of water, a number of approaches have been derived including using the attenuated total reflection (ATR) method, which probes the attached living cell without significant contribution from the water in the culture medium [ 3 , 4 , 6 , 7 ], the use of a small path length liquid cells or microfluidic transmission cell with spacer matching to the thickness of the cell [ 12 – 15 ]. Synchrotron-based FTIR microscopy is also often used to achieve diffraction limited resolution and to enhance the signal to noise ratio [ 5 , 12 , 16 – 21 ].…”

Machine learning utilising spectral derivative data improves cellular health classification through hyperspectral infra-red spectroscopy

“…These also often require specialised cell preparations, including fixing and drying of samples, which have been shown to exhibit a loss in cellular content, effecting the resulting IR spectra and detectable contrast [13]. More recently, the work has moved towards custom imaging systems for live cell analysis, such as the use of narrow viewing windows and IR transparent housing [14]. It has been shown that in both the SWIR and MWIR, spectral regions of interest corresponding to lipids (1200, 1400, 1700, and 3333-3533 nm), collagen (1200 and 1500 nm) and other cellular constituents are detectable [12,15,16].…”

Estimating and correcting interference fringes in infrared spectra in infrared hyperspectral imaging

“…For example, microchannels with wedge shaped windows, that were originally designed for measuring absorbent liquids, 15 have been modified to provide a controlled environment for live cell measurement in transmission mode. 16,17 However, not all microscope geometries allow for such microfluidic chambers, such as high numerical aperture, small working distance objectives that require thin cross section microfluidic chambers. One such chamber consists of submicrometer thick diamond windows that are deposited on Si wafers.…”