Zernike Phase contrast for SPM

27MicroscopyandAnalysis | July 2013

Condenser-free Zernike phase contrast imaging for scanning probe microscopy Kevin F. Webb,1 Giovanna De Filippi 2 and Nicholas S. Johnston1

1. Institute of Biophysics, Imaging and Optical Science, University of Nottingham, UK.

2. Ionscope Ltd, Melbourn, Cambridge, UK.

IntroductionPhase-contrast microscopy is a laboratory workhorse technique particularly suited to cell biology since biological samples are usually quite transparent and yield poor intrinsic contrast under brightfield observation. Zernike phase contrast produces intensity contrast from refractive index variations within the sample by interfering light passing directly through the preparation with light deviated by interaction with the biological material [1].

In commercial instruments an annular ring of illumination is selected by a mask in the aperture plane of a Köhler illumination system [2], which is focused through the sample as a hollow cone by the condenser lens (Figure 1). In a conjugate aperture plane a complementary phase plate is aligned and overlapped with the illuminating annulus to produce l/4 retardation of direct-path light relative to light deviated by the sample. Interference between the direct and deviated light produces phase contrast, in which intensity contrast is produced from tiny alterations in phase caused by passage through structures of differing refractive index.

In classical commercial instruments the phase plate is usually provided inside the back focal plane (BFP) of the objective. A conventional phase condenser to produce the requisite cone of illumination contains selectable annular masks to match the phase plate, designated Ph1, Ph2, Ph3 etc., as suited to the magnification and numerical aperture (NA) of particular objectives. This condenser assembly is bulky and consists of several optomechanical components to align and focus the relevant conjugate planes for Köhler illumination.

While refinements have been made in the interim the core optical design of the phase-contrast microscope has been largely conserved from its development in the 1950s. Key innovations have been the move towards infinite-conjugate optics by commercial manufacturers and the use of apodisation to minimise halo artifacts

[3]. To enable high-quality multimodal imaging, Nikon recently offered an external phase contrast option allowing the use of non-phase-contrast objectives for fluorescence and other applications in conjunction with a phase plate positioned in a conjugate aperture plane within the trinocular head (Nikon Ti-Eclipse). In order to match very high-NA objectives such as the TIRF series (1.49 NA), a very high-NA condenser assembly is required which further restricts the available space above the sample.

Various electrophysiological techniques such as patch clamping and microelectrode recording can be hampered by the presence of the condenser assembly, restricting access to the preparation in the inverted microscope configuration. The usual solution is to approach the preparation at a shallow angle, in the manner of a snooker player; this is practical for static recordings from individual cells but unsuitable for scanning probe systems where orthogonal access to the preparation is required.

One such example is the scanning ion conductance microscope (SICM), where a glass nanoelectrode is manipulated under closed-loop control in order to

map the three-dimensional surface of a sample under feedback from a high-precision current amplifier [4]. SICM is especially suited to imaging live cells because samples are scanned in conductive, physiological saline media and the feedback signal detects impedance changes at the electrode tip prior to contact with the sample. SICM as a rapidly growing non-contact non-invasive SPM modality has been recently applied to the localisation and delineation of individual populations of ion channels [5], to guide and redirect neuronal growth cones [6] as well as being integrated with other advanced SPM modalities including scanning electrochemical microscopy (SECM) [7].

However, SICM, SECM, and AFM share with other SPM modalities the disadvantage that phase and other contrast enhancement methods are not generally possible in commercial systems because the instrument or electrode body obstruct the transmitted light path (Figure 2 a,b). Off-axis, suboptimal illumination (Figure 2 c) and meniscus effects (Figure 2 d) grossly compromise image contrast and make vertical movements of the electrode

Figure 1 Conventional schema for phase-contrast microscopy using infinite conjugate optics. A collimated light source is spatially selected by an annulus in a conjugate aperture plane and is focused by the condenser into the sample as a hollow cone of light. A phase ring in a conjugate aperture plane imposes l/4 retardation on direct path light while interaction with the sample causes a portion of the light to be deviated (Deviated path) and therefore miss the phase ring. Interference between deviated and direct-path light creates contrast in the resulting phase-contrast image.

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difficult to control. A SPM-compatible phase contrast or similar contrast-enhancing method is required to allow robust viewing of samples and accurate electrode positioning for precision SICM scans.

We present here a general schema for condenser-free phase-contrast microscopy that is fully compatible with commercial phase-contrast optics. The chief advantage of this schema is that it abrogates the condenser assembly, allowing the precise alignment of phase-contrast illumination for objectives of any magnification or NA. Phase contrast in a hybrid AFM system was previously demonstrated in one paper using a fixed-geometry LED ring built into the cantilever mount [8]. The alternative schema proposed here circumvents many issues of condenser-based illumination, allowing the seamless integration of phase contrast with SPM and other methods while maintaining excellent phase contrast (Figure 2 e). Presented here is the first implementation of true phase-contrast microscopy simultaneously with SICM, allowing accurate electrode positioning and multimodal imaging in an unprecedented combination of techniques.

Materials and Methods

Cells ARPE-19 (human-derived retinal pigment epithelium) and 3T3 (mouse fibroblast) cultured cell lines were plated on poly-L-lysine-coated glass-bottomed Petri dishes in DMEM/Ham’s F12 or DMEM media supplemented with 2.5 mM L-1 glutamine, 10% foetal bovine serum, 100 U mL-1 penicillin and 100 µg mL-1 streptomycin. Cells were cultured for either 24 h (for individual cells) or 72 h (for confluent cells). For SICM imaging, cells were plated in filtered phosphate buffered saline at 300 mOsm kg-1, pH 7.4, and recordings were taken at room temperature under atmospheric [CO2]. For DNA staining, 1:1000 Hoechst stain stock (Sigma-Aldrich) was added to the bath and imaged using Hg lamp illumination (Ex: 355/25, Em: 420 LP).

MicroscopeS We used a Nikon Ti Eclipse inverted microscope body without the brightfield condenser assembly but fitted with an external phase ring turret. The Nikon objectives used were: 10x/0.3NA Ph1; 20x/0.45NA Ph1 ELWD; 40x/0.6NA Ph2 ELWD; 60x/1.49NA TIRF (with requisite external Ph3/60x phase ring.

ICNanoP (pipette-scan) or ICNanoS2 (sample-scan) SICM systems (Ionscope Ltd, Mebourne, UK) were mounted with the scanned nanoelectrode parallel to the optic axis.

LED illuminator Bespoke LED ring printed circuit boards (PCB) were fabricated using a 13-mm

Figure 2 (a) Scanning ion conductance microscopy on an inverted microscope. A glass microelectrode is moved under closed-loop feedback referenced to current leaving the tip, and is scanned in XYZ to build an image. The electrode holder and motion control apparatus prevent the use of a condenser. (b) Transmitted light path, as obstructed by the SICM instrument. (c) Maximum contrast obtained from a buccal epithelial cell in brightfield illumination. (d) Brightfield image with electrode (arrowhead) present: meniscus effects further degrade contrast. (e) True phase-contrast image obtained using the novel condenser-free phase contrast illuminator described in this article.

diameter closed ring of 24x SMD0603 emitters (Kingbright, lpeak 515 nm, Dl1/2 30 nm) and 14-mm diameter notched ring of 13x SMD1206 emitters (Kingbright, lpeak 515 nm, Dl1/2 30 nm). LEDs were driven by a regulated constant-current supply and positioned using a bespoke manipulator.

Imaging Cells were mounted in glass-bottomed

Petri dishes inside the Faraday cage of the SICM instrument on the stage of the inverted microscope. The SICM signal was referenced to a pair of AgCl electrodes, inserted into both nanoelectrode and the bath. The LED ring illuminator was manually positioned around the electrode close to working position, and aligned by inspecting the back focal plane using a Bertrand lens to overlie the phase ring in the conjugate aperture

Figure 3 (a) Illumination schema for condenser-free phase contrast illumination, wherein a ring of LEDs is positioned around the electrode. The objective lens creates a simple image of the LEDs at its back focal plane, conjugate with the phase ring. (b) Adjustment of LED ring XY position allows alignment with the phase ring. Z adjustment alters the diameter of the ring. (c) LED illuminator PCB in working position around the electrode of an ICNanoS2 SICM instrument. This high dynamic range image has been tone-mapped in Adobe Photoshop.

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Figure 4 (a) Brightfield image of confluent ARPE-19 cells. (b) Closed LED ring (0603 SMD, 24 emitters, 13-mm diameter). (c) Notched LED ring (1206 SMD, 13 emitters, 14-mm diameter). (d) Commercial phase-contrast image of cells in panel A using 10x/0.3NA Ph1 objective and matched condenser annulus (Nikon Ti-Eclipse). (e) Condenser-free phase-contrast image of cells in panel A using closed LED ring in panel B. (f) Condenser-free phase-contrast image of cells in panel A using notched LED ring in panel C. Scale bar = 50 μm.

plane (Figure 3). Widefield phase-contrast and epifluorescence images were acquired using an OpticStar DCM35 camera attached to the side-port of the external phase contrast unit. Hopping-mode SICM scans were obtained to show 3D morphological features of the cell membrane using either sample scanning (ICNanoS2) or pipette scanning (IPNanoP) as described according to the experiment.

Results and discussionSimple lens optics ensures that the ring of LEDs, being further from the objective than its focal length, are imaged into the objective back focal plane (BFP). Inspection of the BFP using a Bertrand lens visualises both the phase ring as well as the ring of LEDs, which may then be aligned using a bespoke manipulator. XY adjustments of the LED ring produce XY movements in the BFP, while vertical adjustments in Z cause the apparent diameter of the LED ring image to vary according to perspective: appearing larger when the ring is moved closer to the sample and smaller when the ring is moved further away (Figure 3).

Due to the requirement for a notch to fit around the electrode in SICM we tested the relative performance of our LED rings in closed (Figure 4 b) as well as notched (Figure 4 c) configurations. Confluent ARPE-19 cells were imaged using standard commercial phase contrast optics (10x/0.3NA Ph1, Figure 4 d), which produced the expected phase-contrast enhancement compared with brightfield (Figure 4 a). Phase contrast was found to be functionally equivalent between our rings (Figure 4 e,f) and conventional condenser-based illumination (Figure 4 d). Furthermore, it was demonstrated that the performance of the “notched” ring (Figure 4 f) was functionally equivalent to the closed ring (Figure 4 e).

Using a fixed sized ring (notched, 14 mm diameter, 13 emitters, Figure 4 c), we then tested the ability of a single illuminating LED ring to compensate for different phase geometries by testing a range of objectives while inspecting the BFP (Figure 5 a). Using a preparation of fresh buccal epithelial cells, it was demonstrated that a single ring of fixed size could be matched to provide true Zernike phase contrast at 10x/0.3NA Ph1 (Figure 5 b), 20x/0.45NA Ph1 (Figure 5 c) and 40x/0.6NA Ph2 (Figure 5 d). Excellent phase contrast was obtained at each position by careful XY and Z adjustments to align the BFP image with the phase ring inside each objective. Note that no adjustment was necessary between 10x and 20x objectives as both were Ph1.

SPM methods, and SICM in particular, have historically required long working distance objectives in order to locate and align the scanned nanoelectrode.

To demonstrate the utility of this improved condenser-free illumination method in high-NA, high-magnification

Figure 5 (a) LED ring aligned with the phase plate, viewed via Bertrand lens focused on objective back focal plane. (b) Phase contrast image of buccal epithelial cells, 10x/0.3NA Ph1 objective. (c) Phase contrast image of cells in panel A, 20x/0.45NA Ph1 ELWD objective. (d) Phase contrast image of cells in panel A, 40x/0.6NA Ph2 ELWD objective. Scale bars = 50 μm.

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Figure 7 (below)(a) ARPE-19 cell under phase contrast illumination (40x/0.6NA Ph2 ELWD), scale box 40 μm. (b) Corresponding back focal plane of panel A, showing alignment of LED ring with phase plate. (c) Scanning ion conductance image, taken using sample scanning in hopping mode (ICNanoS2). Scale box 15 μm. (d) 3D rendering of dataset from panel C (SPIP software, Image Metrology, Hørsholm, Denmark). (e) ARPE-19 cell under phase contrast observation as in panel A, with electrode inserted (arrowhead). (f) Corresponding back focal plane image from panel E. (g) SICM image of area delimited in panel C. (h) 3D rendering of dataset from panel G.

objectives we used a 60x/1.49NA TIRF lens to perform phase contrast (Figure 6) using Nikon’s external phase contrast module. By aligning the LED ring in the BFP then progressively misaligning the phase ring relative to the LEDs using the turret centration we were able to demonstrate perfect phase contrast microscopy (Ph3/60x, Figure 6 a) which collapsed progressively via an apparent “relief” appearance (Figure 6 b) to poorly contrasted brightfield (Figure 6 c) as the rings were deliberately misaligned.

In living cultures of ARPE-19 cells, we demonstrated the utility of our novel condenser-free schema in locating and accurately targeting individual cells by SICM under phase contrast observation (Figure 7). We further demonstrated that introducing the electrode into the preparation had negligible effect on phase contrast (Figure 7 a,e). In addition, observing the BFP (Figure 7 b,f) revealed that subsidiary reflections from the shaft of the electrode could be used to locate and centre the electrode in the optical path when still at some considerable distance from the sample (Figure 7 f). This trick allows short working distance objectives to be used reliably in SICM. Hopping-mode sample-scanning SICM measurements (ICNanoS2) were thus made possible in individual cells targeted from phase contrast images (Figure 7 c,d) as well as subcellular features such as lamellipodia and filopodia in live cells (Figure 7 g,h).

In order to demonstrate the enormous potential for multimodal imaging in the functional investigation of living cells by parallel SICM and high-resolution widefield microscopy, cultures of 3T3

Figure 6 (a) Objective back focal plane viewed through Bertrand lens with Nikon external phase ring (Ph3) in conjugate plane, aligned with LED ring. (b) Corresponding phase contrast image of buccal epithelial cell using 60x/1.49NA TIRF objective. (c) Back focal plane as above but phase plate slightly misaligned with LED ring. (d) Corresponding brightfield image from panel C, showing loss of phase contrast and pseudo-relief appearance. (e) Back focal plane with profound misalignment of phase plate with LED ring. (f) Corresponding brightfield image from panel E, showing complete collapse of contrast. Scale bars 20 μm.

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fibroblasts were labelled to reveal DNA dynamics during mitosis (Figure 8). Mitotic figures representing all stages from chromatin condensation through cytokinesis were evident within the preparation (Figure 8 b,c), which between phase contrast and widefield epifluorescence allowed the precise targeting of cells at particular stages. Pipette-scanning hopping mode SICM (ICNanoP) was performed to demonstrate multimodal phase contrast (Figure 8 a), epifluorescence (Figure 8b), and SICM imaging (Figure 8 d,e) of individually targeted cells. Since pipette scanning avoids multiple issues with plumbing and electrical interconnection, we believe we have a system in which dynamic physiology and cell biology will now be accessible in parallel using optics no longer constrained by the steric hindrance of the SICM instrument.

ConclusionsWe have demonstrated that condenser-free true Zernike phase contrast is obtainable in scanning probe systems by using the objective lens to image a ring of surface-mount LEDs into the aperture plane of a phase-contrast imaging system. This schema provides a novel solution to the problem of simultaneous label-free imaging of live cells while performing SPM measurements, and will be highly beneficial to users of such technologies in many biological systems. By removing the need for a bulky condenser assembly and providing for a centrally-mounted electrode, excellent phase-contrast

biography Kevin Webb received his BSc(Hons) and PhD in physiology from the University of Auckland, New Zealand. He is currently Royal Academy of Engineering Research Fellow at the Institute of Biophysics, Imaging and Optical Science (IBIOS), University of Nottingham. His lab develops novel imaging and electrophysiological methods to study fluid transport and volume regulation in transparent tissues of the eye.

abstractScanning ion conductance microscopy (SICM) is an advanced technique for non-contact 3D live cell imaging which uses a scanned nanoelectrode to map morphology in conductive physiological media. While SICM is very useful, parallel brightfield image contrast in live cells is compromised by interruption of the transmitted light path. Here we present a general schema for condenser-free phase-contrast microscopy that is fully compatible with conventional optics. The chief advantage of this design is that it abrogates the condenser assembly, allowing the precise alignment of true phase-contrast illumination for objectives of any magnification or NA in parallel with scanning probe microscopy. Keywords: scanning ion conductance microscopy, SICM, phase contrast microscopy, live cell imaging, cultured cells, scanning probe microscopy

acknowledgementsThis work was supported by a Royal Academy of Engineering/EPSRC Postdoctoral Fellowship. We thank Dr Emilia Moradi (IBIOS) for preparing cells.

Corresponding author details Dr Kevin F. WebbInstitute of Biophysics, Imaging and Optical Science (IBIOS),School of Electrical and Electronic Engineering,The University of Nottingham, University Park,Nottingham, Notts NG72RD, UKTel/Fax: +44 (0)115 816 6580Email: kevin.webb@nottingham.ac.uk

Microscopy and Analysis 27(5):27-32 (EU), 2013

©2013 John Wiley & Sons, Ltd

Figure 8 (a) 3T3 fibroblasts under condenser-free phase-contrast illumination. Scale box 50 μm. (b) 3T3 cells from panel A stained for DNA with Hoechst stain. Cells with condensed chromatin, as inside the box, are about to divide (arrowhead). (c) Overlay of panels A and B showing target cell in prophase. (d) Hopping-mode ICNanoP SICM image of boxed cell in panels A-C. (e) 3D rendering of dataset in panel D (SPIP software).

illumination is obtained using rings of LEDs for phase-contrast illumination which may be geometrically matched to any objective magnification and NA. We expect that the opportunities afforded by making high-NA non-phase objectives wieldable in parallel with SICM, as well as other SPM modalities, may offer significant potential benefits in studying dynamic cellular processes at the subcellular to nanoscale.

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and experiment for quantitative high resolution spatially resolved voltammetry and simultaneous ion-conductance measurements. Analytical Chemistry 84:2483-2491, 2012.8 Lugmaier, R. A. et al. Phase contrast and DIC illumination for AFM hybrids. Ultramicroscopy 104:255–260, 2005.