Spatial Light Interference Microscopy (SLIM)
For centuries, light microscopy has been the dominant tool for studying cell biology. In the late 1800’s, Abbe formulated the resolution limitation of the light microscope to approximately half the light wavelength, or 200-300 nanometres. Remarkably, in recent years several approaches have been successfully developed to exceed this diffraction barrier in far field fluorescence microscopy. At QLI we have recently developed spatial light interference microscopy (SLIM) as a new optical microscopy technique, capable of measuring nanoscale structures and dynamics in live cells via interferometry. SLIM combines two classic ideas in light imaging: Zernikes phase contrast microscopy and Gabors holography. Thus, SLIM reveals the intrinsic contrast of cell structures and, in addition, renders quantitative optical path-length maps across the sample. The resulting topographic accuracy is comparable to that of atomic force microscopy, while the acquisition speed is 1,000 times higher. Due to the micron-scale coherence length of the illuminating field, SLIM provides high axial resolution optical sectioning. Using a 3D complex field deconvolution operation, we render tomographic refractive index distributions of live, unstained cells. This novel capability is referred to as Spatial Light Interference Tomography (SLIT).
SLIM was developed by producing additional spatial modulation to the image field outputted by a commercial phase contrast microscope. Specifically, in addition to the π/2 shift introduced in phase contrast between the scattered and unscattered light from the sample, we generated further phase shifts, by increments of π/2, and recorded additional images for each phase map. Thus, the objective exit pupil, containing the phase shifting ring, is imaged via lens L1 onto the surface of a reflective liquid crystal phase modulator (LCPM, Boulder Nonlinear). The active pattern on the LCPM is calculated to precisely match the size and position of the phase ring image, such that additional phase delay can be precisely controlled between the scattered and unscattered components of the image field. In this setup, 4 images corresponding to each phase shift are recorded to produce a quantitative phase image that is uniquely determined.
In order to assess the spatial accuracy of SLIM, we imaged an amorphous carbon film deposited on glass and compared the topography measurements against atomic force microscopy (AFM) as shown in the figure above. The topography measurements by SLIM and AFM, respectively, are summarized in Fig. 2a and 2b. The two types of measurement agree within a fraction of a nanometer. Note that both SLIM and AFM are characterized by much smaller errors than suggested by the widths of the histogram modes, as these widths also reflect irregularities in the surface topography due to the fabrication process itself. Of course, AFM is not limited by diffraction in the transverse direction. Unlike AFM, SLIM is non-contact, parallel, and faster by more than 3 orders of magnitude. Thus, SLIM can optically measure an area of 75 × 100 μm in 0.5 s compared to a 10 × 10 μm field of view measured by AFM in 21 minutes. As further comparison, we evaluated background images (i.e., no sample) from SLIM and diffraction phase microscopy (DPM) , an established laser-based technique that was interfaced with the same microscope. Due to the lack of speckle effects granted by its broad spectral illumination, SLIM’s spatial uniformity and accuracy for structural measurements is substantially better than DPM’s. To quantify the spatio-temporal phase sensitivity, we imaged the SLIM background repeatedly to obtain a 256-frame stack. Figure 2f shows the spatial and temporal histograms associated with the optical path-length shifts across a 10 × 10 μm field of view and over the entire stack, respectively. These noise levels, 0.3 nm and 0.03 nm, represent the limit in optical path-length sensitivity across the image and between frames, respectively. The diminished effects of speckles allowed quantitative phase imaging to reveal single atomic layers of carbon, for the first time. Several error sources can potentially be diminished further: residual mechanical vibrations in the system that are not “common path”, minute fluctuations in the intensity and spectrum of the thermal light source, digitization noise from the CCD camera (12 bit in our case), and the stability (repeatability) of the liquid crystal modulator (8 bit).
A distinct feature of SLIM is that the quantitative phase image is overlaid with the other imaging channels of the microscope, such as epi-fluorescence, differential interference contrast, and phase contrast. Simultaneous fluorescence imaging complements SLIM’s structural information with the ability to study cellular constituents with molecular specificity. In Fig. 3a and 3b, we show multimodal SLIM-fluorescence imaging of primary hippocampal neurons cultured for 19 days in vitro (DIV). Nucleus (blue) and dendrites (green) are stained by 4′,6-diamidino-2-phenylindole (DAPI) and the somatodendritic marker microtubuleassociated protein 2 (MAP2), respectively. The red line in the inset of Fig. 4a indicates the axon, as evidenced by the absence of MAP2 staining. In order to quantify the structures observed by SLIM, we traced individual neurites using NeuronJ (Fig. 4c). Each trace shows the optical path-length fluctuations along each different neurite. The standard deviation (σ) of the path-length fluctuation is, on average, twice as large for dendrites as it is for the axon, i.e., 49 nm vs. 25 nm. This result suggests that subtle inhomogeneities are associated with the synaptic structures and these inhomogeneities can be revealed by SLIM as path-length changes. By 3 weeks in dispersed culture, the majority of dendritic spines generally mature to form presynaptic boutons on the dendritic shafts. After 33 DIV, we observe synapsin and MAP2 labeling of putative synaptic elaborations on a mature hippocampal neuron (Fig. 3d).
So far SLIM has been used for a variety of applications including Cell Dynamics, Cell Growth, Intracellular Transport, Topography, Tomography and Refractometry. For more details please refer to the publications listed below.
SLIM related Publications
- Z. Wang, L. J. Millet, M. Mir, H. Ding, S. Unarunotai, J. A. Rogers, M. U. Gillette and G. Popescu, Spatial light interference microscopy (SLIM) , Opt. Exp., 19, 1016 (2011).
- Z. Wang, K. Tangella, A. Balla and G. Popescu, Tissue refractive index as marker of disease, J. Biomed. Opt. 16 (11), 2011.
- M. Mir, K. Tangella and G.Popescu, Blood testing at the single cell level using quantitative phase and amplitude microscopy, Biomed. Opt. Exp., 2 (12), 2011.
- R. Zhu, S. Sridharan, K. Tangella, A. Balla and G.Popescu, Correlation induced spectral changes in tissues, Opt. Lett., 36 (21), 2011.
- R.Wang, Z. Wang, L. Millet, M. U. Gillette, A.J. Levine, and G.Popescu, “Dispersion-relation phase spectroscopy of intracellular transport“, Opt. Exp. 19(21), 2011.
- Z. Wang, D. L. Marks, P. S. Carney, L. J. Millet, M. U. Gillett, A. Mihi, P. V. Braun, Z. Shen, S. G. Prasanth and G. Popescu, “Spatial Light Interference Tomography (SLIT)“, Opt. Exp., 19(21), 2011.
- S. Sridharan, M. Mir and G.Popescu, “Simultaneous Optical Measurement of cell motility and growth“, Biomed. Opt. Exp., 2(1), 2011.
- R. Wang, Z. Wang, J. Leigh, N. Sobh, L. Millet, M. Gillette, A. Levine and G. Popescu, “One dimensional deterministic transport in neurons measured by dispersion-related phase spectroscopy”, J. Phys. Cond. Matt., 23, 2011.
- J. P. Haldar, Z. Wang, G.Popescu, and Z. Liang, Deconvolved Spatial Light Interference Microscopy for Live Cell Imaging, IEEE Trans. Biomed. Eng. , 58(9), 2011.
- M. Mir, Z. Wang, Z. Shen, M. Bednarz, R. Bashir, I. Golding, S. Prasanth and G.Popescu, Optical Measurement of cycle-dependent growth , Proc. Natl. Acad. Sci., 108 (32), (2011).
- Z. Wang, H. Ding, and G.Popescu,Scattering-phase theorem, Opt. Lett., 36(7) (2011).
- J.P Haldar, Z. Wang, G. Popescu and L. Zhi-Pei, Label-free high-resolution imaging of live cells with deconvolved spatial light interference microscopy., Engineering in Medicine and Biology Society (EMBC), 2010 Annual International Conference of the IEEE, 3382-3385 (2010).
- Z. Wang, I.S. Chun, X. Li, Z.Y. Ong, E. Pop, L. Millet, M. Gillette, and G. Popescu, Topography and refractometry of nanostructures using spatial light interference microscopy, Opt. Let., 35, 208-210 (2010).
- Z.Wang and G. Popescu Quantitative phase imaging with broadband fields, Appl. Phys. Lett., 96, (2010).