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Structured illumination microscopy using a photonic chip

Øystein I. Helle, Firehun T. Dullo, Marcel Lahrberg, Jean-Claude Tinguely, Balpreet S. Ahluwalia

arXiv:1903.05512v1physics.opticsphysics.app-ph

TL;DR

Conventional SIM relies on costly, complex free-space optics and its resolution enhancement is limited by the imaging objective. This paper demonstrates chip-based TIRF-SIM, using planar waveguides to create and phase-control illumination patterns. The chip-generated fringes decouple illumination from collection, supporting resolution enhancement with low-NA objectives and larger fields of view.

  • Problem

    Conventional SIM uses costly, complex free-space optics and its resolution enhancement is limited by the collection objective’s numerical aperture.

    Source: Discussion · pp. 11, 13

  • Method

    The photonic chip holds the specimen and uses planar waveguides to create an evanescent standing-wave pattern, with on-chip phase splitting and thermo-optical modulation among the described approaches.

    Source: Introduction · p. 2, Discussion · pp. 13, 15

  • Results

    Chip-generated fringes are independent of the imaging objective, and simulations show that combined waveguide patterns can improve resolution without losing frequency bands for low-NA imaging.

    Source: Introduction · p. 2, Simulation of waveguide based… · p. 5, Discussion · p. 14

  • Takeaways & Limitations

    The chip-based setup can retrofit a standard optical microscope and supports low-NA, low-magnification imaging over larger fields while retaining illumination-defined resolution improvement.

    Source: Discussion · pp. 13–14

    Unequal light guidance between waveguide arms reduces fringe visibility; the best reported structures achieved around 80% visibility.

    Source: Discussion · p. 15

Abstract

from arXiv · show

Structured illumination microscopy (SIM) enables live cell, super-resolution imaging at high speeds. SIM uses sophisticated optical systems to generate pre-determined excitation light patterns, and reconstruction algorithms to enhance the resolution by up to a factor of two. The optical set-up of SIM relies on delicate free-space optics to generate the light patterns, and a high numerical aperture objective lens to project the pattern on the sample. These arrangements are prone to miss-alignment, often with high costs, and with the final resolution-enhancement being limited by the numerical aperture of the collection optics. Here, we present a photonic-chip based total internal reflection fluorescence (TIRF)-SIM that greatly reduces the complexity of the optical setup needed to acquire TIRF-SIM images. This is achieved by taking out the light delivery from the microscope and transferring it to a photonic-chip. The conventional glass slide is replaced by the planar photonic chip, that both holds and illuminates the specimen. The chip is used to create a standing wave interference pattern, which illuminates the sample via evanescent fields. The phase of the interference pattern is controlled by the use of thermo-optical modulation, leaving the footprint of the light illumination path for the SIM system to around 4 by 4 cm$^2$. Furthermore, we show that by the use of the photonic-chip technology, the resolution enhancement of SIM can be increased above that of the conventional approach. In addition, by the separation of excitation and collection light paths the technology opens the possibility to use low numerical objective lenses, without sacrificing on the SIM resolution. Chip-based SIM represents a simple, stable and affordable approach, which could enable widespread penetration of the technique and might also open avenues for high throughput optical super-resolution microscopy.

Introduction

Conventional SIM is fast and compatible with bright fluorophores, but its free-space pattern-generation optics are complex and its resolution enhancement is limited by the collection objective. Chip-based SIM moves illumination to planar waveguides, whose interference patterns support resolution enhancement independent of the imaging objective and can be phase-stepped on chip.

Introduction

  • Conventional SIM uses free-space optics to generate patterns and is costly, bulky, alignment-prone, and limited to a twofold resolution enhancement set by the objective’s numerical aperture.SIM otherwise works with most bright fluorophores and is inherently fast as a widefield technique.Source: Introduction · p. 2
  • Waveguide-generated illumination decouples pattern frequency from the collection objective, allowing low-NA, low-magnification imaging over large fields while retaining resolution set by the illumination fringe spacing.High-index waveguides can produce fringe spacing smaller than that achievable with a high-NA oil-immersion objective.Source: Introduction · p. 2
  • On-chip thermo-optical heating changes one waveguide arm’s refractive index to shift the interference phase, while alternatives use fixed path lengths or off-chip splitting and phase control.The fixed-path design uses three waveguides for three phase steps; dynamic on-chip phase stepping uses a heating element on one arm.Source: Introduction · pp. 2–3

Simulation of waveguide based interference patterns for SIM

  • Counter-propagating waveguides produce a 188 nm simulated pattern period, compared with 445 nm for waveguides crossing at 50°.The simulations model the fundamental TE mode in Si3N4 waveguides with effective index n = 1.7552 at λ = 660 nm.Source: Simulation of waveguide based… · pp. 4–5, Figure 3 · p. 5
  • For an N.A. = 0.6 objective, combining illumination patterns from 60°, 120°, and 180° improves resolution without losing frequency bands, supporting large-field imaging with low-magnification objectives.At N.A. = 1.2, increasing the interference angle from 60° to 120° and 180° increases modulation frequency and expected resolution improvement.Source: Simulation of waveguide based… · pp. 5, 7, Figure 5 · p. 6

Materials and Methods

The chip uses shallow-rib Si3N4 waveguides designed for single-mode guiding and uniform interference fringes, then adds fabrication, heating, and imaging components for cSIM. The methods also describe dSTORM field mapping of the waveguide surface.

Chip Design

  • Shallow-rib waveguides 1–1.5 µm wide support fundamental-mode guiding; 4 nm ribs taper to 25 or 50 µm over 2 or 4 mm, respectively.The design avoids residual higher-order modes that would create an undesirable mode-beat pattern.Source: Chip Design · p. 7

Chip fabrication

  • The chip process forms 150 nm Si3N4 waveguides on oxidized silicon, adds an absorption layer to limit crosstalk, and applies cladding and imaging windows.Waveguides are patterned by photolithography and etched by reactive-ion etching; the imaging windows are opened through the oxide.Source: Chip fabrication · p. 7
  • Silver resistors on a PDMS or Su-8 cladding layer provide cSIM phase stepping by heating one waveguide arm for each SIM angle.Photolithography removes silver residue from the openings, leaving the resistor on one arm.Source: Deposition of thermo-optical heating… · p. 8

Experimental setup

  • A vacuum chuck holds the chip on an xyz stage, while a nine-fiber array couples light into waveguides spaced 127 µm apart.A 50:50 fiber splitter is used for the two-input design; a voltage supply drives the on-chip heating elements for phase stepping. Images were analyzed with Fiji and FairSIM, with custom software for 180° interference data.Source: Experimental setup · p. 9

dSTORM field mapping

  • For dSTORM field mapping, CellMask deep red was applied to the waveguide surface and 100,000 images were acquired at 30 ms exposure.The surface was excited at 660 nm, and the image stack was reconstructed with ThunderStorm.Source: dSTORM field mapping · p. 9

Experiments and Results

The experiments demonstrate stable, controllable chip-based phase stepping and cSIM super-resolution imaging. At 180° interference, cSIM resolved beads 117 nm apart with a 2.3× resolution enhancement.

Modulation index

  • At 50° interference, the measured modulation index was around 78%.It was calculated from fluorescent-bead intensity variation as the interference pattern phase changed.Source: Experiments and Results · pp. 9–10

Phase stability

  • On-chip y-branch splitting maintained stable phase over 80 s, whereas the off-chip fibre split showed phase drift.Phase stability was assessed indirectly by tracking normalized fluorescence from a bead.Source: Experiments and Results · p. 10, Figure 9 · p. 10

Reproducible phase stepping

Three-angle cSIM imaging

  • The cSIM reconstruction resolved beads 206 nm apart that were unresolved in the diffraction-limited image.The overlapping region from three pattern orientations was reconstructed after equidistant phase steps were identified.Source: Experiments and Results · pp. 10–11

Thermo-optical phase modulation

  • Chip-based phase modulation resolved two beads 194 nm apart in the cSIM reconstruction.The thermo-optical element changed phase by heating and expanding Su-8 in the window openings.Source: Experiments and Results · p. 11

180° interference and resolution enhancement

  • 2.3× resolution enhancement resolved beads 117 nm apart with 180° interference, surpassing conventional SIM.The experiment used 660 nm excitation and a 1.2 NA objective.Source: Experiments and Results · p. 11

Discussion

The discussion presents cSIM as a simpler photonic-chip alternative to free-space SIM, with illumination patterns generated independently of the collection objective. It reports resolution beyond conventional linear SIM while identifying phase-control and fabrication-related challenges, and leaves 2D performance for future work.

Simplifying the optical setup

  • The photonic chip supplies light delivery, pattern generation, and beam steering, simplifying retrofit of super-resolution SIM to a standard microscope.The authors describe the setup as stable with few bulk optical elements and note that fibre pigtailling can add stability and integration.Source: Discussion · pp. 11, 13

Future 2D cSIM

  • The authors plan to study development of 2D SIM with 2.3-2.4X resolution enhancement in future work by designing suitable waveguide geometries.Source: Discussion · p. 15

Resolution and collection optics

  • Chip-generated fringes decouple illumination from collection, allowing a low-NA, low-magnification objective to retain resolution improvement while imaging a larger field of view.Conventional SIM's resolution enhancement is limited to 2× by the objective's numerical aperture and excitation wavelength.Source: Discussion · pp. 13–14

Phase-control strategies

  • Off-chip fibre splitting drifts and lacks speed and repeatability, while on-chip splitting removes the observed environmental phase instability.The alternatives include fixed path-length differences for phase shifts and thermo-optical modulation, which may support fast phase shifting.Source: Discussion · pp. 14–15

Resolution enhancement

  • At 180° interference, cSIM resolved 117 nm-separated beads with a 2.3× resolution enhancement, experimentally verifying resolution beyond conventional SIM.The discussion attributes the potential gain to chip-generated fringes with smaller spacing than objective-generated patterns.Source: Discussion · pp. 13–14, Figure 14 · p. 15

Thermo-optical phase-control trade-offs

  • Thermo-optical heating can shift the sample position and cause coupling loss; the authors propose cooling and smaller silver circuits to mitigate this.The projected heating and cooling rise/fall time is in the microsecond range, but this speed was not demonstrated here.Source: Discussion · p. 15

Visibility limitation

  • Best fringe visibility was around 80%; small losses in either interfering arm can greatly reduce visibility and may cause reconstruction failure if it becomes too low.Potential loss sources include hot spots, waveguide imperfections, and losses associated with PDMS/Su-8 deposition in phase-change openings.Source: Discussion · p. 15

Future directions

  • The chip permits integrated light manipulation and may support new illumination strategies for biological applications.The authors present these possibilities as future avenues rather than demonstrated applications.Source: Discussion · p. 16
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