Journal of Microscopy, Vol. 260, Issue 1 2015, pp. 62–72
doi: 10.1111/jmi.12267
Received 11 June 2014; accepted 26 April 2015
Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components M A R T I N V O G E L ∗ , A X E L W I N G E R T †, R A I N E R H . A . F I N K †, C H R I S T I A N H A G L ‡, F E R U Z G A N I K H A N O V § & C H R I S T I A N P . P F E F F E R ‡, ∗ Center for Nanoscale Systems, Harvard University, Cambridge, Massachusetts, U.S.A.
†Medical Biophysics Group, University of Heidelberg, Heidelberg, Germany ‡Department of Cardiac Surgery, Ludwig-Maximilians-Universitaet, Munchen, Germany §Department of Physics, University of Rhode Island, Kingston, Rhode Island, U.S.A. Department of Craniofacial and Developmental Biology, Harvard Medical School, Boston, Massachusetts, U.S.A.
Key words. Coherent anti-Stokes Raman scattering, condenser, multimodal nonlinear microscopy, third harmonic generation, third-order sum frequency generation, ultraviolet transmission. Summary Using an optical system made from fused silica catalogue optical components, third-order nonlinear microscopy has been enabled on conventional Ti:sapphire laser-based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of 300 nm and in multimodal nonlinear optical imaging experiments using third-order sum frequency generation and coherent anti-Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from 300 nm to 660 nm. Introduction In the last decades, multiphoton excited fluorescence microscopy (2PEF, 3PEF; Denk et al., 1990; Hell et al., 1996; Maiti et al., 1997) has seen a continuing growth in applications. This is also true for multiharmonic microscopy, for example, second harmonic generation microscopy (SHG; Hellwarth & Christensen, 1974; Gannaway & Sheppard, 1978) and third Correspondence to: Martin Vogel, Max Planck-Institute of Biophysics, 60438 Frankfurt, Germany; Tel: +49 69 6303 4640; fax: +49 69 6303 4602; e-mail: [email protected]
harmonic generation microscopy (THG; Barad et al., 1997; Mueller et al., 1998; Millard et al., 1999), sum frequency generation microscopy (SFG; Fl¨orsheimer et al., 1999) or coherent anti-Stokes Raman scattering microscopy (CARS; Duncan et al., 1982; Zumbusch et al., 1999) that allow, among others, the characterization of surfaces and nanoparticles (e.g. Shen, 1989; Gauderon et al., 1998; Hsieh et al., 2010; Pantazis et al., 2010). These techniques are used to investigate the physiology or pathophysiology of biological material (Peleg et al., 1996, 1999; Campagnola et al., 1999; Rama et al., 2010) and for morphological (Yelin & Silberberg, 1999; Potma et al., 2001; Campagnola et al., 2002; Cheng et al., 2002; Both et al., 2004; Oron et al., 2004; Evans et al., 2005; D´ebarre et al., 2006; L´egar´e et al., 2007; Plotnikov et al., 2008; Friedrich et al., 2010; Pfeffer et al., 2011; Raghunathan et al., 2011; Recher et al., 2011) or for functional tissue imaging (Nuc¨ ciotti et al., 2010; Schurmann et al., 2010), even in humans (Llewellyn et al., 2008). These imaging modalities probe different and nonoverlapping characteristics of samples calling for multimodal imaging approaches (Chu et al., 2001, 2004, 2005; Nemet et al., 2004; Fu et al., 2007; Hsieh et al., 2008; Wallenburg et al., 2010; Rehberg et al., 2011), so that the availability of as many imaging modalities as possible on a given multiphoton microscopy system is a useful and important asset to many fields of research. A high numerical aperture (NA) objective lens is a common element for the mentioned imaging modalities. The focused laser beams are being scanned across the sample, whereas generated signal photons can be collected using different geometries, with high-efficiency photo detectors. The nonlinear character of a multiphoton process leads to further sharpening of the probed microscopic volume within the sample foci (Sun et al., 2002).
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DETECTION OF UV SIGNAL IN MULTIMODAL NONLINEAR MICROSCOPY
Nonlinear microscopies put further requirements to optical systems: They have to perform over a wider spectral range compared to the classical optical microscope. This is especially true for imaging modalities of higher order (THG, SFG) where excitation and emission wavelength differ by a factor of up to three. This is obviously a much larger difference than, for example, a Stokes shift of only few nanometers known for many fluorescent dyes (Lakowicz, 2010). Diffraction limited performance and minimized aberrations are required for the objective lens. These can be loosened for the condenser lens as it only serves to collect signal photons. However, for scanning laser microscopy, the condenser should show minimal vignetting. In addition, for multimodal studies using multicolour lasers, the performance of the condenser should not depend on the collected signal wavelengths so that adjustments during the experiment can be avoided. A significant constraint in the optical design of such a condenser is the fact that most glasses are almost opaque for the near ultraviolet (NUV) range of wavelengths (λ < 350 nm). Concerning the laser source for two-photon excited fluorescence microscopy, mode-locked titanium sapphire (Ti:S) lasers are a common and preferred choice. This is due to its relatively broad tuning range (680 nm to 1100 nm) and its wavelength range compatibility with two-photon absorption bands of many standard fluorescent dyes. Within the same system, SHG signals can be easily generated and detected using driving field laser wavelengths within the 800–1000 nm range as the corresponding SHG signals then fall within the visible range at which standard microscope optics is perfectly optimized. For SHG wavelengths between 360 nm and 400 nm, the selection of suitable microscope optics already becomes less straightforward whereas for wavelengths below 350 nm, the inherent opacity of many glasses comes into effect. Both theoretical predictions and experimental evidences show that second-order nonlinearity for important media of both inorganic and biological origins like nanoparticles or protein collagen is significantly increased for the wavelength range below 350 nm (Oudar & Chemla, 1977; Zipfel et al., 2003; Harnagea et al., 2010; Butet et al., 2012; Hall et al., 2014). Thus, an extension of the standard instrumental capabilities to shorter wavelengths is highly desirable. With a Ti:S laser, third harmonic signals will have wavelengths between 230 nm and 330 nm. The signals will always get strongly attenuated by both microscope optics and even by the microscope glass slide or the cover slip as transmission data for Schott cover glass (type ‘D 263M’ of thickness 150 μm) suggest indicating only 10% transmission at 300 nm. Therefore THG and SFG microscopies are usually carried out with lasers of longer wavelengths as it was demonstrated with ytterbium doped fibre oscillators tuned to 1030 nm (Gualda et al., 2008; Tserevelakis et al., 2011), optical parametric am¨ plifiers and oscillators tuned to 1200 nm (Muller et al., 1998; Squier et al., 1998; B´elisle et al., 2008; Chang et al., 2010; C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
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Rehberg et al., 2011), and chromium doped forsterite laser emitting at 1230 nm (Chu et al., 2001, 2005; Yu et al., 2008; Lee et al., 2010). In the referenced studies, authors argue that photo damage and scattering are minimized due to the longer wavelength for the driving field. However, transmission of microscope optics can be poor within the mentioned wavelength ranges and the capability to simultaneously excite blue/green fluorescent dyes is lost, unless three-photon excited fluorescence is used with a laser source that is tuned to even longer wavelengths in the 1400 nm to 1500 nm range (Yelin & Silberberg, 1999; McConnell, 2007). Yet, the wavelength tuning may come at a cost of increased absorption of light in water (Hale & Querry, 1973) that is ubiquitous in life biological sample. Thus, it is tempting to develop THG imaging capabilities with a Ti:S laser that is still the standard laser source for many commercial multiphoton microscopes. Yelin and co-workers (Yelin et al., 2002) have demonstrated two photon excited fluorescence and THG with a Ti:S laser, but unfortunately their approach has not triggered broader application. They have custom-built and modified an inverted microscope for THG imaging with a laser scanning system and a UV-transmissive lens combination in the detection path (assumingly G063011000, then sourced from LINOS, Gottingen, Germany). From the latter, we expect worse achromatic properties and less optimization for multimodal imaging (see the “Discussion” section). Our approach was therefore to design and build a condenser system with improved achromatic properties that is purely made from NUV to near infrared transmissive standard lens elements. Designed as a drop-in replacement for the condenser, our system was made to directly fit the stand of our laser scanning microscope system and we were careful to maintain all bright field imaging capabilities. We show that our condenser system enables both THG imaging with a Ti:S laser and multimodal multiharmonic imaging on a standard Zeiss multiphoton laser scanning microscope setup (LSM510META). In addition, using a Leica multiphoton system (SP2) as an example, we show that our condenser can be readily adapted and used with other microscopy brands and designs. Experimental work Design considerations Although the optical industry offers the manufacturing of customized lens components, we decided to design the condenser from standard catalogue elements to limit cost, to expedite the setup in time and to ease cloning and adaptation to different microscope setups. Standard NUV-transmissive components are usually made from calcium fluoride (CaF2 ), magnesium fluoride (MgF2 ) or UV-grade fused silica. It was our impression at the time of design that more suitable and less expensive components were available made from fused silica. The lenses
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and mechanical mounts have been sourced from Thorlabs (Thorlabs, Inc., Newton NJ, U.S.A.) and LINOS (now: Qioptiq Photonics GmbH & Co. KG, Gottingen, Germany) to maintain mechanical compatibility with the predominant percentage of the components at the ‘Advanced Microscopy Laboratory’ at the ‘Center for Nanoscale Systems’ at Harvard University. We are nevertheless certain that components from other sources would have performed in a comparable way. The fused silica glass used to produce those lenses (Corning 7980) is highly transmissive for light between 250 nm and 2 μm. With the exception of a small dip at 1400 nm, it shows an almost flat transmission of >90% for material 1 mm in thickness between these wavelength limits (see data sheet ‘HPFS Fused Silica Standard Grade’, Corning Inc., Corning, NY, U.S.A.). To maintain this broad and flat transmission characteristic, we have not used components coated with anti reflection layers. However, our design has several glass–air interfaces so that an overall on-axis transmission of 0.97 50% can be expected; a revised design could therefore benefit from such coatings for specific wavelength ranges. As design goals, we have defined the following conditions: The condenser must be able to collect all the light from a telecentric source field, 1 mm in diameter, with an NA as large as possible. On the image side, the dimension of the light ray footprint should be minimized for improved light detection. To fit with the geometry of our microscope, a Zeiss Axiovert 200 with a LSM510META scan head (Carl Zeiss Microimaging, Thornwood, NY, U.S.A.), and to enable bright field illumination through the condenser, the condenser should produce a real image at 240 mm away from the object plane. However, being a condenser lens, diffraction-limited imaging quality was no primary design criterion. Given the restricted choice of UV transmitting but IR laser blocking filters, we have set the primary design wavelengths to 300 nm, corresponding to THG signals excited with a Ti:S laser tuned to 900 nm, and to 660 nm where CARS signals were generated with synchronized 817 nm and 1064 nm laser sources, the wavelength configuration that enables thirdorder resonant signal associated with an important CH2 bond vibration resonance at 2850 cm-1 . Secondary design wavelengths were chosen at 450 nm (SHG from the 900 nm laser source) and at 408 nm, where SHG signals from the 817 nm laser source are expected. The lens was designed with the help of ZEMAX (ZEMAX Development Corporation, Bellevue, WA, USA) and OpTaliX (Optenso Optical Engineering Software, Heerbrugg, Switzerland) software, respectively. From the supplied software catalogues, ‘F_SILICA’ and ‘SILICA’, respectively, have been used as glass models and component prescriptions were loaded from the ‘Thorlabs’ and ‘Linos’ component catalogues respectively. Panel A of Figure 1 shows a typical ray trace through the optical elements of the condenser system. Red, blue and green rays are sourced from the central field point and from field points 0.5 mm left and right off-centre, respectively.
Fig. 1. Setup of the condenser system. Panel A shows a ray trace through the optical elements of the lens rendered with OpTaliX software. Red rays source from the central field point, blue and green rays source from field points 0.5 mm left and right off-centre, respectively. Scale bar: 100 mm. In panel B, a sectional view of the mechanical model of the condenser is shown (Alibre Design software), including retaining rings and other elements that introduce additional optical apertures, see text. In panel C, we finally show a photograph of the condenser as it was built and used (photograph courtesy of Arthur McClelland, Center for Nanoscale Systems, Harvard University).
Optical lens design After an initial optical design was established, the mechanical setup was realized and subsequently modelled with CAD software (Alibre Design, Alibre Inc., Richardson, TX, U.S.A.). Off-the-shelf parts for lens mounting were added to the model, C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
DETECTION OF UV SIGNAL IN MULTIMODAL NONLINEAR MICROSCOPY
and the latter caused a slight adjustment of the optical distances and a final refinement of the optical model. The surface data of this final condenser system are listed in Table 1. The design consists of two lens groups, group I and group II, and an aperture iris placed between these groups. Lens group I is common to all designs and it is compiled from four components, LC4357 (Thorlabs, nominal focal length f1 = –50 mm, d1 = 12.7 mm in diameter), LA4647 (Thorlabs, f2 = 20 mm, d2 = 12.7 mm), LC4888 (Thorlabs, f3 = –100 mm, d3 = 25.4 mm) and LA4306 (Thorlabs, f4 = 40 mm, d4 = 25.4 mm). Lens group II – specifically designed for the Zeiss stand – is made from three components, LA4904 (Thorlabs, f5 = 150 mm, d5 = 50.8 mm), LB4842 (Thorlabs, f6 = 200 mm, d6 = 50.8 mm) and LA4984 (Thorlabs, f7 = 200 mm, d7 = 50.8 mm). Optical design analysis At a wavelength of 300 nm and for a stop aperture 12 mm in diameter (see surface #19 and #20 in Table 1), optical modelling with an object field 1 mm in diameter yields an effective focal length of 26 mm and an object side NA of 0.29. The image side footprint of optical rays is minimal in size (8 mm in diameter) for a working distance of 1 mm. In image space, the maximum ray angle with respect to the optical axis is 3.5°, which is ideal to preserve the spectral performance of filters (Yeh, 2005). Surfaces #19 and #20 remain the defining stop apertures in the system for a diameter of up to 24 mm; then the defining stop aperture changes to surface #10 (11.4 mm in diameter). In this limiting case, an object side NA of 0.56 is reached with a slightly enlarged image side footprint size of 10 mm, image space ray angles of up to 6° and an unchanged working distance of 1 mm. A change of the primary wavelength has a significant impact on the working distance only where a minimum image side footprint must be reached: At 250 nm, the image side footprint is minimal for a working distance of 0.4 mm, 1 mm at 300 nm, 1.7 mm at 410 nm and 2.2 mm at 730 nm. Yet, keeping the working distance fixed at 1 mm for multimodal imaging will have only a minor impact on the image side footprint dimensions: Again for a telecentric object field 1 mm in diameter (0.56 NA), the footprint will be 15 mm at 250 mm, 10 mm at 300 nm and 410 nm and 12 mm at 730 nm. Although the stop aperture is not exactly at the back focal position of lens group I, chief ray angles are all smaller than 0.2° (@ 250 nm) or 0.3° (@ 730 nm) in the object space. Consequently, the condenser should work well both with telecentric sources, as for example, multiharmonic signals excited in a laser scanning microscope where the scan mirror planes are conjugate to the back focal plane of the objective lens, or with isotropic sources as, for example, multiphoton-induced fluorescence. C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
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Table 1. Surface data of the condenser system model, including all apertures defined by the mechanical setup of the lens, with the working distance (surface #1) optimized for λ = 300 nm, see text. With a stop radius of 6 mm (surfaces #19 and #20), this system has an effective object side NA of 0.29. Note that negative thickness values are purely virtual; they are only used to include mechanical apertures in the ray trace.
Surface
Surface radius (mm)
Material Thickness (mm)
Object plane 0.0000 1: Working distance 1.0349 2: Lens tube 0.8000 3 0.8900 4: LC4357 (Thorlabs) –23.0410 3.5000 5 0.0000 6: LA4647 4.3400 (Thorlabs) 7 –9.2058 0.0000 8 –1.9860 9: Retaining ring 3.5060 10 2.5120 11: Lens tube 1.7880 12: LC4888 –45.9730 3.5000 (Thorlabs) 13 0.0000 14: LA4306 7.0800 15 –18.3680 0.0000 16 –3.9750 17: Retaining ring 2.0000 18: iris mount 13.7250 19: Stop aperture 1.6510 20 4.2050 21: iris mount 0.0000 22: LA4904 7.8400 (Thorlabs) 23 –69.0340 0.0000 24 –4.3560 25: Retaining ring 5.9540 26 –1.5980 27: LB4842 183.0200 6.5400 (Thorlabs) 28 –183.0200 0.0000 29 –1.5980 30: Retaining rings 5.1690 31 0.0000 32 -3.2200 33: LA4984 92.0580 6.5700 (Thorlabs) 34 0.0000 35: Retaining ring 2.5000 0.0000 36 37 171.6281 38: Image Total system length=240
Material
Aperture radius (mm) 0.50 5.71
SILICA SILICA
6.35 6.35 6.35 6.35
SILICA
SILICA
5.71 5.71 12.00 12.70 12.70 12.70 12.70
11.43 10.16 6.00 As surface #19 9.27 SILICA 25.40 25.40
SILICA
24.13 24.13 25.40 25.40 24.13 24.13
SILICA
25.40 25.40 24.13
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This condition has been tested: In empty areas of the sample, the difference between grey values has been measured with and without the laser scanning the object, that is, laserinduced versus residual background values, respectively: With proper alignment and centring of the condenser lens, this laserinduced background level showed overall variations smaller than 15% for an object field 0.5 mm in diameter. Mechanical lens setup The mechanical setup of the condenser system is shown in panels B and C of Figure 1: The lenses are all retained in optical tubes and the latter are assembled using a four-rod cage system. This setup has very good mechanical stability but still allows a relative movement of lens group I with respect to lens group II. The iris aperture is mounted to the star-like adapter plate that also holds the tubes of lens group I. It can easily be removed or exchanged with a different aperture, for example, that is larger in diameter. The tubes are blackened on the inside to reduce effects of stray light. All mechanical parts were purchased and used off-the-shelf with two exceptions, the plate that attaches to the microscope stand (which may now be available as a standard product) and the front tube: The front tube (Thorlabs part #SM05L03) had an additional external threading towards the nose of the lens, which would have forced a working distance of at least 2.8 mm, causing a substantial loss of aperture and light collecting capability. This otherwise useless threading and some additional material have therefore been removed. Optical filters can be inserted between the two largediameter plates (seen on the right of the photograph, panel C of Fig. 1), and the optical path can be sealed there with appropriate blackened light sealing foil wrapped around the cage rods. If needed, additional sealing can be mounted in the opening between the iris aperture and the optical tube of lens group II. Laser scanning setup For this study, both THG imaging and combined CARS/thirdorder sum frequency generation (TOSFG) imaging have been carried out to test the custom-built condenser. A beam emitted from a Ti:S (Mira 900, Coherent, Santa Clara CA, U.S.A.) served as the fundamental wave in the THG imaging. It was tuned to a central wavelength of approximately 900 nm and set to deliver a train of pulses 85–90 fs in width with a repetition rate of 76 MHz. The dispersive microscope optics somewhat broadened the pulses so that typical pulse widths of 120 fs are obtained after propagation through the objective lens (UPLSApo 20x/0.75NA air immersion lens or UPlanApo 60x/1.2NA water immersion lens, Olympus America, Center Valley PA, USA). CARS microscopy has been realized with two spatiotemporally overlapping laser beams of different wavelengths. The
first beam has been sourced from a picosecond Nd:Vanadate laser at 1064 nm and the second beam from a tuneable optical parametric oscillator (OPO, APE GmbH, Berlin, Germany) synchronously pumped by the second harmonic (532 nm) of the first laser. By setting the OPO wavelength to, for example, 817 nm, the Raman-shifted anti-Stokes signal pulses then are produced at 660 nm. In parallel to CARS and similar to THG, two photons of 817 nm and one photon of 1064 nm can also nonlinearly combine to produce photons of the third-order summed frequency at 295 nm (Segawa et al., 2012). All the three processes are governed by the real part of the wavelength-dependent thirdorder nonlinear susceptibility tensor χ (3) , a property of the scattering material in the laser foci. Characteristically for harmonic imaging, all signal photons emitted from the focus are in phase, yet, conservation of momentum (phase matching) yields different phase relations between source and signal photons for forward scattered CARS and THG/TOSFG, resulting in varying coherent add up and net signal output intensity. As a rule of thumb, we expect forward scattered CARS from bulk material (Volkmer et al., 2001), whereas THG can be expected to govern regions of optical discontinuity (Barad et al., 1997). A flip mirror was used to select either the Ti:S beam or the OPO/1064 nm beam pair, and all laser beams were coupled to the LSM510 scan head via its near infrared port. Typical average power levels at the sample were 60–70 mW at 817 nm, 50 mW at 1064 nm and 40 mW for 900 nm, and all beams entered the back aperture of the objective lens in linear polarization parallel to each other. Microscope setup The UV condenser system is a drop-in replacement for the standard condenser of the microscope (Zeiss Axiovert 200 with Zeiss LSM510META scan head). Yet in our experimental setup, additional optics had to be replaced to ensure UV signal transmission: the collector lens of the bright field illumination system and the light collecting optics in front of one of the PMTs. To ensure high UV transmission, the collector lens (focal length 125 mm, diameter 40 mm) of the Zeiss Axiovert 200 stand was replaced with an optical sibling made from fused silica (G311194000, LINOS). For single channel THG imaging, a single band pass filter (D300/10x, Chroma Technology Corporation, Rockingham VT, U.S.A.) has been used to separate THG signal from residual laser light. For two-channel multimodal imaging, the light path has been separated and filtered by a dichroic mirror (FF310, Semrock Inc., Rochester NY, U.S.A.) and a subsequent band pass filter of type D300/10x or two filters of type HQ710/100-2p (Chroma) in stack, respectively. In front of each PMT photo detector module, light collection optics is mounted. We had no detailed description of this optical system, and yet estimated that it functions as fast focusing C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
DETECTION OF UV SIGNAL IN MULTIMODAL NONLINEAR MICROSCOPY
optics with only small regard to imaging quality. We therefore replaced that optic system with a composition of fused silica components (lenses, windows) that had similar outer dimensions and optical properties. Multiphoton fluorescence or multiharmonic signals are typically separated from residual laser light with optical filters, see earlier. To support optical filtering in the dedicated NUV channel, we have therefore replaced the standard PMT (R6357, Hamamatsu Corporation, Bridgewater NJ, U.S.A.), which is specified for wavelengths between 185 nm and 900 nm with a compatible model of the same series (R6352, Hamamatsu) that is specified for 185 nm to 750 nm only and that is therefore virtually ’blind’ for the laser light. At 300 nm, sensitivity and quantum efficiency of both models are approximately the same. Prior to each experiment, the condenser has been aligned to optimize Koehler illumination conditions and the focus of the condenser has been adjusted for maximum signal collection efficiency in the NUV. Test sample preparation Peripheral nerve samples for THG imaging were gained from C57/B6 wild-type mice. A piece of the saphenous nerve 500 μm in length was excised under a Nikon dissection microscope and freed from additional fatty tissue as well as the collagenous nerve sheath. The myelinated nerve tissue was fixed in 4% PFA or 10% formalin for 3–5 h and subsequently mounted on cover slips (VWR International, Radnor, PA, U.S.A.) treated with 3-aminopropyltriethoxysilane or a chromium potassium sulphate solution (gelatine Type A, chromium potassium sulphate; both from Sigma-Aldrich, Inc., St. Louis, MO, U.S.A.) for optimal tissue adhesion. After fixation and mounting, moisture in the sample was preserved with 1x PBS solution to prevent drying artefacts during the imaging session. As sample burning has been observed even with deposition of reduced levels of energy, the samples were kept wet for improved heat dissipation. Next, we covered the sample with a cover slip made from UV transmissive silica (CGQ066001, Fisher Scientific, Suwanee, GA, U.S.A.). For CARS/TOSFG experiments, a drop of emulsion containing unstained polystyrene beads (flow cytometry size calibration kit, F-13838, Invitrogen, Carlsbad, CA, U.S.A.) was applied to a cover slip and air dried in order to concentrate and fix the beads on the glass surface. Test measurement In Figure 2, we show a pseudo-coloured image of intrinsic forward scattered THG of peripheral nerve tissue recorded at a signal wavelength of 300 nm. As THG is sensitive to optical interfaces (Boyd, 2008), that is, regions where the third-order nonlinearity changes within the typical size of the laser spot, C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
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Fig. 2. Pseudo-coloured intrinsic forward scattered third harmonic generation (THG) imaging of peripheral nerve tissue. THG has been excited with a field scanning Ti:S laser tuned to 900 nm, focused with the Olympus 60× 1.2NA water immersion lens. Signals are then emitted in the NUV range at 300 nm, collected with the custom-built condenser, and directed to a PMT installed in the transmission path of the Zeiss microscope; 512 by 512 pixels; pixel dwell time: 1.0 μs; scale bar: 5 μm.
the optically inhomogeneous core of the axon across and along the nerve is clearly detectable in high resolution. For the multimodal CARS/TOSFG two-channel test measurement in this study, we have recorded signals Si,i i in z stacks (0.34 μm spacing, 72 slices) from the aforementioned bead sample under three conditions: (i) both laser sources uni,i i i,i i blocked, S817/1064 , (ii) 1064 nm laser source blocked, S817 , i,i i and (iii) 817 nm laser source blocked, S1064 . All other experimental conditions, especially PMT voltage and laser power have been carefully checked to remain constant. Net signals I i,i i from only the interaction of both laser sources with the sample have then been derived with i,i i i,i i i,i i I i,i i = (S817/1064 − Bi,i i ) − (S817 − Bi,i i ) − (S1064 − Bi,i i ), i,i i where B are background signals with both laser sources i,i i blocked. The stacks I i,i i have been maximum projected, Ima x, and, to account for different signal levels, both channels have been scaled to the same range of grey values, see panels A and B of Figure 3. To evaluate whether both channels show identical features, we have calculated the channel dominance ratio to ii i ii R = (I imax − Imax )/(Imax + Imax ). If both channels are identical, we expect R = 0, else R will tend to positive or negative values for a local dominance of channel 1 or channel 2, respectively. For the pseudo-coloured RGB map MR of R that is shown in panel C of Figure 3, we have therefore filled the red channel with the positive values of R, and the green channel has been filled with the negative of all values < 0, respectively: MR;red = (R+ |R|)/2, MR;green = (−R+ |R|)/2 and MR;blue = 0. Regions coloured in red are therefore
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Fig. 3. Pseudo-coloured multimodal third-order multiharmonic imaging of unstained polystyrene beads, 4 μm in diameter. Intrinsic signals have been excited by two spatially and temporally overlapping laser beams with wavelengths of 817 nm and 1064 nm, focused to the sample with the 20×/0.75 NA objective lens. In panels A and B, we show images where these signals were recorded in the forward scattered ‘CARS’ and ‘THG/TOSFG’ channel, respectively. In panel C, a ‘ratiometric’ image of both channels is shown, where red and green spots indicate a dominance of the ‘CARS’ and the ‘THG/TOSFG’ channel, respectively. We conclude that both channels have differing morphology, see text; 512 by 512 pixels; pixel dwell time: 1.3 μs; scale bar: 20 μm.
dominated by signals from the CARS channel whereas regions coloured in green are dominated by signals from the TOSFG channel. We clearly see that both channels are very unlikely to be identical and we conclude that we probe the sample with different harmonic modalities, see ‘Discussion’.
Adaptation to a different microscope stand To test the general applicability of our approach, we have adapted our condenser system to fit into an inverted microscope stand of a different brand (DM IRBE, Leica Microsystems, Mannheim, Germany), see Figure 4. Here, as previously ¨ described (Schurmann et al., 2010; Wingert et al., 2013), a beam sourced from a picosecond (τ 2 ps) pulsed Ti:S laser (Tsunami, Spectra Physics, Irvine, CA, U.S.A.) is coupled into an SP2 scan head (Leica) and focused down to the sample with a high NA objective lens; for our tests with this system, a 63 × 1.2 NA water immersion objective lens has been used. The optical design of the revised condenser system is shown in panel A of Figure 4: Lens group I is the same as described earlier, and the whole group is attached to the condenser mount of the Leica stand, see panel B of Figure 4. Lens group II, however, has changed to accommodate for the different experimental setting and merely exists of one component only (Thorlabs LA4874, f = 150 mm, d = 25.4 mm). Our theoretical optical design suggests mounting this lens as close as 2 mm to lens group I. This small distance, however, would render the filter wheel in the Leica condenser immotile and we would lose other imaging modalities. We therefore decided to go for a nonperfect solution with a larger distance (5–10 mm) and installed the lens into a free spot of the filter wheel, with the aperture iris stop located directly above. There are two more lenses in the optical path that we have again replaced with optical siblings made from fused silica, a plano-convex lens (LINOS G312415522, f = 100 mm, d
= 31.5 mm) that is installed in the tube on the top side of the condenser, and a biconvex lens (LINOS G311207522, f = 30 mm, d = 22.4 mm) that is mounted in front of the PMT detector, see dashed marks in panel B of Figure 4. Next, we removed the original laser blocking BG39 filter in front of the PMT with two narrow (10 nm) band pass filters centred at 300 nm (XBPA300, Asahi Spectra USA Inc., Torrance, CA, U.S.A.) in stack. For ease of handling, we mounted this filter stack in a mount (86-018, Edmund Optics Inc., Barrington, NJ, U.S.A.) that is fixed to the vertically flexible tubing of the Leica condenser, see panel B of Figure 4, so that the condenser focusing capability is maintained. With the Ti:S laser tuned to 890 nm, we tested THG signal detection by focusing the beam to a cover slip–air interface (Boyd, 2008). We were able to record a distinct THG signal that was clearly related to the focus position of the objective lens. In addition, driving the laser in cw mode made the signal disappear.
Discussion In the present study, we have shown that it is possible to build a NUV- to near infrared transmissive condenser lens from catalogue elements and to use this lens system as a drop-in replacement for the condenser that comes with a commercial inverted microscope like the Zeiss Axiovert 200 or the Leica DM IRBE. In detail, we have described the condenser system and all other necessary steps to enable THG and third-order SFG imaging in the NUV range with our commercial Zeiss LSM510 or Leica SP2 confocal/two-photon laser scanning microscopes. Finding appropriate filters for NUV imaging was a second challenge besides the design and built-up of the optics itself: It was our impression that many filters that transmit in a THG wavelength band also do so slightly in the fundamental range of wavelengths, providing not enough optical density C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
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Fig. 4. Adaptation of the condenser to the Leica DM IRBE inverted microscope stand. In panel A, we show the optical design with identical lens group I and a lens group II that has been modified to contain one lens only to fit the physical constraints of the Leica stand. Additional lenses in the optical pathway are an intrinsic part of group II and they have been replaced with optical siblings made from fused silica. Scale bar: 100 mm. In panel B, we show the actual installation of lens group I (at the bottom, near the sample), and the position of the lenses of group II marked in light blue.
to block residual laser light where, in our experience, at least OD6 or OD7 is necessary. Two appropriate filters with the band pass centralized at 300 nm, the D300/10x (Chroma) and the XBPA300 (Asahi Spectra) were used in this study. It should be noted that filters from the same series, but, for example, with different central wavelengths, do not necessarily permit comparable performance. To test the condenser lens we have carried out both single and multimodal harmonic imaging on our modified LSM510 laser scanning microscope. Two-channel multimodal imaging has been performed using nonfluorescent bare polystyrene beads illuminated with two synchronized picosecond-pulsed laser sources at central wavelengths of 817 nm and 1064 nm. One channel is configured to record forward scattered CARS signals @ 660 nm corresponding to the spectral resonance at 2850 cm-1 (Zumbusch et al., 1999). The other channel is configured for TOSFG signals where three photons (two at 817 nm, one at 1064 nm) combine to one signal photon at 295 nm (Segawa et al., 2012). Polystyrene shows only small absorption in that wavelength range (Nurmukhametov et al., 2006). A normalized overlay of both channels (see panel C of Fig. 3) shows that the signals in both channels source from different morphological features as expected: CARS signals are dominant in the bead centres whereas TOSFG signals show up predominantly at the bead edges.
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As a second application test, we have successfully carried out THG imaging on peripheral nervous tissue where three source photons from a Ti:S laser tuned to 900 nm combine to one signal photon at 300 nm via a degenerate third-order nonlinear process. As was mentioned, Ti:S laser sources had been previously used for THG microscopy imaging (Yelin et al., 2002). In that work, the problem of the NUV signal transmission has been solved with a commercial condenser system (assumingly G063011000, LINOS). On comparing that system with the one described in this paper, we found that both lens systems can, in principle, be used at an object-side NA of 0.57, for light at 300 nm and at a total path length of 240 mm as given by our microscope layout. Under these conditions, the LINOS system has a preferable working distance of 9.3 mm, which is much longer than the one in our case (1 mm). Both systems are made from fused silica singlets, and both will therefore show close to flat transmission spectra for wavelengths between 250 nm and 2 μm. But the LINOS system is made from three singlets only, compared to seven singlets in our design, so that on-axis transmission values of 0.93 70% versus 0.97 50% can be expected. However, for a telecentric object field 1 mm in diameter, the image side ray foot print diameter would be 42 mm at 250 mm, 57 mm at 300 nm, 90 mm at 410 nm and
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180 mm at 730 nm. These footprint dimensions are much larger than the
doi: 10.1111/jmi.12267
Received 11 June 2014; accepted 26 April 2015
Enabling the detection of UV signal in multimodal nonlinear microscopy with catalogue lens components M A R T I N V O G E L ∗ , A X E L W I N G E R T †, R A I N E R H . A . F I N K †, C H R I S T I A N H A G L ‡, F E R U Z G A N I K H A N O V § & C H R I S T I A N P . P F E F F E R ‡, ∗ Center for Nanoscale Systems, Harvard University, Cambridge, Massachusetts, U.S.A.
†Medical Biophysics Group, University of Heidelberg, Heidelberg, Germany ‡Department of Cardiac Surgery, Ludwig-Maximilians-Universitaet, Munchen, Germany §Department of Physics, University of Rhode Island, Kingston, Rhode Island, U.S.A. Department of Craniofacial and Developmental Biology, Harvard Medical School, Boston, Massachusetts, U.S.A.
Key words. Coherent anti-Stokes Raman scattering, condenser, multimodal nonlinear microscopy, third harmonic generation, third-order sum frequency generation, ultraviolet transmission. Summary Using an optical system made from fused silica catalogue optical components, third-order nonlinear microscopy has been enabled on conventional Ti:sapphire laser-based multiphoton microscopy setups. The optical system is designed using two lens groups with straightforward adaptation to other microscope stands when one of the lens groups is exchanged. Within the theoretical design, the optical system collects and transmits light with wavelengths between the near ultraviolet and the near infrared from an object field of at least 1 mm in diameter within a resulting numerical aperture of up to 0.56. The numerical aperture can be controlled with a variable aperture stop between the two lens groups of the condenser. We demonstrate this new detection capability in third harmonic generation imaging experiments at the harmonic wavelength of 300 nm and in multimodal nonlinear optical imaging experiments using third-order sum frequency generation and coherent anti-Stokes Raman scattering microscopy so that the wavelengths of the detected signals range from 300 nm to 660 nm. Introduction In the last decades, multiphoton excited fluorescence microscopy (2PEF, 3PEF; Denk et al., 1990; Hell et al., 1996; Maiti et al., 1997) has seen a continuing growth in applications. This is also true for multiharmonic microscopy, for example, second harmonic generation microscopy (SHG; Hellwarth & Christensen, 1974; Gannaway & Sheppard, 1978) and third Correspondence to: Martin Vogel, Max Planck-Institute of Biophysics, 60438 Frankfurt, Germany; Tel: +49 69 6303 4640; fax: +49 69 6303 4602; e-mail: [email protected]
harmonic generation microscopy (THG; Barad et al., 1997; Mueller et al., 1998; Millard et al., 1999), sum frequency generation microscopy (SFG; Fl¨orsheimer et al., 1999) or coherent anti-Stokes Raman scattering microscopy (CARS; Duncan et al., 1982; Zumbusch et al., 1999) that allow, among others, the characterization of surfaces and nanoparticles (e.g. Shen, 1989; Gauderon et al., 1998; Hsieh et al., 2010; Pantazis et al., 2010). These techniques are used to investigate the physiology or pathophysiology of biological material (Peleg et al., 1996, 1999; Campagnola et al., 1999; Rama et al., 2010) and for morphological (Yelin & Silberberg, 1999; Potma et al., 2001; Campagnola et al., 2002; Cheng et al., 2002; Both et al., 2004; Oron et al., 2004; Evans et al., 2005; D´ebarre et al., 2006; L´egar´e et al., 2007; Plotnikov et al., 2008; Friedrich et al., 2010; Pfeffer et al., 2011; Raghunathan et al., 2011; Recher et al., 2011) or for functional tissue imaging (Nuc¨ ciotti et al., 2010; Schurmann et al., 2010), even in humans (Llewellyn et al., 2008). These imaging modalities probe different and nonoverlapping characteristics of samples calling for multimodal imaging approaches (Chu et al., 2001, 2004, 2005; Nemet et al., 2004; Fu et al., 2007; Hsieh et al., 2008; Wallenburg et al., 2010; Rehberg et al., 2011), so that the availability of as many imaging modalities as possible on a given multiphoton microscopy system is a useful and important asset to many fields of research. A high numerical aperture (NA) objective lens is a common element for the mentioned imaging modalities. The focused laser beams are being scanned across the sample, whereas generated signal photons can be collected using different geometries, with high-efficiency photo detectors. The nonlinear character of a multiphoton process leads to further sharpening of the probed microscopic volume within the sample foci (Sun et al., 2002).
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Nonlinear microscopies put further requirements to optical systems: They have to perform over a wider spectral range compared to the classical optical microscope. This is especially true for imaging modalities of higher order (THG, SFG) where excitation and emission wavelength differ by a factor of up to three. This is obviously a much larger difference than, for example, a Stokes shift of only few nanometers known for many fluorescent dyes (Lakowicz, 2010). Diffraction limited performance and minimized aberrations are required for the objective lens. These can be loosened for the condenser lens as it only serves to collect signal photons. However, for scanning laser microscopy, the condenser should show minimal vignetting. In addition, for multimodal studies using multicolour lasers, the performance of the condenser should not depend on the collected signal wavelengths so that adjustments during the experiment can be avoided. A significant constraint in the optical design of such a condenser is the fact that most glasses are almost opaque for the near ultraviolet (NUV) range of wavelengths (λ < 350 nm). Concerning the laser source for two-photon excited fluorescence microscopy, mode-locked titanium sapphire (Ti:S) lasers are a common and preferred choice. This is due to its relatively broad tuning range (680 nm to 1100 nm) and its wavelength range compatibility with two-photon absorption bands of many standard fluorescent dyes. Within the same system, SHG signals can be easily generated and detected using driving field laser wavelengths within the 800–1000 nm range as the corresponding SHG signals then fall within the visible range at which standard microscope optics is perfectly optimized. For SHG wavelengths between 360 nm and 400 nm, the selection of suitable microscope optics already becomes less straightforward whereas for wavelengths below 350 nm, the inherent opacity of many glasses comes into effect. Both theoretical predictions and experimental evidences show that second-order nonlinearity for important media of both inorganic and biological origins like nanoparticles or protein collagen is significantly increased for the wavelength range below 350 nm (Oudar & Chemla, 1977; Zipfel et al., 2003; Harnagea et al., 2010; Butet et al., 2012; Hall et al., 2014). Thus, an extension of the standard instrumental capabilities to shorter wavelengths is highly desirable. With a Ti:S laser, third harmonic signals will have wavelengths between 230 nm and 330 nm. The signals will always get strongly attenuated by both microscope optics and even by the microscope glass slide or the cover slip as transmission data for Schott cover glass (type ‘D 263M’ of thickness 150 μm) suggest indicating only 10% transmission at 300 nm. Therefore THG and SFG microscopies are usually carried out with lasers of longer wavelengths as it was demonstrated with ytterbium doped fibre oscillators tuned to 1030 nm (Gualda et al., 2008; Tserevelakis et al., 2011), optical parametric am¨ plifiers and oscillators tuned to 1200 nm (Muller et al., 1998; Squier et al., 1998; B´elisle et al., 2008; Chang et al., 2010; C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
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Rehberg et al., 2011), and chromium doped forsterite laser emitting at 1230 nm (Chu et al., 2001, 2005; Yu et al., 2008; Lee et al., 2010). In the referenced studies, authors argue that photo damage and scattering are minimized due to the longer wavelength for the driving field. However, transmission of microscope optics can be poor within the mentioned wavelength ranges and the capability to simultaneously excite blue/green fluorescent dyes is lost, unless three-photon excited fluorescence is used with a laser source that is tuned to even longer wavelengths in the 1400 nm to 1500 nm range (Yelin & Silberberg, 1999; McConnell, 2007). Yet, the wavelength tuning may come at a cost of increased absorption of light in water (Hale & Querry, 1973) that is ubiquitous in life biological sample. Thus, it is tempting to develop THG imaging capabilities with a Ti:S laser that is still the standard laser source for many commercial multiphoton microscopes. Yelin and co-workers (Yelin et al., 2002) have demonstrated two photon excited fluorescence and THG with a Ti:S laser, but unfortunately their approach has not triggered broader application. They have custom-built and modified an inverted microscope for THG imaging with a laser scanning system and a UV-transmissive lens combination in the detection path (assumingly G063011000, then sourced from LINOS, Gottingen, Germany). From the latter, we expect worse achromatic properties and less optimization for multimodal imaging (see the “Discussion” section). Our approach was therefore to design and build a condenser system with improved achromatic properties that is purely made from NUV to near infrared transmissive standard lens elements. Designed as a drop-in replacement for the condenser, our system was made to directly fit the stand of our laser scanning microscope system and we were careful to maintain all bright field imaging capabilities. We show that our condenser system enables both THG imaging with a Ti:S laser and multimodal multiharmonic imaging on a standard Zeiss multiphoton laser scanning microscope setup (LSM510META). In addition, using a Leica multiphoton system (SP2) as an example, we show that our condenser can be readily adapted and used with other microscopy brands and designs. Experimental work Design considerations Although the optical industry offers the manufacturing of customized lens components, we decided to design the condenser from standard catalogue elements to limit cost, to expedite the setup in time and to ease cloning and adaptation to different microscope setups. Standard NUV-transmissive components are usually made from calcium fluoride (CaF2 ), magnesium fluoride (MgF2 ) or UV-grade fused silica. It was our impression at the time of design that more suitable and less expensive components were available made from fused silica. The lenses
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and mechanical mounts have been sourced from Thorlabs (Thorlabs, Inc., Newton NJ, U.S.A.) and LINOS (now: Qioptiq Photonics GmbH & Co. KG, Gottingen, Germany) to maintain mechanical compatibility with the predominant percentage of the components at the ‘Advanced Microscopy Laboratory’ at the ‘Center for Nanoscale Systems’ at Harvard University. We are nevertheless certain that components from other sources would have performed in a comparable way. The fused silica glass used to produce those lenses (Corning 7980) is highly transmissive for light between 250 nm and 2 μm. With the exception of a small dip at 1400 nm, it shows an almost flat transmission of >90% for material 1 mm in thickness between these wavelength limits (see data sheet ‘HPFS Fused Silica Standard Grade’, Corning Inc., Corning, NY, U.S.A.). To maintain this broad and flat transmission characteristic, we have not used components coated with anti reflection layers. However, our design has several glass–air interfaces so that an overall on-axis transmission of 0.97 50% can be expected; a revised design could therefore benefit from such coatings for specific wavelength ranges. As design goals, we have defined the following conditions: The condenser must be able to collect all the light from a telecentric source field, 1 mm in diameter, with an NA as large as possible. On the image side, the dimension of the light ray footprint should be minimized for improved light detection. To fit with the geometry of our microscope, a Zeiss Axiovert 200 with a LSM510META scan head (Carl Zeiss Microimaging, Thornwood, NY, U.S.A.), and to enable bright field illumination through the condenser, the condenser should produce a real image at 240 mm away from the object plane. However, being a condenser lens, diffraction-limited imaging quality was no primary design criterion. Given the restricted choice of UV transmitting but IR laser blocking filters, we have set the primary design wavelengths to 300 nm, corresponding to THG signals excited with a Ti:S laser tuned to 900 nm, and to 660 nm where CARS signals were generated with synchronized 817 nm and 1064 nm laser sources, the wavelength configuration that enables thirdorder resonant signal associated with an important CH2 bond vibration resonance at 2850 cm-1 . Secondary design wavelengths were chosen at 450 nm (SHG from the 900 nm laser source) and at 408 nm, where SHG signals from the 817 nm laser source are expected. The lens was designed with the help of ZEMAX (ZEMAX Development Corporation, Bellevue, WA, USA) and OpTaliX (Optenso Optical Engineering Software, Heerbrugg, Switzerland) software, respectively. From the supplied software catalogues, ‘F_SILICA’ and ‘SILICA’, respectively, have been used as glass models and component prescriptions were loaded from the ‘Thorlabs’ and ‘Linos’ component catalogues respectively. Panel A of Figure 1 shows a typical ray trace through the optical elements of the condenser system. Red, blue and green rays are sourced from the central field point and from field points 0.5 mm left and right off-centre, respectively.
Fig. 1. Setup of the condenser system. Panel A shows a ray trace through the optical elements of the lens rendered with OpTaliX software. Red rays source from the central field point, blue and green rays source from field points 0.5 mm left and right off-centre, respectively. Scale bar: 100 mm. In panel B, a sectional view of the mechanical model of the condenser is shown (Alibre Design software), including retaining rings and other elements that introduce additional optical apertures, see text. In panel C, we finally show a photograph of the condenser as it was built and used (photograph courtesy of Arthur McClelland, Center for Nanoscale Systems, Harvard University).
Optical lens design After an initial optical design was established, the mechanical setup was realized and subsequently modelled with CAD software (Alibre Design, Alibre Inc., Richardson, TX, U.S.A.). Off-the-shelf parts for lens mounting were added to the model, C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
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and the latter caused a slight adjustment of the optical distances and a final refinement of the optical model. The surface data of this final condenser system are listed in Table 1. The design consists of two lens groups, group I and group II, and an aperture iris placed between these groups. Lens group I is common to all designs and it is compiled from four components, LC4357 (Thorlabs, nominal focal length f1 = –50 mm, d1 = 12.7 mm in diameter), LA4647 (Thorlabs, f2 = 20 mm, d2 = 12.7 mm), LC4888 (Thorlabs, f3 = –100 mm, d3 = 25.4 mm) and LA4306 (Thorlabs, f4 = 40 mm, d4 = 25.4 mm). Lens group II – specifically designed for the Zeiss stand – is made from three components, LA4904 (Thorlabs, f5 = 150 mm, d5 = 50.8 mm), LB4842 (Thorlabs, f6 = 200 mm, d6 = 50.8 mm) and LA4984 (Thorlabs, f7 = 200 mm, d7 = 50.8 mm). Optical design analysis At a wavelength of 300 nm and for a stop aperture 12 mm in diameter (see surface #19 and #20 in Table 1), optical modelling with an object field 1 mm in diameter yields an effective focal length of 26 mm and an object side NA of 0.29. The image side footprint of optical rays is minimal in size (8 mm in diameter) for a working distance of 1 mm. In image space, the maximum ray angle with respect to the optical axis is 3.5°, which is ideal to preserve the spectral performance of filters (Yeh, 2005). Surfaces #19 and #20 remain the defining stop apertures in the system for a diameter of up to 24 mm; then the defining stop aperture changes to surface #10 (11.4 mm in diameter). In this limiting case, an object side NA of 0.56 is reached with a slightly enlarged image side footprint size of 10 mm, image space ray angles of up to 6° and an unchanged working distance of 1 mm. A change of the primary wavelength has a significant impact on the working distance only where a minimum image side footprint must be reached: At 250 nm, the image side footprint is minimal for a working distance of 0.4 mm, 1 mm at 300 nm, 1.7 mm at 410 nm and 2.2 mm at 730 nm. Yet, keeping the working distance fixed at 1 mm for multimodal imaging will have only a minor impact on the image side footprint dimensions: Again for a telecentric object field 1 mm in diameter (0.56 NA), the footprint will be 15 mm at 250 mm, 10 mm at 300 nm and 410 nm and 12 mm at 730 nm. Although the stop aperture is not exactly at the back focal position of lens group I, chief ray angles are all smaller than 0.2° (@ 250 nm) or 0.3° (@ 730 nm) in the object space. Consequently, the condenser should work well both with telecentric sources, as for example, multiharmonic signals excited in a laser scanning microscope where the scan mirror planes are conjugate to the back focal plane of the objective lens, or with isotropic sources as, for example, multiphoton-induced fluorescence. C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
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Table 1. Surface data of the condenser system model, including all apertures defined by the mechanical setup of the lens, with the working distance (surface #1) optimized for λ = 300 nm, see text. With a stop radius of 6 mm (surfaces #19 and #20), this system has an effective object side NA of 0.29. Note that negative thickness values are purely virtual; they are only used to include mechanical apertures in the ray trace.
Surface
Surface radius (mm)
Material Thickness (mm)
Object plane 0.0000 1: Working distance 1.0349 2: Lens tube 0.8000 3 0.8900 4: LC4357 (Thorlabs) –23.0410 3.5000 5 0.0000 6: LA4647 4.3400 (Thorlabs) 7 –9.2058 0.0000 8 –1.9860 9: Retaining ring 3.5060 10 2.5120 11: Lens tube 1.7880 12: LC4888 –45.9730 3.5000 (Thorlabs) 13 0.0000 14: LA4306 7.0800 15 –18.3680 0.0000 16 –3.9750 17: Retaining ring 2.0000 18: iris mount 13.7250 19: Stop aperture 1.6510 20 4.2050 21: iris mount 0.0000 22: LA4904 7.8400 (Thorlabs) 23 –69.0340 0.0000 24 –4.3560 25: Retaining ring 5.9540 26 –1.5980 27: LB4842 183.0200 6.5400 (Thorlabs) 28 –183.0200 0.0000 29 –1.5980 30: Retaining rings 5.1690 31 0.0000 32 -3.2200 33: LA4984 92.0580 6.5700 (Thorlabs) 34 0.0000 35: Retaining ring 2.5000 0.0000 36 37 171.6281 38: Image Total system length=240
Material
Aperture radius (mm) 0.50 5.71
SILICA SILICA
6.35 6.35 6.35 6.35
SILICA
SILICA
5.71 5.71 12.00 12.70 12.70 12.70 12.70
11.43 10.16 6.00 As surface #19 9.27 SILICA 25.40 25.40
SILICA
24.13 24.13 25.40 25.40 24.13 24.13
SILICA
25.40 25.40 24.13
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This condition has been tested: In empty areas of the sample, the difference between grey values has been measured with and without the laser scanning the object, that is, laserinduced versus residual background values, respectively: With proper alignment and centring of the condenser lens, this laserinduced background level showed overall variations smaller than 15% for an object field 0.5 mm in diameter. Mechanical lens setup The mechanical setup of the condenser system is shown in panels B and C of Figure 1: The lenses are all retained in optical tubes and the latter are assembled using a four-rod cage system. This setup has very good mechanical stability but still allows a relative movement of lens group I with respect to lens group II. The iris aperture is mounted to the star-like adapter plate that also holds the tubes of lens group I. It can easily be removed or exchanged with a different aperture, for example, that is larger in diameter. The tubes are blackened on the inside to reduce effects of stray light. All mechanical parts were purchased and used off-the-shelf with two exceptions, the plate that attaches to the microscope stand (which may now be available as a standard product) and the front tube: The front tube (Thorlabs part #SM05L03) had an additional external threading towards the nose of the lens, which would have forced a working distance of at least 2.8 mm, causing a substantial loss of aperture and light collecting capability. This otherwise useless threading and some additional material have therefore been removed. Optical filters can be inserted between the two largediameter plates (seen on the right of the photograph, panel C of Fig. 1), and the optical path can be sealed there with appropriate blackened light sealing foil wrapped around the cage rods. If needed, additional sealing can be mounted in the opening between the iris aperture and the optical tube of lens group II. Laser scanning setup For this study, both THG imaging and combined CARS/thirdorder sum frequency generation (TOSFG) imaging have been carried out to test the custom-built condenser. A beam emitted from a Ti:S (Mira 900, Coherent, Santa Clara CA, U.S.A.) served as the fundamental wave in the THG imaging. It was tuned to a central wavelength of approximately 900 nm and set to deliver a train of pulses 85–90 fs in width with a repetition rate of 76 MHz. The dispersive microscope optics somewhat broadened the pulses so that typical pulse widths of 120 fs are obtained after propagation through the objective lens (UPLSApo 20x/0.75NA air immersion lens or UPlanApo 60x/1.2NA water immersion lens, Olympus America, Center Valley PA, USA). CARS microscopy has been realized with two spatiotemporally overlapping laser beams of different wavelengths. The
first beam has been sourced from a picosecond Nd:Vanadate laser at 1064 nm and the second beam from a tuneable optical parametric oscillator (OPO, APE GmbH, Berlin, Germany) synchronously pumped by the second harmonic (532 nm) of the first laser. By setting the OPO wavelength to, for example, 817 nm, the Raman-shifted anti-Stokes signal pulses then are produced at 660 nm. In parallel to CARS and similar to THG, two photons of 817 nm and one photon of 1064 nm can also nonlinearly combine to produce photons of the third-order summed frequency at 295 nm (Segawa et al., 2012). All the three processes are governed by the real part of the wavelength-dependent thirdorder nonlinear susceptibility tensor χ (3) , a property of the scattering material in the laser foci. Characteristically for harmonic imaging, all signal photons emitted from the focus are in phase, yet, conservation of momentum (phase matching) yields different phase relations between source and signal photons for forward scattered CARS and THG/TOSFG, resulting in varying coherent add up and net signal output intensity. As a rule of thumb, we expect forward scattered CARS from bulk material (Volkmer et al., 2001), whereas THG can be expected to govern regions of optical discontinuity (Barad et al., 1997). A flip mirror was used to select either the Ti:S beam or the OPO/1064 nm beam pair, and all laser beams were coupled to the LSM510 scan head via its near infrared port. Typical average power levels at the sample were 60–70 mW at 817 nm, 50 mW at 1064 nm and 40 mW for 900 nm, and all beams entered the back aperture of the objective lens in linear polarization parallel to each other. Microscope setup The UV condenser system is a drop-in replacement for the standard condenser of the microscope (Zeiss Axiovert 200 with Zeiss LSM510META scan head). Yet in our experimental setup, additional optics had to be replaced to ensure UV signal transmission: the collector lens of the bright field illumination system and the light collecting optics in front of one of the PMTs. To ensure high UV transmission, the collector lens (focal length 125 mm, diameter 40 mm) of the Zeiss Axiovert 200 stand was replaced with an optical sibling made from fused silica (G311194000, LINOS). For single channel THG imaging, a single band pass filter (D300/10x, Chroma Technology Corporation, Rockingham VT, U.S.A.) has been used to separate THG signal from residual laser light. For two-channel multimodal imaging, the light path has been separated and filtered by a dichroic mirror (FF310, Semrock Inc., Rochester NY, U.S.A.) and a subsequent band pass filter of type D300/10x or two filters of type HQ710/100-2p (Chroma) in stack, respectively. In front of each PMT photo detector module, light collection optics is mounted. We had no detailed description of this optical system, and yet estimated that it functions as fast focusing C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
DETECTION OF UV SIGNAL IN MULTIMODAL NONLINEAR MICROSCOPY
optics with only small regard to imaging quality. We therefore replaced that optic system with a composition of fused silica components (lenses, windows) that had similar outer dimensions and optical properties. Multiphoton fluorescence or multiharmonic signals are typically separated from residual laser light with optical filters, see earlier. To support optical filtering in the dedicated NUV channel, we have therefore replaced the standard PMT (R6357, Hamamatsu Corporation, Bridgewater NJ, U.S.A.), which is specified for wavelengths between 185 nm and 900 nm with a compatible model of the same series (R6352, Hamamatsu) that is specified for 185 nm to 750 nm only and that is therefore virtually ’blind’ for the laser light. At 300 nm, sensitivity and quantum efficiency of both models are approximately the same. Prior to each experiment, the condenser has been aligned to optimize Koehler illumination conditions and the focus of the condenser has been adjusted for maximum signal collection efficiency in the NUV. Test sample preparation Peripheral nerve samples for THG imaging were gained from C57/B6 wild-type mice. A piece of the saphenous nerve 500 μm in length was excised under a Nikon dissection microscope and freed from additional fatty tissue as well as the collagenous nerve sheath. The myelinated nerve tissue was fixed in 4% PFA or 10% formalin for 3–5 h and subsequently mounted on cover slips (VWR International, Radnor, PA, U.S.A.) treated with 3-aminopropyltriethoxysilane or a chromium potassium sulphate solution (gelatine Type A, chromium potassium sulphate; both from Sigma-Aldrich, Inc., St. Louis, MO, U.S.A.) for optimal tissue adhesion. After fixation and mounting, moisture in the sample was preserved with 1x PBS solution to prevent drying artefacts during the imaging session. As sample burning has been observed even with deposition of reduced levels of energy, the samples were kept wet for improved heat dissipation. Next, we covered the sample with a cover slip made from UV transmissive silica (CGQ066001, Fisher Scientific, Suwanee, GA, U.S.A.). For CARS/TOSFG experiments, a drop of emulsion containing unstained polystyrene beads (flow cytometry size calibration kit, F-13838, Invitrogen, Carlsbad, CA, U.S.A.) was applied to a cover slip and air dried in order to concentrate and fix the beads on the glass surface. Test measurement In Figure 2, we show a pseudo-coloured image of intrinsic forward scattered THG of peripheral nerve tissue recorded at a signal wavelength of 300 nm. As THG is sensitive to optical interfaces (Boyd, 2008), that is, regions where the third-order nonlinearity changes within the typical size of the laser spot, C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
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Fig. 2. Pseudo-coloured intrinsic forward scattered third harmonic generation (THG) imaging of peripheral nerve tissue. THG has been excited with a field scanning Ti:S laser tuned to 900 nm, focused with the Olympus 60× 1.2NA water immersion lens. Signals are then emitted in the NUV range at 300 nm, collected with the custom-built condenser, and directed to a PMT installed in the transmission path of the Zeiss microscope; 512 by 512 pixels; pixel dwell time: 1.0 μs; scale bar: 5 μm.
the optically inhomogeneous core of the axon across and along the nerve is clearly detectable in high resolution. For the multimodal CARS/TOSFG two-channel test measurement in this study, we have recorded signals Si,i i in z stacks (0.34 μm spacing, 72 slices) from the aforementioned bead sample under three conditions: (i) both laser sources uni,i i i,i i blocked, S817/1064 , (ii) 1064 nm laser source blocked, S817 , i,i i and (iii) 817 nm laser source blocked, S1064 . All other experimental conditions, especially PMT voltage and laser power have been carefully checked to remain constant. Net signals I i,i i from only the interaction of both laser sources with the sample have then been derived with i,i i i,i i i,i i I i,i i = (S817/1064 − Bi,i i ) − (S817 − Bi,i i ) − (S1064 − Bi,i i ), i,i i where B are background signals with both laser sources i,i i blocked. The stacks I i,i i have been maximum projected, Ima x, and, to account for different signal levels, both channels have been scaled to the same range of grey values, see panels A and B of Figure 3. To evaluate whether both channels show identical features, we have calculated the channel dominance ratio to ii i ii R = (I imax − Imax )/(Imax + Imax ). If both channels are identical, we expect R = 0, else R will tend to positive or negative values for a local dominance of channel 1 or channel 2, respectively. For the pseudo-coloured RGB map MR of R that is shown in panel C of Figure 3, we have therefore filled the red channel with the positive values of R, and the green channel has been filled with the negative of all values < 0, respectively: MR;red = (R+ |R|)/2, MR;green = (−R+ |R|)/2 and MR;blue = 0. Regions coloured in red are therefore
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Fig. 3. Pseudo-coloured multimodal third-order multiharmonic imaging of unstained polystyrene beads, 4 μm in diameter. Intrinsic signals have been excited by two spatially and temporally overlapping laser beams with wavelengths of 817 nm and 1064 nm, focused to the sample with the 20×/0.75 NA objective lens. In panels A and B, we show images where these signals were recorded in the forward scattered ‘CARS’ and ‘THG/TOSFG’ channel, respectively. In panel C, a ‘ratiometric’ image of both channels is shown, where red and green spots indicate a dominance of the ‘CARS’ and the ‘THG/TOSFG’ channel, respectively. We conclude that both channels have differing morphology, see text; 512 by 512 pixels; pixel dwell time: 1.3 μs; scale bar: 20 μm.
dominated by signals from the CARS channel whereas regions coloured in green are dominated by signals from the TOSFG channel. We clearly see that both channels are very unlikely to be identical and we conclude that we probe the sample with different harmonic modalities, see ‘Discussion’.
Adaptation to a different microscope stand To test the general applicability of our approach, we have adapted our condenser system to fit into an inverted microscope stand of a different brand (DM IRBE, Leica Microsystems, Mannheim, Germany), see Figure 4. Here, as previously ¨ described (Schurmann et al., 2010; Wingert et al., 2013), a beam sourced from a picosecond (τ 2 ps) pulsed Ti:S laser (Tsunami, Spectra Physics, Irvine, CA, U.S.A.) is coupled into an SP2 scan head (Leica) and focused down to the sample with a high NA objective lens; for our tests with this system, a 63 × 1.2 NA water immersion objective lens has been used. The optical design of the revised condenser system is shown in panel A of Figure 4: Lens group I is the same as described earlier, and the whole group is attached to the condenser mount of the Leica stand, see panel B of Figure 4. Lens group II, however, has changed to accommodate for the different experimental setting and merely exists of one component only (Thorlabs LA4874, f = 150 mm, d = 25.4 mm). Our theoretical optical design suggests mounting this lens as close as 2 mm to lens group I. This small distance, however, would render the filter wheel in the Leica condenser immotile and we would lose other imaging modalities. We therefore decided to go for a nonperfect solution with a larger distance (5–10 mm) and installed the lens into a free spot of the filter wheel, with the aperture iris stop located directly above. There are two more lenses in the optical path that we have again replaced with optical siblings made from fused silica, a plano-convex lens (LINOS G312415522, f = 100 mm, d
= 31.5 mm) that is installed in the tube on the top side of the condenser, and a biconvex lens (LINOS G311207522, f = 30 mm, d = 22.4 mm) that is mounted in front of the PMT detector, see dashed marks in panel B of Figure 4. Next, we removed the original laser blocking BG39 filter in front of the PMT with two narrow (10 nm) band pass filters centred at 300 nm (XBPA300, Asahi Spectra USA Inc., Torrance, CA, U.S.A.) in stack. For ease of handling, we mounted this filter stack in a mount (86-018, Edmund Optics Inc., Barrington, NJ, U.S.A.) that is fixed to the vertically flexible tubing of the Leica condenser, see panel B of Figure 4, so that the condenser focusing capability is maintained. With the Ti:S laser tuned to 890 nm, we tested THG signal detection by focusing the beam to a cover slip–air interface (Boyd, 2008). We were able to record a distinct THG signal that was clearly related to the focus position of the objective lens. In addition, driving the laser in cw mode made the signal disappear.
Discussion In the present study, we have shown that it is possible to build a NUV- to near infrared transmissive condenser lens from catalogue elements and to use this lens system as a drop-in replacement for the condenser that comes with a commercial inverted microscope like the Zeiss Axiovert 200 or the Leica DM IRBE. In detail, we have described the condenser system and all other necessary steps to enable THG and third-order SFG imaging in the NUV range with our commercial Zeiss LSM510 or Leica SP2 confocal/two-photon laser scanning microscopes. Finding appropriate filters for NUV imaging was a second challenge besides the design and built-up of the optics itself: It was our impression that many filters that transmit in a THG wavelength band also do so slightly in the fundamental range of wavelengths, providing not enough optical density C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
DETECTION OF UV SIGNAL IN MULTIMODAL NONLINEAR MICROSCOPY
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Fig. 4. Adaptation of the condenser to the Leica DM IRBE inverted microscope stand. In panel A, we show the optical design with identical lens group I and a lens group II that has been modified to contain one lens only to fit the physical constraints of the Leica stand. Additional lenses in the optical pathway are an intrinsic part of group II and they have been replaced with optical siblings made from fused silica. Scale bar: 100 mm. In panel B, we show the actual installation of lens group I (at the bottom, near the sample), and the position of the lenses of group II marked in light blue.
to block residual laser light where, in our experience, at least OD6 or OD7 is necessary. Two appropriate filters with the band pass centralized at 300 nm, the D300/10x (Chroma) and the XBPA300 (Asahi Spectra) were used in this study. It should be noted that filters from the same series, but, for example, with different central wavelengths, do not necessarily permit comparable performance. To test the condenser lens we have carried out both single and multimodal harmonic imaging on our modified LSM510 laser scanning microscope. Two-channel multimodal imaging has been performed using nonfluorescent bare polystyrene beads illuminated with two synchronized picosecond-pulsed laser sources at central wavelengths of 817 nm and 1064 nm. One channel is configured to record forward scattered CARS signals @ 660 nm corresponding to the spectral resonance at 2850 cm-1 (Zumbusch et al., 1999). The other channel is configured for TOSFG signals where three photons (two at 817 nm, one at 1064 nm) combine to one signal photon at 295 nm (Segawa et al., 2012). Polystyrene shows only small absorption in that wavelength range (Nurmukhametov et al., 2006). A normalized overlay of both channels (see panel C of Fig. 3) shows that the signals in both channels source from different morphological features as expected: CARS signals are dominant in the bead centres whereas TOSFG signals show up predominantly at the bead edges.
C 2015 The Authors C 2015 Royal Microscopical Society, 260, 62–72 Journal of Microscopy
As a second application test, we have successfully carried out THG imaging on peripheral nervous tissue where three source photons from a Ti:S laser tuned to 900 nm combine to one signal photon at 300 nm via a degenerate third-order nonlinear process. As was mentioned, Ti:S laser sources had been previously used for THG microscopy imaging (Yelin et al., 2002). In that work, the problem of the NUV signal transmission has been solved with a commercial condenser system (assumingly G063011000, LINOS). On comparing that system with the one described in this paper, we found that both lens systems can, in principle, be used at an object-side NA of 0.57, for light at 300 nm and at a total path length of 240 mm as given by our microscope layout. Under these conditions, the LINOS system has a preferable working distance of 9.3 mm, which is much longer than the one in our case (1 mm). Both systems are made from fused silica singlets, and both will therefore show close to flat transmission spectra for wavelengths between 250 nm and 2 μm. But the LINOS system is made from three singlets only, compared to seven singlets in our design, so that on-axis transmission values of 0.93 70% versus 0.97 50% can be expected. However, for a telecentric object field 1 mm in diameter, the image side ray foot print diameter would be 42 mm at 250 mm, 57 mm at 300 nm, 90 mm at 410 nm and
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180 mm at 730 nm. These footprint dimensions are much larger than the
