A novel dynamic flow system for chemical analysis of live biological cells

Related publication: Doherty J., Raoof A., Hussain A., Wolna M., Cinque G., Brown M., Gardner P. & Denbigh J. Live single cell analysis using synchrotron FTIR microspectroscopy: Development of a simple dynamic flow system for prolonged sample viability. Analyst 144, 997–1007 (2019). DOI: 10.1039/c8an01566j
 
Publication keywords: Live cells; Infrared spectroscopy; Dynamic flow; Water correction; Ovarian cancer 

Analysing cells on a cell-by-cell basis using infrared microspectroscopy can reveal important biochemical information, providing insight into diseases, and drug-cell interactions. However, for visible and infrared microscopy, the cells have to be preserved and dried; also, water is a strong infrared absorber and obscures the spectrum of the cells under investigation. Analysing dried fixed cells reduces the relevance of the results since they are not in their natural environment. 

Researchers investigated building a sample environment that would allow the cells to be kept alive in water during infrared microspectroscopy analysis. The sample chamber needed an amount of water small enough to not be a major problem, but large enough to allow the correct flow of nutrients to the cells, to keep them alive for a prolonged period of time. It also had to be temperature-controlled.

They carried out their work on the Multimode Infrared Imaging And Microspectroscopy (MIRIAM) beamline (B22), modifying a commercially-available liquid sample holder with a narrow gap for the cells to sit in, and just enough water to stay alive, without inducing mechanical stress. They also used a special hydrophobic pen to draw a channel in the chamber that allowed the nutrients to flow though the chamber and keep the cells alive. They tested the dynamic system by introducing a labelled molecule and monitoring the uptake by the cells, and also by looking at temperature-induced degradation of cells. Their tests were successful, and the dynamic cell system is now available to other infrared users of Diamond Light Source. 
 
Figure 1: Diagram of the assembly of the modified liquid sample holder. The heating jacket is shown around the sample chamber, and the inlet and outlet flow from the sample holder is indicated.
Inside the sample chamber, the 10 μm spacer, hydrophobic barriers and the flow channel are all highlighted.
Figure 1: Diagram of the assembly of the modified liquid sample holder. The heating jacket is shown around the sample chamber, and the inlet and outlet flow from the sample holder is indicated. Inside the sample chamber, the 10 μm spacer, hydrophobic barriers and the flow channel are all highlighted.
The study of biological cells using micro-Fourier Transform Infrared (micro-FTIR) spectroscopy has historically been limited to the use of fixed, dried samples. While there are clear benefits to this type of sample preparation, for example the ability to return to the same sample for repeat measurement, chemical fixatives are known to affect various cellular structures, limiting the interpretation of data obtained1.
 
Cell dehydration can affect the position, intensity, and ratio of bands across the spectrum, in particular DNA bands that become harder to distinguish from those of proteins, RNA, and carbohydrates. In contrast, studies of living cells provide biological detail that was previously lost when using fixed samples, especially when live cell analysis has been combined with the increased infrared (IR) brilliance of a synchrotron radiation (SR) source2.
 
The study of living cells, however, presents a range of challenges to bioanalysts. Live cells require an aqueous environment to remain viable for any significant length of time, and water presents two significant problems for IR spectroscopists. Firstly, the strength of the water absorption prevents sufficient IR from reaching the sample to give a good signal and consequently produce high-quality data. Secondly, the position of the O–H bending and stretching modes, at ∼1650 and 3000–3500 cm−1 respectively, obscures key biological information relating to protein and lipid bands. This makes the extraction of biochemical information difficult1.
 
In a recent publication, a collaborative team from the groups of Dr J. L. Denbigh (University of Salford), Prof P. Gardner (University of Manchester), and Dr G. Cinque (Diamond Light Source), have developed a liquid sample chamber for live cell analysis that balances the requirements of a water layer that is thin enough for the transmission of infrared radiation, but thick enough to keep the cells fully hydrated in a constant flow of nutrients, enabling them to be studied for up to 24 hours3.
 
Figure 2: Second derivative mean spectra (1500-1580 cm<sup>-1</sup> region) of SKOV3 cells, indicating
the changes around the Amide II band as a function of increasing temperature.
Figure 2: Second derivative mean spectra (1500-1580 cm-1 region) of SKOV3 cells, indicating the changes around the Amide II band as a function of increasing temperature.
The sample chamber is based on a modified commercial (Harrick) liquid sample holder, consisting of two 2 mm thick CaF2 windows separated with a 10 μm metal spacer. The spacer has been modified to allow liquid (in this case cell culture media) to flow through the chamber. A key feature of the design is the creation of a ~2 mm channel on the bottom window, using a PAP pen which deposits a hydrophobic barrier to direct the flow over the cells (Fig. 1). The cells under study are deposited on to the bottom CaF2 window, in the channel, prior to the assembly of the rest of the chamber. The sample chamber is fitted with a temperature-controlled heating jacket to maintain the cells at 37 °C, or to cool/ heat the sample as required. The cell culture media is delivered to the sample chamber using a Hamilton Syringe connected to a syringe pump.
 
In this study, it was important to demonstrate the capabilities of the sample environment, and this was achieved using two types of test IR measurement; one monitoring the heat-induced denaturing of cells, and another looking at uptake of an important dietary fatty acid implicated in cancer progression. For both studies, data were collected in transmission mode using the 36× objective/condenser optics on a (Bruker) Hyperion 3000 microscope coupled to a Vertex 80 FTIR spectrometer on B22 at Diamond. This used a liquid–nitrogen cooled mercury–cadmium–telluride high sensitivity 50 micron pitch detector4. The raw spectra were subjected to an ad hoc water correctional algorithm that enabled the single cell spectrum to be recovered from the substantial water background5.
 
Fig. 2 shows the second derivative of infrared spectra from SKOV3 ovarian cancer cells in media, being subjected to thermal stress. In particular, the collapse of Amide II-related bands at 60 °C between 1547 and ∼1520 cm−1 is a significant structural change, and consistent with published work on temperature-induced denaturing of proteins.
 
Figure 3: Mean micro-FTIR spectra of deuterated palmitic acid uptake in cells at each
incubation time.
Figure 3: Mean micro-FTIR spectra of deuterated palmitic acid uptake in cells at each incubation time.
Monitoring of the uptake of deuterated palmitic acid (D31PA) over time involved the collection of a large number of spectra (∼200) from single SKOV3 ovarian cancer cells over the course of several hours, while the sample was maintained in a healthy condition in the dynamic flow system. Fig. 3 shows the infrared spectra of the CD2/CD3 symmetric and asymmetric stretches as a function of cell exposure. As can be seen, D31PA is observed after just 15 minutes and continues to build up in the cell over a 24 hour period.
 
By demonstrating cell viability up to 24 hours, we have shown that the dynamic flow chamber is potentially suitable for a range of live cell applications of IR microspectroscopy monitoring cellular changes following exposure to a stimulus. Combined with effective water correction, and the brilliance of SR, we have acquired high-quality micro-FTIR spectra from living cells over any extended period of time. This paves the way for further dynamic flow studies of cells at the MIRIAM beamline.
 

 References:

  1. Doherty J. et al. Single-cell analysis using Fourier transform infrared microspectroscopy. Appl. Spectrosc. Rev. 52, 560–587 (2017). DOI: 10.1080/05704928.2016.1250214
  2. Diem M. et al. Monitoring the reversible B to A-like transition of DNA in eukaryotic cells using Fourier transform infrared spectroscopy. Nucleic Acids Res. 39, 5439–5448 (2011). DOI: 10.1093/nar/gkr175
  3. Doherty J. et al. Live single cell analysis using synchrotron FTIR microspectroscopy: Development of a simple dynamic flow system for prolonged sample viability. Analyst 144, 997–1007 (2019). DOI: 10.1039/c8an01566j
  4. Cinque G. et al. Multimode infrared imaging and microspectroscopy (MIRIAM) beamline at diamond. Synchrotron Radiat. News 24, 24–33 (2011). DOI: 10.1080/08940886.2011.618093
  5. Doherty J. et al. Increased optical pathlength through aqueous media for the infrared microanalysis of live cells. Anal. Bioanal. Chem. 410, 5779–5789 (2018). DOI: 10.1007/s00216-018-1188-2
Funding acknowledgement:
Diamond Light Source; University of Manchester; Kidscan Children’s Cancer Research.
 
Corresponding author:
Dr Joanna Denbigh, University of Salford, j.l.denbigh@salford.ac.uk