Synchrotron radiation FTIR imaging in minutes: a first step towards real-time cell imaging

Abstract

FTIR microscopy with a focal plane array (FPA) of detectors enables routine chemical imaging on individual cells in only a few minutes. The brilliance of synchrotron radiation (SR) IR sources may enhance the signal obtained from such small biosamples containing small amounts of organic matter. We investigated individual cells obtained from a cell culture specifically developed for transmission FTIR imaging using either a Globar or an SR source coupled to the same instrumentation. SR-IR source focussing was optimized to control the energy distribution on the FPA of detectors. Here we show that accessing the IR absorption distribution from all the organic contents of cells at 1 x 1 microm pixel resolution was possible only with high circulating current (> or = 1.2 A) illuminating a limited number of the FPA's detectors to increase the signal-to-noise ratio of IR images. Finally, a high-current SR ring is mandatory for collecting FTIR images of biosamples with a high contrast in minutes.

Methodology for FTIR Imaging of Individual Cells

“…To conclude about biocompatibility, the best substrates are Si 3 N 4 , Si, LaF 3 , and Ge. It must be stressed here that, except Ge, regularly used for reflection measurements in the mid-IR region due to its high refractive index (4.0) compared to biosamples (≈1.2-1.4), the other biocompatible substrates are rarely used by spectroscopists [11,[20][21][22][23][24][25].…”

“…To the contrary, FTIR imaging is now routinely used for analyzing biosamples, notably tissue sections [10]. It is however still in an experimental development phase for a routine use for cell imaging [1,11,12], suffering principally from the lack of spatial lateral resolution, typically a few µm, which is not sufficient for sub-cellular analyses.…”

“…In vitro imaging has been also shown to work with globar and SR sources, thus promising routine and high spectral quality results, respectively [15]. To benefit from SR source performances, a specific experimental setup was proposed, where the IR beam was tuned in such a way to maximize the photon flux homogeneity between pixels rather than an intense small photon spot [11]. This was a real advancement if compared with previous image acquisition based on single element detector, that requires long acquisition time affected also by beam stability and homogeneity of SNR [16].…”

The Future of Infrared Spectroscopy in Biosciences: In Vitro, Time-Resolved, and 3D

“…At this state of technological development, FTIR imaging was considered as cutting-edge technology for biosciences since the reduction of time for spectra acquisitions [12] allowed applications to large tissue areas and thus opened IR to the medical field [13]. The use of high-intensity (or high-photon flux) sources could be also considered as a major advantage to obtain high S/R levels with shorter acquisition durations [14][15][16][17]. Cell imaging applications were also demonstrated taking into account a few analytical requirements to fit analytical standards in biology [14,16,[18][19][20].…”

“…The use of high-intensity (or high-photon flux) sources could be also considered as a major advantage to obtain high S/R levels with shorter acquisition durations [14][15][16][17]. Cell imaging applications were also demonstrated taking into account a few analytical requirements to fit analytical standards in biology [14,16,[18][19][20]. However, in the current competition for proposing high-performance analytical means for biosciences, it seems that FTIR instrumentations suffer from a major lack of R&D efforts, and traditional manufacturers have not released major innovations for almost 20 years.…”