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Integrated Nanophotonic Excitation and Detection of Fluorescent Microparticles Sarp Kerman, Dries Vercruysse, Tom Claes, Andim Stassen, MD. Mahmud-Ul-Hasan, Pieter Neutens, Vignesh Mukund, Niels Verellen, Xavier Rottenberg, Liesbet Lagae, and Pol Van Dorpe ACS Photonics, Just Accepted Manuscript • DOI: 10.1021/acsphotonics.7b00171 • Publication Date (Web): 28 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017

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Integrated Nanophotonic Excitation and Detection of Fluorescent Microparticles

Sarp Kerman1,2, Dries Vercruysse1, Tom Claes1, Andim Stassen1, 2, Md. Mahmud ul Hasan1,2, Pieter Neutens1, Vignesh Mukund1,2, Niels Verellen1,2, Xavier Rottenberg1, Liesbet Lagae1,2, Pol Van Dorpe1,2

1

2

imec, Kapeldreef 75, Leuven, B-3001, Belgium

KU Leuven, Department of Physics and Astronomy, Celestijnenlaan 200D, B-3001 Leuven, Belgium

KEYWORDS: Integrated photonics, Silicon Nitride, Fluorescence, Focusing, Grating, Diffraction, On-chip, Detection, Lab-on-Chip, Point-of-Care, Cytometry

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ABSTRACT: Biological micro-entities, such as cells and bacteria, are often detected and identified by bulky optical setups such as fluorescent microscopes and flow cytometers. Integrated photonics provides the prospect of dramatically miniaturizing these setups, enabling highly parallelized on-chip detection. In this work we designed, fabricated and characterized a SiN photonic circuit for fluorescence detection of micro-entities. A tailored diffraction grating excites fluorescent particles, whose emitted light is collected in a single mode waveguide by a second neighboring diffraction grating. Both fluorescent polystyrene microparticles and labeled peripheral blood mononuclear cells were detected in a microfluidic channel integrated on top of the waveguide circuit. We quantified both numerically and experimentally the efficiency of this grating system and obtained a good agreement. The presented on-chip fluorescent detection structure and the obtained results will strongly contribute to the development of on-chip cytometry and spectroscopy applications.


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Integrated photonics has contributed to the miniaturization of conventional optical detection devices that use detection schemes based on scattering, optical trapping, fluorescence and Raman spectroscopy1–20. For the detection of moving fluorescent particles, lab-on-chip systems have mainly relied on optical fibers and large on-chip polymer waveguides 1,3,5,17-20. Optical fibers are nevertheless hard to scale up in fabrication and often require careful manual alignment. For such applications, planar waveguide technologies are therefore attractive for their robustness and ease of parallel fabrication. Previously, glass and polymer waveguides have been used in lab-on-chip devices5,18–20. However, their relatively low refractive index contrast has not allowed large pattern densities. Smaller and denser photonic structures can be achieved using higher index materials such as Si or SiN. In contrast to Si, SiN is a transparent material in the visible spectral range with a relatively high refractive index (~2). Previously, both the deposition and patterning of SiN for integrated photonics applications at visible wavelengths were optimized21. This has paved the way to miniaturized biosensors based on evanescent excitation and collection of fluorescence and Raman spectroscopy4. Nevertheless, the use of the evanescent field limits the volume of the detection region, therefore, the range of possible objects. Addressing objects located at a distance more than ~100 nm away from the waveguides requires a component that can efficiently couple the light out of the waveguide into the far-field region and collect the signal back into the waveguide structures. Usually this task in conventional optical setups is taken up by lenses or microscope objectives. They can focus the excitation light into a diffraction limited spot, and collect the optical signal (i.e. fluorescence) from the same focal point. This functionality can also be integrated on chip by means of micro-lenses or

diffractive surfaces

22–25

.

A similar concept based on tailored


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diffraction gratings can be exploited in integrated photonics applications. In this case, the light is carried using single mode waveguides and coupled out to free space through grating couplers, which diffract the light out of plane. Usually these grating couplers are designed & optimized for interfacing with fibers. In a similar way, the diffraction can be tailored to result in a focused beam, which leads to the name “Focusing grating couplers (FGCs)”. FGCs are structures that diffract the light carried by a waveguide and focus at a designated location above the sample surface26–31. This makes them ideally suited for an efficient excitation of flowing fluorescent micro-particles in a microfluidic channel integrated on top of the waveguide circuit. FGCs are not only capable of focusing the light above the sample surface but, in a similar way as a conventional lens, they can also be used for collecting optical signals (i.e. fluorescence) from a localized region. Using FGCs, we aim to pave the path to a compact photonic device for the detection and identification of bio-entities without a need of a microscope. In this report, we demonstrate onchip fluorescent microparticle excitation and detection using FGCs. A waveguide fed FGC focuses the light out-of-plane, excites the fluorescent micro particles and three FGCs collect the fluorescence into a set of single mode waveguides as illustrated in Fig. 1 a. This structure achieved a completely on-chip excitation and collection of the fluorescence of 1 µm and 15 µm polystyrene particles, and labeled peripheral blood mononuclear cells (PBMCs). The focusing properties of this structure are characterized and the efficiency of the fluorescence excitation and collection for varying size of particles are analyzed by Finite-Difference Time-Domain (FDTD) simulations and experiments.


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Focusing Grating Couplers for Fluorescence Excitation and Detection

Fig. 1a schematically shows the FGCs used for excitation and detection of micro-particles. As illustrated in the left panel, a FGC fed by a single mode waveguide couples the excitation light (green) into a spot 11 µm from the sample surface. When a fluorescent particle passes through the spot, it emits fluorescent light (red) in all directions. Part of this fluorescent light falls on a second grating, i.e. the collection FGC, that couples the light into a single mode waveguide. The collected light can then be routed to a detector on- or off-chip. In our experimental system, the fluorescent light is coupled out of the collection waveguide with linear diffraction gratings positioned only 100 µm away from the collection FGCs (Figure 1c). This allows us to image the fluorescent particles and the out-couplers of the collection waveguides in the same field of view of the microscope. To maximize the light collection, we have put multiple collection gratings around the excitation FGC. A first FGC is located opposite to the excitation FGC. Two additional side collection FGCs were positioned perpendicular to the excitation FGC. The collection FGCs were designed to focus near the focal spot of the excitation FGC for a center wavelength close to the fluorescence emission peak. The collected signal is diffracted out by the linear output couplers into a microscope objective. This entire entity, consisting of the excitation and collection FGCs combined with the linear diffraction gratings, is referred as the “camera FGC structure” in this article. In addition to this geometry, we also defined a different structure where the collection waveguide was routed further away, and interfaced with a photomultiplier tube (PMT) via a multimode optical fiber (see Fig. S-2). This structure was used to demonstrate the fluorescent detection of micro-particles without a need for a microscope.


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Fig. 1b depicts the experimental setup consisting of a microfluidic system and a microscope that images the ‘camera FGC structure’. To assure that the particles flow through the excitation light spot, the sample flow is laterally focused in the microfluidic channel by sheet flows from two additional inlets. The microfluidic channels are defined in photo-patternable adhesive (PA) photoresist32 on top of the SiN chip (Supporting Information S3). The design of the FGCs was based on engineering the phase of the scattered light at each grating line to focus at an out of plane position27 (Supporting Information S1). All gratings were designed in 220 nm thick SiN and optimized to couple to a 400 nm wide single mode waveguide for TE polarization. Fig. 1c shows an image of the ‘camera FGC structure’ consisting of: an excitation FGC, FGCE; an in-line collection FGC, FGCCM; and two side collection FGCs, FGCCL and FGCCR. The origin of a FGC is defined as the junction point of the single mode input or output waveguides and the slab waveguide on the SiN surface as illustrated on Fig. 1c by the green dots. All FGCs were designed to focus 11 µm above the sample surface and 20 µm away from their respective origins. FGCE and FGCCM were designed to share a common focal point as shown by the yellow dot in Fig. 1c. The side FGCs were identical with the in-line collection FGC (FGCCM). They were anticipated to focus close to the excitation focus which resulted in an overlap of the side collection FGCs with the excitation grating. Therefore, the side collection FGCs were intentionally shifted 10 µm further in a lateral direction to avoid any overlap, resulting in expected focal positions that are 10 µm away from the common focus as shown by the orange dots. Therefore, their detection regions partially overlap with the excitation volume and is separated from each other and from the one of the in-line collection FGC. This separation decreases the efficiency of the side collection FGCs.


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Figure 1. FGC structure for fluorescence detection. (a) The ‘camera FGC structure’ when a fluorescent particle is not in the focus (left) and when it is in the focus (right). The focused beam (green) excites the fluorescence (red), which is collected by the FGCs and diffracted to the microscope by the linear grating couplers. (b) Photonic chip with integrated microfluidic channels for particle flow and the experimental setup of the fluorescence microscope. A microscope was used to image the particles and the output couplers of the collection FGCs. (c) FGC structure with multiple collection spots where the focal point of the excitation and in-line collection FGC were coinciding (yellow dot) and the focal point of the side collection FGCs (orange dots) were 10 µm away. The fluorescence was guided to the linear output couplers. The origins of the FGCs, where the single mode waveguides merge with the slab waveguide (the FGCs are etched on), are indicated by the green dots.


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FDTD Simulations of the FGC Structure

3D finite-difference time-domain (FDTD) simulations were performed to study the excitation and collection characteristics of the waveguide fed FGCs. We mapped output electric field intensity profile, |E|2, of both the excitation FGC and the collection FGCs in three dimensions for their respective designed wavelengths. The use of the output intensity profile of the collection FGCs is representative as well for the collection profile due to the reciprocity of out-coupling and in-coupling. The region where fluorescence can be efficiently excited and collected is subsequently obtained by a multiplication of the excitation and collection profiles. Fig. 2a shows the simulated intensity profile of the excitation beam. The profile is simulated by launching a fundamental TE mode in the single mode waveguide that feeds the FGC. At the focal point, we observe that the beam width is 1.3 µm in the longitudinal direction (x axis), while the Rayleigh length is 5.7 µm. The power in the focal point is 30.6% of the power injected into the single mode waveguide according to the FDTD simulations. Compared to the design, the excitation focal position was slightly shifted in the -x and +z directions (~2 µm), which could be attributed to a slight difference in the design refractive index (nSiN = 2) and the simulated SiN refractive index (nSiN = 1.89). The simulation was set according to the latest measurements for the SiN refractive index of the fabricated sample in order to make the FDTD simulations comparable with the experimental results. In the FDTD simulations, we also calculated the collection efficiency of a FGC. We define the collection ( ( , , ) =

efficiency

of

( , , ) ( , , ))


a as

FGC the

ratio

of

the

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fluorescence emission collected in the waveguide ( ( , , )) to the total emission from a point source ( ( , , )) at a specific source position ( , , ). As it is computationally cumbersome to calculate the coupling efficiency in three dimensions by simulating the response of individual point sources separately, we have implemented a reciprocal approach. A broadband mode source is launched in the single mode waveguide feeding the collection FGC with its TE00 mode to profile the relative collection efficiency map. The collection efficiency amplitude of this map was calibrated by using a series of point source incoupling simulations. A collection efficiency profile was calculated by weighted averaging of the collection efficiency map (obtained from reciprocity) of individual wavelengths using a weighing factor based on the emission spectrum of the experimentally used Crimson dye (Supporting Information S4). Fig. 2b illustrates the Crimson dye collection efficiency profile of FGCCM. The collection efficiency tops at 0.055% of the total emission for a point source near the excitation focus. The full-width at half-maximum (FWHM) of the collection efficiency peak in the x and z axis are 2.6 µm and 5.5 µm, respectively. The collection volume is larger than the excitation volume since collection occurs over the large emission bandwidth of the Crimson dye and the collection peak position varies by the wavelength.


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Figure 2. FDTD simulation results of the FGC structure: (a) The schematics of the simulated structure and the excitation beam electric field intensity profile on y=0 plane for the wavelength 638 nm. (b) The collection efficiency profile of the in-line collection FGC on the same vertical plane. (c) The multiplication of the excitation and collection efficiency profiles on the vertical plane for the symmetric collection FGC.

The FGC structure can be compared with a conventional microscope in terms of how much of fluorescence signal can be expected for a given input excitation power. The excitation efficiency of a conventional microscope, as defined as the percentage of the power that is in the focal position approaches 100% while the collection efficiency for a point source depends on the numerical aperture (NA), and is e.g. 10% for a water immersion objective with NA = 0.8. The resulting fluorescence signal is proportional to the local laser power density and to both the absorption cross-section and the quantum yield. Comparing the FGC structure with this 10 ACS Paragon Plus Environment

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microscope objective, this yields an excitation efficiency of 30% (FGC) vs 100% (objective) and collection efficiencies of 0.03% (FGC, see Fig. 2b) and 10% (objective), making the microscope objective ~900 times more efficient than the FGC structure in exciting and collecting fluorescence from a point source. Fig. 2c depicts the multiplication of the excitation profile from the excitation FGC with the collection efficiency profile of the in-line collection FGC. The resulting excitation × collection efficiency peaks at the region where the excitation and collection beams overlap the most efficiently. The FWHM of this peak was 0.7 µm and 1.8 µm in the x and z axes. The resulting photon yield is predominantly originating from this small region. This is similar to the situation in confocal systems, where the collection is purposefully limited to a small volume. Apart from the collection efficiency for a point source, we also define, the system collection efficiency , a new efficiency term, which facilitates comparison between the simulations and the experiments with the fluorescent particles. The system collection efficiency ( = ) is defined as the ratio of the fluorescence observed from the output linear grating coupler of the collection FGCs ( ) to the fluorescence observed from the particle by a microscope objective ( ). Using the FDTD results, the system collection efficiency can be calculated for a particle at a specific position. Pparticle is proportional to the excitation profile integrated over a particle volume times the collection efficiency of the experimental objective (NA = 0.8). Plinear

grating

is

proportional to the multiplication of the excitation × collection profile integrated over a particle volume and the diffraction grating’s efficiency (which can be collected by the experimental objective NA = 0.8). The maximum system collection efficiencies for 1 µm, 5 µm and 15 µm


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particles are calculated to be 0.130%, 0.027% and 0.009% respectively. As expected, the efficiency is lower for larger particles since the collection grating only samples a small volume. Once a particle is larger than the collection spot shown in Fig. 2 b, additional particle volume will increase Pparticle, but not affect the collected power Plinear grating, and thus lower the system collection efficiency. For the 15um particles, lateral and vertical variations have little impact on the efficiency. A part of the particle overlaps with the detection spot in Fig. 2b even if there are variations in the position of the particle. For 1um particles this is not the case, small variations in the particles position can move the particle completely out of the collection spot.

Experimental Characterization of the FGC Structure

Figure 3. Experimental characterization of the FGC structure and the fluorescent particle detection. (a) The experimental intensity profile of the excitation FGC (left) and the in-line collection FGC (right) for 638 nm and 657 nm respectively as mapped by a confocal microscope. (b) A microscope image of the structure when the particle


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crosses the focal points of the FGCs. The particle is indicated in the white box below. The three output couplers of the collection FGCs are shown above the particle.

The beam profiles of the excitation and collection FGCs were experimentally characterized by 3D confocal scans above the grating. Fig. 3a depicts the two superimposed confocal scans of the beam profiles of FGCE and FGCCM when fed by the single mode waveguides with 638 nm and 657 nm light respectively. The focal beams of the FGCE (left) and FGCCM (right) match the simulated profile of Figure 2a. The FWHM of the excitation focus in x and z axes were 0.75 µm and 3.64 µm. It was also observed that the focal position shifted for varying wavelength (see Fig. S-6). The power in the focal point of FGCE was measured as 31 ± 3% of the power in the single mode waveguide; this result is in good agreement with the FDTD simulation result 30.6% (see Methods). To experimentally characterize the FGC structure’s system collection efficiency, Crimson red stained polystyrene particles with a diameter of 1 µm and 15 µm as well as Vybrant DiD stained PBMCs were flowed in the microfluidic channel. Fig. 3b shows an image of the FGC structure when a 15 µm particle passed through the detection region. At the bottom of the image the fluorescence of the particle in the excitation beam is seen while at the top the three linear output couplers can be observed coupling out the collected fluorescent light. Pparticle and Plinear grating were evaluated by integrating the pixel amplitudes inside the boxes around the particle and gratings as indicated in Fig 3b. Figure 4a shows the measured Pparticle and Plinear grating (FGCCM) over a 5 s and 50 s (inset) period for the 15 µm fluorescent polystyrene particles and PBMCs. It’s observed that every 15 µm particle passing through the excitation beam is detected at the waveguide end. However, it was not the case for the smaller particles (for PBMCs and 1 µm particles, see Fig. S-7). For the 1 µm 13 ACS Paragon Plus Environment

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particles a peak in Plinear grating is observed only 9% of the times when there is a peak in Pparticle. This is caused by a variation in the position of the particles. The particles flowing in the channel are focused as much as possible by the sheet flow. Yet, small variations in both vertically and laterally are still possible. As observed in Fig. S-7, this variation strongly influences the collection efficiencies of small particles.

Figure 4. The time traces of the fluorescence signal of the 15 µm particles and the system collection efficiencies. (a) The time trace of the fluorescence signal observed by the camera from the output coupler of FGCCM (Plinear grating) and the particle (Pparticle) over 5 s and 50 s (inset). (b) The system collection efficiency of FGCCM for the 1 µm particle (black), PBMC (red) and 15 µm particle (blue) flow. The efficiency of each event is shown by the circles. The corresponding probability distributions (90% and the median) are indicated next to the circles with their efficiency values.

The system collection efficiency can be calculated based on the peak values seen in Pparticle and Plinear grating in Figure 4a (and Fig. S-7). Fig. 4b shows the experimental collection efficiencies of each fluorescence detection event at the linear grating of the in-line collection FGC for the 1 µm 14 ACS Paragon Plus Environment

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and 15 µm polystyrene particles and PBMCs. The efficiencies are indicated by the circles and the median and 90% values of their probability distribution are shown next to them. The maximum efficiency was observed for the 1 µm particles. The occurrence probability distribution reached to 90% at the fluorescence collection efficiency of 0.088%, while it was 0.010% for the 15 µm particles and 0.033% for the PBMCs. These experimental values and the observed decrease in the efficiency with increasing particle size are in accordance with the simulations. The increasing system collection efficiency by decreasing particle size indicates that the region where the excitation beam and the collection volume overlap is smaller than the PBMCs and the 15 µm particles, as expected by the FDTD simulations. The median of the distribution for the 1 µm and 15 µm particles were 0.013% and 0.006%. The majority of the events had the efficiencies close to the median values for the 15 µm particles. Moreover, it was observed that the 1 µm particles had a wider range of fluorescence collection efficiency due to their small size. The system collection efficiency of the side FGCs can also be analyzed using images of detection events (Figure 3b). However, the signal of the side collection gratings is considerably smaller than that of the in-line collection grating. The camera’s dynamic range prevents us to image a detection event at the side collection FGCs without saturating the camera at the excitation spot. Rather than calculating the system collection efficiency directly, we compare the power at the linear grating of the in-line collection FGC with that of the side FGCs. Figure 5a shows an example of the Plinear grating for all three FGCs. The maximum power of the detection events captured by side FGC (right axis) is four to seven times smaller than that of the in-line collection FGC. Figure 5b shows the power detected at every linear grating on a logarithmic


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scale. The detection events of the 15 µm particles in the in-line collection FGC correlate well with the detection events at the side FGCs. Finally, we used the FGC structure to detect fluorescent particles without the microscope. A separate FGC structure was used where the light collected by the in-line collection FGC is routed near the input coupler where it is coupled to a multimode fiber which guides the fluorescence to a PMT. Fig. 5c depicts the fluorescence signal of the 15 µm particles measured by the PMT. A clear signal could be seen for both the 1 µm and 15 µm polystyrene particles (see Fig. S-10). For PBMCs, on the other hand, no signal could be detected. As observed in the measurements with the microscope, the fluorescence of the PBMCs is about 4 times smaller than that of the 1 µm particles, making them hard to distinguish from the noise of the PMT.


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Figure 5. The time trace of the fluorescence signal from the out-couplers of the in-line and side collection FGCs, the system collection efficiency of the in-line collection FGC in comparison with the side collection FGCs and the PMT fluorescence signal during the 15 µm particle flow (a) The time trace of the fluorescence signal observed by the camera from the output couplers of all three collection FGCs over 2 s and 50 s (inset). (b) The fluorescence amplitude from the output couplers of the side collection FGCs (FGCCL and FGCCR) vs the in-line collection FGC (FGCCM) for the 15 µm particle flow. The amplitude of each event for FGCCL and FGCCR are indicated by black squares and blue rectangles respectively. (c) The time trace of the fluorescence signal recorded by a PMT (demonstration of the detection without a microscope) which was coupled to the in-line collection FGC (FGCCM) over 20 s and 150 s (inset).


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Conclusion An integrated SiN photonics circuit was investigated for the excitation and collection of fluorescence of micro-particles flowing in a microfluidic channel above the photonics structure. The structure consisted of a FGC for fluorescence excitation and three FGCs for collection, and was characterized experimentally and the experimental results were compared with the FDTD simulations. The FWHM of the focal spot of FGCs were experimentally measured to be 0.75 µm x 3.64 µm in the vertical plane, comparable to the spot size of a 0.33 NA objective. The combination of the excitation and in-line collection FGC formed a confocal like detection region of 1.8 x 0.7 µm about 11 µm above the sample surface where the detection efficiency peaked. 1 µm and 15 µm polystyrene particles and PBMCs were successfully detected using this structure. Due the small detection volume, the maximum system collection efficiency was inversely proportional to the size of the particle. The system collection efficiency for the in-line collection FGC reached up to 0.088%, 0.010% and 0.033% for 1 µm and 15um particles and PBMCs, respectively, in accordance with the FDTD simulations. Despite the low efficiency, we were able to detect fluorescent micro-particles with this structure without the use of a microscope, i.e. by guiding the collection waveguide’s output directly to a PMT. The strongly fluorescent 1 µm and 15 µm particles could be detected in this set-up, although the PBMC detection was unsuccessful. There are still many opportunities to improve the collection efficiency of the presented FGC structure. Better overlap of the excitation and collection FGC intensity profiles would improve the detection efficiency. Also, collection by a multimode waveguide or a long-tapered waveguide could possibly increase the efficiency by making the structure more broadband and less sensitive 18 ACS Paragon Plus Environment

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to position variations. Future work will first and foremost strive for an improved efficiency, and can secondly focus on combining the detection structure with other on-chip photonics systems, e.g. spectrometers, in order to form fully integrated optical detection devices.

ASSOCIATED CONTENT The Supporting Information is available free of charge on the ACS Publications website at. Details on the design of the focusing grating coupler structure, microfluidic channel integration, finite-difference time-domain simulations for the excitation and collection efficiency of the structure, system collection efficiency of the in-line and side collection focusing grating couplers for 1 µm polystyrene particles and peripheral blood mononuclear cells, experimental detection of 1 µm polystyrene particles and peripheral blood mononuclear cells

by a

photomultiplier tube (PDF)

AUTHOR INFORMATION Corresponding Authors *Email: (S. K.) [email protected] and (P.V.) [email protected] ACKNOWLEDGMENT N.V. acknowledges financial support from the FWO Flanders. We thank J. O’Callaghan for PA-glass bonding, A. Dusa for the preparation of PBMCs, and K. de Wijs for his help with PMT experiments. We acknowledge JSR Corporation, Japan for providing PA. COMPETING FINANCIAL INTERESTS


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The authors declare no competing financial interests. AUTHOR CONTRIBUTIONS The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ABBREVIATIONS FGC, focusing grating coupler; FDTD, finite-difference time-domain; PBMC, peripheral blood mononuclear cell; PMT, photo-multiplier tube; PS, polystyrene.

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