Light Sheet Illumination on a Chip

Enabling Diagnostic Measurements of Vesicles in Biofluids

  • Light Sheet Illumination on a Chip - Enabling Diagnostic Measurements of Vesicles in BiofluidsLight Sheet Illumination on a Chip - Enabling Diagnostic Measurements of Vesicles in Biofluids
  • Light Sheet Illumination on a Chip - Enabling Diagnostic Measurements of Vesicles in Biofluids
  • Fig. 1: Photographs of microfluidic chips with integrated planar waveguide for light sheet illumination in a microchannel. (A) Image of a silicon wafer with 20 planar waveguides made from SU-8 after photolithography. (B) Light microscopy image of a microfluidic chip showing the 100 µm wide microchannel. (C) Image of a microfluidic chip with a glass substrate sealed with PDMS. (D) Image of a microfluidic chip with a silicon substrate covered with a microscope cover slip.
  • Fig. 2: Experimental characterization of the on-chip light sheet using a dispersion of 0.2 µm fluorescent polystyrene nanospheres. (A) Average nanosphere intensity along the optical axis of the objective lens for light sheet (red points) and epi-fluorescence (blue points) illumination. (B) On the right, the nanosphere contrast values in function of the concentration of Cy5 dye added to the dispersion, using light sheet (red crosses) and epi-fluorescence (blue crosses) illumination. On the left, example images obtained with a silicon chip. The error bars represent standard deviations.
  • Fig. 3: On-chip fSPT concentration and size measurements of cell-derived MVs. (A) fSPT size distribution and number concentration of MVs in cell culture medium, using light sheet (blue line) and epi-fluorescence (red line) illumination. (B) fSPT size distribution and number concentration of MVs in human tumors interstital fluid. Meaningful measurements could only be obtained with light sheet illumination (blue line).

Cell-derived membrane vesicles (MVs) that are released in body fluids, like blood or saliva, are potential biomarkers for diseases, such as cancer. Techniques capable of measuring MV properties, like size and concentration, directly in biofluids are thus needed. We developed a microfluidic chip with integrated light sheet illumination, and used it to perform fluorescence Single Particle Tracking (fSPT) size and concentration measurements of MVs in human tumor interstitial fluid.

Membrane Vesicle Based Diagnostics

The relation between disease progression and submicron cell-derived MVs that are released in body fluids is a topic that receives a lot of attention, because certain MV properties, such as size and concentration, are believed to have diagnostic and prognostic value [1,2]. Due to a lack of standardized isolation and purification protocols and in order to avoid manipulation artefacts, techniques for MV characterization directly in biofluids are urgently needed [1,3]. fSPT is such a technique, as it allows size distribution and number concentration measurements of fluorescently labelled nanoparticles in biofluids [4,5].

Being based on epi-fluorescence illumination, a limitation of fSPT is the reduced contrast due to fluorescence coming from out of focus particles or unbound fluorescent dye. Especially the latter aspect can be problematic for MV characterization, as the concentration in a patient sample is unknown a priori, so that a surplus of fluorescent labels has to be added in order to be certain that all MVs will be stained. Light sheet illumination offers superior contrast, usually by employing two objective lenses positioned perpendicular to one another, one for imaging and the other for illumination. This is compatible with fSPT if a sample holder is used that has two separate high-quality optical windows for imaging and illumination [6]. However, as such sample holders are expensive and difficult to manufacture [7], they are not suitable for high-throughput diagnostic assays for which disposable sample holders are preferred to avoid cleaning procedures and sample contamination. We therefore created a mass-producible microfluidic chip with integrated light sheet illumination [8].

Microfluidic Chip with Integrated Light Sheet Illumination

The chip consists of a glass or silicon substrate with a planar waveguide on top [8].

The ~5 µm thick middle core layer of the waveguide consists of SU-8, while the ~25 µm thick bottom and top cladding layers are made of SU-8 mixed with the epoxy resin D.E.R. 353, in order to lower the refractive index [9]. A 100 µm wide microchannel containing in- and outlet reservoirs is created in the SU-8 by photolithography (fig. 1A-B). The entire process is carried out on a wafer, obtaining 20 chips in parallel. Chips based on the glass substrate are sealed with PDMS (fig. 1C) and imaging of the sample is performed through the substrate. Chips based on the silicon substrate are covered with a microscopy cover slip through which the sample can be imaged (fig. 1D). The chips are mounted on a microscope for image acquisition and laser light is coupled into the waveguide using an optical fiber attached to a high precision alignment stage.

Characterization of the Light Sheet

The light sheet intensity profile along the optical axis was determined by recording a z-stack through the microchannel filled with a dispersion of 0.2 µm fluorescent polystyrene nanospheres, and calculating the average intensity of the nanospheres visible in each frame [8]. For both chip types, an average thickness of ~9 µm FWHM over the microchannel width was found (fig. 2A). The smaller intensity peaks in the intensity profiles indicate that the waveguide is likely multimodal.

The contrast with which the nanospheres were visible was also quantified for both chip types [8]. To simulate different background intensities coming from unbound dye, various amounts of Cy5 dye were added to the nanosphere dispersion. Compared to epi-fluorescence illumination, a contrast improvement of 1.5-2.4× was obtained in the glass chip, and 1.9-6.4× in the silicon chip (fig. 2B). This improvement approaches the performance of light sheet illumination created with a high quality objective lens [6]. The better relative increase with the silicon chip is due to the light intensity almost going to zero at the edges of the light sheet (fig. 2A). However, the lower absolute contrast obtained with the silicon chip is due to the higher reflectivity of the substrate which produces a higher background intensity.

Size and Concentration Measurements of Membrane Vesicles

On-chip fSPT size and concentration measurements were performed on cell-derived MVs in the cell culture medium of human breast cancer cells [8]. An excess of fluorescently labelled Annexin V was used to label the MVs [10], followed by fSPT measurements without additional purification to remove the unbound label. Using sheet illumination, the majority of the MVs were found to be in the 50-700 nm size range, and the overall number concentration was 8.4 × 108 # per ml (fig. 3A). Using epi-fluorescence, a 4× lower concentration was found with a size distribution shifted towards larger values.

To demonstrate the potential of the chip as a diagnostic tool, on-chip fSPT measurements were performed on cell-derived MVs secreted in the interstitial fluid harvested from human breast cancer specimens [8]. Again an excess of fluorescently labelled Annexin V was added and subsequent fSPT measurements were performed without extra purification steps. A broad distribution of MV sizes was found with the majority situated in the 90-900 nm range, and the overall number concentration was 4.1 × 108 # per ml (fig. 3B). Using epi-fluorescence illumination, the background fluorescence was so high that only few MVs were visible and no meaningful size distribution or concentration could be determined.

Conclusion

We realized for the first time light sheet illumination in a mass-producible microfluidic chip, by coupling laser light into a planar waveguide in which a microchannel is provided that contains the sample. The contrast with which fluorescent nanospheres can be visualized was shown to improve substantially compared to classic epi-fluorescence illumination. fSPT size and concentration measurements were performed on fluorescently labeled MVs in cell culture medium and in interstitial fluid collected from human breast tumors. Because of the high background coming from unbound fluorescent labels, the on-chip light sheet illumination was found to be essential for correct MV characterization. The chip has the potential to be used as a diagnostic tool that combines low cost, ease of use, and sensitivity.

Acknowledgements
Financial support of the Agency for Innovation by Science and Technology in Belgium, the Ghent University Special Research Fund, and the Research Foundation - Flanders in Belgium is acknowledged.

Reference [8] - Reproduced by permission of The Royal Society of Chemistry.

References
[1] C. D'Souza and J. W. Clancy: Gene Dev. 26, 1287-1299 (2012)
[2] J. Skog et al.: Nat. Cell Biol. 10, 1470-1476 (2008)
[3] E. van der Pol et al.: Pharmacol. Rev. 64, 676-705 (2012)
[4] K. Braeckmans et al.: Nano Lett. 10, 4435-4442 (2010)
[5] M. Röding et al.: Phys. Rev. E 84, 031920 (2011)
[6] J. G. Ritter et al.: Opt. Express 16, 7142-7160 (2008)
[7] F. Cutrale and E. Gratton: Microsc. Res. Tech. 75, 1461-1466 (2012)
[8] H. Deschout et al.: Nanoscale 6, 1741-1747 (2014)
[9] J. M. Ruano-Lopez et al.: Sens. Actuators B 114, 542-551 (2006)
[10] C. Thery et al.: Nat. Rev. Immunol. 9, 581-593 (2009) 

Author
Dr. Hendrik Deschout

Postdoctoral researcher
EPFL
Laboratory of Nanoscale Biology
Lausanne, Switzerland 

Contact

EPFL Ecole Polytechnique Federale de Lausanne

1015 Lausanne
Switzerland
Phone: +41 21 69 31162

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