Research Interest

1. Super-resolution bio-imaging

The group is interested in developing structured illumination microscopy and chip-based structured illumination microscopy for live cell imaging application in Tromsø. I have been involved with a few projects involving super-resolution imaging of live cells in collaboration with Prof Thomas Huser (University of Bielefeld) and Prof Frank Chuang. I used OMX, (GE Heathcare) Structured illumination microscopy installed at University of California, Davis. Some of these projects are briefly described below.

1.1 Physics of structured illumination microscopy

The optical transfer function (OTF) of an objective lens is, in the frequency domain, given as a circle with radius corresponding to the cut-off frequency, as shown in Fig 1(a). Spatial frequencies within the circle are resolved and those outside correspond to information that is lost, which limits the optical resolution of the objective. In structured illumination microscopy (SIM), the target is illuminated with a grid-like interference pattern. By taking several images with different interference patterns, super-resolved images are obtained. The working principle of SIM is usually described in the spatial frequency domain as schematically shown in Fig 1. In SIM, higher frequency information is pushed inside the cut-off frequency of the conventional OTF (as shown in Fig 1e) using Moiré fringes. When two frequencies are mixed, Moiré fringes are created as shown in Fig 1(b). Higher frequency (f₁ and f₃) information is made available by the Moiré fringes at lower frequency (f₂) as depicted in Fig 1(b). The lower frequency Moiré fringes can thus be imaged by the objective lens. By measuring Moiré fringes and from one known frequency (i.e. the interference pattern) it is possible to get information about the other high frequency (i.e. the target). When the target is illuminated by an interference pattern, the fluorescence signal is collected only from the bright regions, thus it is necessary to take additional acquisitions at different phase steps (by moving fringes by 1/3 or 1/5) of a period in each direction. For 2D:SIM images are acquired for 3 angles and 3 phase steps (in total 9 images) and for 3D:SIM a total of 15 images are acquired for 3 angles and 5 phase steps for each Z-step as shown in Fig 1(c). The enhancement in optical resolution is achieved by pushing higher un-resolved frequencies into the OTF of a conventional objective (effectively increasing the radius of the OTF), as shown in Fig 1(d, e).

Figure 1 Optical transfer function (OTF) of the aperture with cut-off frequency. (b) Generation of lower frequency Moiré fringes (f₂) by interfering higher frequency signals (f₁, f₃). (c) For 3D SIM, images are acquired for 3 angles and 5 phases. (d-e) The enhancement in optical resolution is obtained by pushing higher frequencies inside OTF cut-off.

1.2 Super-resolution imaging of live cells

High-resolution imaging of Mitochondria in living keratinocytes: Replying on sub-diffraction resolution (100nm) of structured illumination microscopy and selective stain binding, I imaged sub-mitochondrial regions in living keratinocytes cells. Mito-Tracker stain was trapped inside the mitochondria consequently imaging mitochondrial compartments, whereas GFP-BacMan stains the mitochondrial membrane. Using dual stain on the same cell, sub-mitochondrial region was imaged. Figure 2 and Fig 3 shows a comparison of SIM images with conventional DV fluorescence images. It is evident that the resolution enhancement provided by SIM (i.e. 2X in each dimension) enables efficient imaging of mitochondria in keratinocytes. Using 488 nm excitation the optical resolution of 120 nm was obtained as shown in Fig. 4.

Imaging Autophagy phenomena using structured illumination microscopy: Autophagy is a ubiquitous process that enables cells to degrade and recycle proteins and organelles. We apply advanced fluorescence microscopy to visualize and quantify the small, but essential, physical changes associated with the induction of autophagy, including the formation and distribution of autophagosomes and lysosomes, and their fusion into autophagolysosomes. Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of autophagy induction. Lateral resolution is improved to at least 120nm with structured-illumination, and there is an 8-fold enhancement of contrast resulting from the structured-illumination microscopy reconstruction algorithm. Figure 3 shows spatial details of the fusion between autophagosomes and lysosomes (as indicated in white color). We can clearly see the interaction between green LC3 signals and red Lysosome signals, and the merge of the two signals to produce yellow signals. The co-localization is not evident in conventional DV images [1].

Figure 4A shows a side-by-side comparison of images acquired and reconstructed in DV mode (simulated widefield deconvolution, left) vs. structured-illumination image using the OMX microscope (right). Small-scale colocalization of autophagosomes and lysosomes (Fig 4B, indicated by arrows) are clearly apparent with super-resolution microscopy, whereas they are hardly evident with conventional fluorescence imaging.

Imaging HIV transfer at virological synapses using structured illumination microscopy:In this project, I used structured illumination microscopy for real-time super-resolved imaging of HiV transfection. The transfer of sub-diffraction HiV viral protein was successfully imaged from an infected Jurkat cell (Gag-iGFP expressing) to the primary CD4+T cells. The entire process of HiV transfection was imaged i.e. accumulation of viral protein at the cell membrane and at the viral synapse between the host and target cell, followed by the transfection of viral protein to the target cell. Figure 5(a) shows bright field image of infected Jurkat cells with 5 CD4+T cells. The accumulation of Gag (stained with iGFP) at the synapse and the transfer of viral protein can be noticed in Fig 5b.

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2. Waveguide trapping: Harnessing Optical Forces for Micro-manipulation

We have developed a new waveguide platform based on tantalum pentoxide waveguide material (Section 2.1) and used it for optical trapping application. Different targets including polystyrene spheres, red blood cells, nanowires, nano-particles and hollow spheres (Section 2.2) were trapped by the waveguides. We explored different waveguide designs (loop with a gap, y-junctions) and geometries (rib and strip) for optical trapping application as covered in Section 2.3.

Background: The gradient optical force generated by a focused beam in laser tweezers is used to trap and manipulate particles freely in three dimensions. Similarly, the optical forces generated by the evanescent field from a waveguide can be used to trap and propel a particle. The integrated platform based on optical waveguides enables on-chip particle manipulation. Waveguide trapping offers a considerable scope for integration that optical tweezers cannot match. The evanescent field of an optical waveguide is dominant about 150 nm into the medium above the surface. Particles interacting with the evanescent field are pulled down towards the waveguide surface due to the gradient generated by the exponentially decaying evanescent field. The gradient of the evanescent field generated at the edge of the waveguide holds the particle on top of the waveguide and radiation pressure propels these particles forward along the waveguide surface.

2.1 Integrated platform:

We developed waveguide platform based on Tantalum Pentoxide (Ta₂O₅) in collaboration with Prof James Wilkinson at Optoelectronics Research Centre, University of Southampton. Waveguide trapping use evanescent field to trap particles thus it is desirable to have high intensity in the evanescent field for efficient trapping. A high refractive index contrast material allows strong confinement of light and by fabricating sub-micron thick optical waveguides, the intensity of the evanescent field can be maximised. The combination of thin waveguides (150-200 nm) made from high refractive index material (n=2.1 @1070 nm for Ta₂O₅) was opted for planar trapping application. Waveguide fabrications steps were meticulously optimized, including, magnetron sputtering, photolithography, etching and annealing. Low propagation loss waveguides are desirable for optical trapping application [2-3].

2.2 Optical manipulation:

We applied tantalum pentoxide (Ta₂O₅) for planar waveguide trapping. Owing to high surface intensity that be obtained on top of Ta₂O₅ surface, efficient trapping of polystyrene particles see and blood cells were obtained, see Fig. 6. We trapped both fixed (dead) and live red blood cells on top of waveguide surface [4-7]. Waveguide trapping uses only evanescent field for optical trapping, thus it is well suited to trap the rim of hollow spheres [8-9]. Waveguide trapping is well-suited for trapping nano-sized targets and we have successfully trapped nano-wires and gold nanoparticles as shown in Fig. 7.

2.3 Novel waveguide designs

Waveguide Loop: a particle is trapped on top of waveguide and is continuously propelled along the waveguide. In addition to propulsion, it is imperative to develop a method to stably hold the particle at a fixed position, e.g. for observation, analysis or interaction with other particles. A waveguide design with a loop is used to generate two counter-propagating beams from a single input. An intentional gap at the centre of the loop is used to generate counter-diverging laser beams as shown in Fig. 7. The counter-diverging field is used to precisely stop and hold particles/cells that arrive from the two sides of the loop. The loop design allows precise optical trapping at a fixed location on a chip. In the gap, there are two rapidly diverging fields emerging from each of the waveguide ends. Two counter-diverging beams will interfere and the visibility of the interference fringes depends on waveguide parameters (width, thickness), gap separations and waveguide type (strip or rib). Fig. 8 shows waveguide loop with a gap separation of 10 µm and resulted interference fringes.

Figure 7: Bright field optical image of a strip waveguide with a loop structure and a gap of separation (a-c) 2 μm and (d-f) 30 μm. At the gap the guided light strongly diverge. Laser is switched on for images shown in (c, f).

Figure 8 Bright field optical image of a waveguide loop structure with a gap of separation 10 μm. (b) Interference fringes generated by counter-diverging laser beams in the gap.

Waveguide loop design was used for transporting and stably trapping polystyrene microparticles and red blood cells. Figure 9 shows trapping of 5 μm spheres in the loop with a gap separation of 30 μm. Fig. 10(a) shows a sphere trapped at the end of waveguide (bottom arm), while another sphere is been delivered at the gap as shown in subsequent images, Fig. 9(a-d). Finally, a third sphere is delivered at the gap as shown in Fig. 9(e-h). While, a 30 μm gap separation was used to trap chain of 5 μm spheres, smaller gap separation can be used to tightly trap spheres with smaller diameter. A loop with 2 μm gap was employed to firmly trap a sphere of diameter 1 μm with a precision of ±0.2 μm along the gap exploiting the interference fringes [10-11].

Reference

1. C A. Changou, D L. Wolfson, B S Ahluwalia, R J. Bold, H-J Kung, F Y. S. Chuang, “Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy”, Journal of Visualized Experiments (75), e50047, (2013).
2. B. P. S. Ahluwalia,; Olav Gaute Hellesø; Ananth Z. Subramanian; Nicolas M. B. Perney; Neil P. Sessions; James S. Wilkinson, ” Fabrication and optimization of Tantalum pentoxide waveguides for optical micro-propulsion Proceedings of the SPIE Vol. 7604 (2010).
3. B. P. S. Ahluwalia, Ananth Z. Subramanian, Olav Gaute Hellesø, Nicolas M. B. Perney, Neil P. Sessions and James S Wilkinson, “Fabrication of sub-micron high refractive index tantalum pentoxide waveguides for optical propulsion of microparticles”, Photonics Technological Letters, 20757, (2009). Citation: 5 (IF=2.191).
4. Pal Lovhaugen, Balpreet S. Ahluwalia, Thomas R. Huser, Peter McCourt and Olav Gaute Helleso, “Optical trapping forces on biological cells on a waveguide surface”, Proceedings of the SPIE, 7902, 79020N (2011).
5. B. S. Ahluwalia, Peter McCourt, Thomas Huser and Olav Gaute Hellesø, “Optical Trapping and propulsion of red blood cells on waveguide surfaces”, Optics Express, 18, 21053 (2010). Citation: 4. Highlighted in Optics & Photonics News (OPN) Issue 2010.
6. B. P. S. Ahluwalia,; Olav Gaute Hellesø; Ananth Z. Subramanian; Nicolas M. B. Perney; Neil P. Sessions; James S. Wilkinson, ” Fabrication and optimization of Tantalum pentoxide waveguides for optical micro-propulsion Proceedings of the SPIE Vol. 7604 (2010).
7. Balpreet S Ahluwalia, O.G. Hellesø, A. Z. Subramanian, James S. Wilkinson, Jie Chen and Xuyuan Chen, “Integrated platform based on high refractive index contrast waveguide for optical guiding and sorting”, Proceedings of the SPIE 7613, 76130R (2010).
8. Balpreet Singh Ahluwalia, P Løvhaugen, and O. G. Hellesø, “Waveguide trapping of hollow glass spheres,” Optics Letters, 36, 3347-3349 (2011). Selected for Spotlight of Optics.
9. Løvhaugen, Pål; Ahluwalia, Balpreet S.; Hellesø, Olav G., “Optical waveguide trapping forces on hollow glass spheres”, Proceedings of the SPIE, Volume 7950, pp. 79500P-79500P-9 (2011).
10. Balpreet S. Ahluwalia, Olav G. Hellesø “Optical waveguide loop for planar trapping of blood cells and microspheres”, Proceedings of the SPIE (NanoScience + Engineering), In Press (2013).
11. Olav Gaute Hellesø, Pal Løvhaugen, Ananth Z. Subramanian, James S. Wilkinson and Balpreet Singh Ahluwalia*, “Surface transport and stable trapping of particles and cells by an optical waveguide loop”, Lab-on-a-chip, 12(18), 3436-40, 2012.