Spatial modulation microscopy is a technique originally developed for
quantitative spectroscopy of individual nano-objects. Here, a parallel
implementation of the spatial modulation microscopy technique is demonstrated
based on a line detector capable of demodulation at kHz frequencies. The
capabilities of the imaging system are shown using an array of plasmonic
nanoantennas and dendritic cells incubated with gold nanoparticles.Comment: 3 pages, 4 figure

A. G. Kanaras

Anker

Arbouet

Gaiduk

Johnston

Kukura

M. C. Pitter

M. G. Somekh

Muskens

N. Fairbairn

O. L. Muskens

R. A. Light

R. Carter

R. Fernandes

Smith

Sonnichsen

T. J. Elliott

van Dijk

English

arXiv

Spatial modulation microscopy (SMM) is a technique originally developed for quantitative spectroscopy of individual nano-objects. Here, a parallel implementation of the SMM technique is demonstrated based on a line detector capable of demodulation at kHz frequencies. The capabilities of the imaging system are shown using an array of plasmonic nanoantennas and dendritic cells incubated with gold nanoparticle

Fairbairn, N.

Light, R.A.

Carter, R.

Fernandes, R.

Kanaras, A.G.

Elliott, T.J.

Somekh, M.G.

Pitter, M.C.

Muskens, O.L.

Southampton (e-Prints Soton)

arXiv:1203.5966v1  [physics.optics]  27 Mar 2012Spatial Modulation Microscopy for Real-Time Imaging of Plasmonic Nanoparticlesand CellsN. Fairbairn,1 R. A. Light,2 R. Carter,3 R. Fernandes,1 A. G. Kanaras,1T. J. Elliott,3 M. G. Somekh,2 M. C. Pitter,2 and O. L. Muskens11Institute for Life Sciences and Faculty of Physical and Applied Sciences,University of Southampton, Highfield, Southampton SO17, 1BJ, United Kingdom2Applied Optics Group, Faculty of Engineering, University of Nottingham, Nottingham NG7 2RD, United Kingdom.3Institute for Life Sciences and Faculty of Medicine, University of Southampton, United Kingdom(Dated: March 28, 2012)Spatial modulation microscopy is a technique originally developed for quantitative spectroscopyof individual nano-objects. Here, a parallel implementation of the spatial modulation microscopytechnique is demonstrated based on a line detector capable of demodulation at kHz frequencies.The capabilities of the imaging system are shown using an array of plasmonic nanoantennas anddendritic cells incubated with gold nanoparticles.PACS numbers:With the increasing employment of nanomaterials inphysical and biomedical science, sensitive methods capa-ble of screening and characterizing these materials areneeded. Currently there is a range of optical microscopytechniques capable of imaging individual nanoparticles[3]. For nonfluorescent particles, interactions take placethrough absorption and/or scattering. Detection ofscattered intensity, generally known as darkfield mi-croscopy, is the most widely used technique, combiningbackground-free imaging with a high spectral selectivityfor e.g. optical sensing [1, 2]. The efficiency of light scat-tering is greatly reduced with decreasing particle size,proportional to the square of the particle volume, whichcan be overcome using more advanced interferometric de-tection schemes [3, 4]. Also scattering-based techniquesare not suitable for materials with an a small dielectriccontrast, i.e. index-matched particles in solution.A fundamentally different method of detection relies ondetection of absorption rather than scattering of light.The most sensitive of these techniques is photothermalimaging, where a thermal response caused by local ab-sorption can be detected with single-molecule sensitivity[5]. Photothermal imaging methods require a relativelyhigh laser intensity (MW/cm2) over a diffraction lim-ited spot, which limits the scalability of photothermalimaging in real-time and live cell applications. Spatialmodulation microscopy (SMM) has been introduced asa technique for quantitative analysis of the optical ex-tinction cross-section of small metal nanoparticles [6, 7].The method is based on recovery of a small modulationcomponent in the transmitted (or reflected) light usinglock-in detection. This spatial modulation is achieved bymeans of a periodical displacement of the specimen ina Gaussian laser focus. While the technique is less sen-sitive than photothermal imaging, it allows for precisein-situ quantitative spectroscopy of nanomaterials whichcan then be correlated to other properties such as theirultrafast response [8]. In addition, the SMM techniqueis scalable as it works at moderate optical intensities, i.e.a few W/cm2 per pixel, and requires only a small focalwidth in one dimension. No reports have been made yeton the potential of SMM for real-time imaging in biolog-ical systems.Here, we demonstrate the integration of SMM into aviable imaging system covering an area of tens of mi-crometers at a rate of around one image per second. Thefast scanning spatial modulation method, makes use of arecently developed CMOS camera technology combiningmultiple wells and a phase stepping technique to detecta modulating signal [9, 10]. The line camera replacesconventional lock-in amplification to detect the modulat-ing signal produced by the spatial modulation technique.The camera features a pixel well depth of 2 billion elec-trons, providing a theoretical shot noise level of 2× 10−5per well. In addition, a series of four on-chip wells perpixel allows fast acquisition of four frames in each modu-lation period, which can be used to recover the first andsecond harmonic spatial modulation at kHz frequencies.The resulting intensities are converted into 16-bit inte-gers, each count representing 3× 104 electrons.The experimental setup is shown in Fig. 1. A trans-mission microscope was used consisting of two 100×, 0.9NA microscope objectives. A vertical line focus was cre-FIG. 1: Experimental setup for spatial modulation mi-croscopy.2FIG. 2: a) SEM images of nanoantennnas, with close up ofsingle antenna. (b) Experimental data for a single antenna forintensity T−〈T 〉, and SMM profiles at first (∆T1f ) and secondharmonic (∆T2f ) of the modulation frequency, together withfits (lines) to the derivatives of a Gaussian beam profile of0.8± 0.01 µm width.ated from a 690 nm beam by imaging of the focus of acylindrical lens onto the sample. The transmitted lightwas subsequently collected by the second objective andimaged onto the CMOS line array. Spatial modulationwas implemented by periodically tilting of a custom-builtpiezo-actuated flexure mirror at a frequency of 1.3 kHz.The microscopy objective acts as a fourier element con-verting the angular tilt into a spatial displacement of theline focus. This method greatly improves the flexibilityof the SMM method in combination with bulky or liquidspecimens compared to previous implementations basedon a piezoelectric sample displacement [6]. In order tooptimally exploit the dynamic range of the 256-pixel ar-ray at a typical frame rate of 5200 frames per second,a total illumination power of around 2 mW is required.Given the optical resolution of 0.2 µm per pixel this re-sults in an average local intensity of around 10 W/cm2.The SMM imaging performance was tested by mappingan array of lithographically defined gold nanoantennason an ITO coated glass substrate. The nanoantennas,shown in Fig. 2(a), comprise of two arms of 200 nm inlength and 100 nm wide, with a a thickness of 25 nm, andwere designed to have a resonant dipole mode at around850 nm [8]. Two-dimensional SMM maps were made ofthe transmitted intensity T as well as the first and secondharmonic components of the spatial modulation intensity,respectively denoted as ∆T1f and ∆T2f [6]. For the in-tensity graph, an average line frame in absence of anten-nas (denoted as 〈T 〉) was subtracted to remove a constantvariation in pixel illumination. Figure 2(b) shows typicalcross-sections through a single antenna selected from alarger image shown in Fig. 3. The signals can be wellFIG. 3: Spatial modulation microscopy maps of intensity dif-ference from average (i), first harmonic (ii) and second har-monic components (iii) of the spatial modulation signal. Linegraphs (I-III) are cross sections of the maps normalized tothe average intensity, with (grey lines) noise background af-ter subtraction of moving average.fitted to a Gaussian beam profile for T and its first andsecond derivatives for ∆T1f and ∆T2f , as expected fromtheory [6]. The fits yield a full-width-at-half-maximumof 0.8± 0.1 µm. This is larger than the diffraction limitdue to aberrations in the imaging system. The depth offocus of the SMM signals follows the divergence of theGaussian beam and results in a decay over ∼ 1.5 µm.The absence of an out-of-focus background signal is ofpotential use in imaging of three-dimensional specimens.Figure 3 shows the SMM image obtained for a 20 ×25 µm2 area containing nanoantennas. Each line wasaveraged over 4 frames, resulting in a total acquisitiontime of 2 seconds. This is an improvement in collectiontime by more than two orders of magnitude comparedto conventional point-scanning SMM [6]. Only a central120-pixel part of the 256-pixel array in the y-direction isdisplayed containing the antenna array. Horizontal cross-sections through a row of antennas (indicated by arrows)are shown in Fig. 3(b), where the 1f and 2f componentshave been normalized to the average intensity 〈T 〉 to ob-tain the relative spatially modulated transmission.3FIG. 4: i - Intensity, ii - 1f and iii - 2f SMM images of dendriticcells along with a cross section.The noise level of the measurement was calculated bysubtracting a 5 point moving average from the originaltraces taken at a horizontal step size of 50 nm. Result-ing noise traces are given by the grey lines in the crosssections of Fig. 3(b). We obtained average noise levelsof 3 × 10−4 and 1 × 10−4 for the 1f and 2f signals re-spectively. The noise level is exceeds the shot-noise dueto the presence of some technical noise in our system atkHz frequencies. The signal to noise ratio exceeds 100for the antennas under study at the first harmonic fre-quency, demonstrating the capability of the system toimage individual nanostructures.In a second experiment, a sample of dendritic cells wasincubated with 5µl of a CTAB-coated gold nanorod so-lution for 2 hours. The gold nanorods, of 16 × 42 nm2average dimensions, have a strong optical resonance ataround 690 nm corresponding to a longitudinal surfaceplasmon resonance. Figure 4 shows an image of a den-dritic cell obtained using the intensity, 1f and 2f com-ponents of the SMM signal, along with a cross section ofeach image taken at the position indicated by the arrows.The expected SMM signal from individual nanorods isaround 10−3 [7], i.e. much smaller than the cellular back-ground signals in Fig. 4. The lack of specific contrastthus limits the efficacy of SMM in imaging nanoparticlesin cells compared other methods such as photothermalimaging. The 1f and 2f components give the first andsecond derivatives of the light transmitted through thecell, which results in an enhancement of edges and point-like objects. This sensitivity of the SMM signal to smallvariations in optical density in the cell may be of interestas an alternative to phase-sensitive imaging, i.e. phase-contrast or Nomarski, under conditions that phase infor-mation is distorted, e.g. for very thick or turbid samples.In conclusion, a parallel implementation of spatialmodulation microscopy has been demonstrated. The the-oretical sensitivity of the CMOS demodulation is com-parable to state-of-the-art lock-in amplifiers, and thecurrent system allows resolving an array of plasmonicnanoantennas of 200 nm with a signal-to-noise ratio of100. This may be improved by reducing sources of clas-sical noise in the system, optimization of the diffraction-limited focus, and averaging over pixels and/or time. Keyaspects of SMM are retained by the setup while improv-ing the acquisition time by several orders of magnitude,therefore opening up new applications requiring simul-taneous imaging and measurement of multiple nanopar-ticles e.g. in solid or microfluidic environments. Theabsence of out-of-focus signals and availability of higher-order derivatives make SMM a potentially useful alter-native or addition to phase-contrast techniques.The authors acknowledge M. Abb for fabrication ofnanoantennas and H. Warren for help with cell prepara-tion.[1] C. Sonnichsen, B. M. Reinhard, J. Liphardt, A. P.Alivisatos, Nat. Biotechnol. 23, 741 (2005).[2] J. N. Anker, W. P. Hall1, O. Lyandres, N. C. Shah, J.Zhao, R. P. Van Duyne, Nat. Mater. 7, 442 (2008).[3] M. A. van Dijk, A. L. Tchebotareva, M. Orrit, M. Lip-pitz, S. Berciaud, D. Lasne, L. Cognet, B. Lounis, Phys.Chem. Chem. Phys. 8 3486 (2006).[4] P. Kukura, H. Ewers, C. Mu¨ller, A. Renn, A. Helenius,V. Sandoghdar, Nat. Methods 6, 923 (2009).[5] A. Gaiduk, M. Yorulmaz, P. V. Ruijgrok, M. Orrit, Sci-ence 330, 353 (2010).[6] A. Arbouet, , D. Christofilos, N. Del Fatti, F. Valle´e, J.R. Huntzinger, L. Arnaud, P. Billaud, M. Broyer, Phys.Rev. Lett. 93, 127401 (2004)[7] O. L. Muskens, G. Bachelier, N. Del Fatti, F. Valle´e, A.Brioude, X. Jiang, M. Pileni, J. Phys. Chem. C 112, 8917(2008).[8] M. Abb, P. Albella, J. Aizpurua, O. L. Muskens, NanoLett. 11, 2457, (2011).[9] R. J. Smith, R. A. Light, S. D. Sharples, N. S. Johnston,M. C. Pitter, M. G. Somekh, Rev. Sci. Inst. 81, 024901(2010).[10] N.S. Johnston, C. Stewart, R.A. Light, B.R. Hayes-Gill,M.G. Somekh, S.P. Morgan, J.R. Sambles, M.C. Pitter,Electron. Lett. 45, 1090 (2009).

Spatial modulation microscopy for real-time imaging of plasmonic nanoparticles and cells

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Optics Letters

Spatial Modulation Microscopy for Real-Time Imaging of Plasmonic
  Nanoparticles and Cells

Spatial Modulation Microscopy for Real-Time Imaging of Plasmonic Nanoparticles and Cells

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