sil-based confocal fluorescence microscope for investigating individual nanostructures

Cent. Eur. J. Phys. • 9(2) • 2011 • 293-299DOI: 10.2478/s11534-010-0098-5

Central European Journal of Physics

SIL-based confocal fluorescence microscope forinvestigating individual nanostructures

Research Article

Bartosz Krajnik1∗, Tim Schulte2, Dawid Piątkowski1, Nikodem Czechowski1, Eckhard Hofmann2,Sebastian Mackowski1†

1 Institute of Physics, Nicolaus Copernicus University,Grudziadzka 5, Torun, Poland

2 Department of Biology and Biotechnology, Ruhr-University Bochum,Bochum, Germany

Received 22 July 2010; accepted 29 September 2010

Abstract: We developed a fluorescence confocal microscope equipped with a hemispherical solid immersion lens(SIL) and apply it to study the optical properties of light-harvesting complexes. We demonstrate that thecollection efficiency of the SIL-equipped microscope is significantly improved, as is the spatial resolution,which reaches 600 nm. This experimental setup is suitable for detailed studies of physical phenomena inhybrid nanostructures. In particular, we compare the results of fluorescence intensity measurements for alight-harvesting peridinin-chlorophyll-protein (PCP) complex with and without the SIL.

PACS (2008): 33.50.-j, 87.14.E-, 87.64.kv, 87.64.M-

Keywords: confocal microscopy • light-harvesting complex • solid immersion lens • fluorescence imaging© Versita Sp. z o.o.

1. Introduction

The spatial resolution of all optical systems is limited bydiffraction, which results from the wave nature of light.Resolution of the conventional optical microscope is ap-proximately λ0

2NA , where λ0 is the free space wavelengthand NA is the numerical aperture of the objective definedby the angular semiaperature in object space θa, and therefractive index n, through the relation NA = n·sinθa. Fora microscope objective with NA = 0.45 and wavelengthλ0 = 670 nm, the resolution in air with n = 1 is approxi-

∗E-mail: [email protected]†E-mail: [email protected] (Corresponding author)

mately 750 nm. Another important drawback of using stan-dard collection optics, in particular microscope objectiveswith large working distances, is very low collection effi-ciency, which prevents high-accuracy studies of individualnanostructures. There are several ways to overcome thislimitation. One possibility is to fill the space between anobjective and a sample with high-refraction-index liquid,as commonly applied in oil-immersion microscopy. The oilused for that purpose is characterized with a refractive in-dex of 1.53 in order to match the refractive index of micro-scope coverslips. However despite its many advantages,oil immersion microscopy has certain limitations: first, itcannot be used for measurements of samples sensitive tothe contact with immersion oil; second, oil-immersion mi-croscopy is not suitable for low-temperature experimentsthat could provide valuable information about biological

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SIL-based confocal fluorescence microscope for investigating individual nanostructures

systems [1].In recent years SIL-based microscopy has been used to in-vestigate the optical properties of semiconductor quantumwells [2] and quantum dots [3–5]. It has been shown thatthe numerical aperture of 2 can be achieved when the re-fraction index of the SIL is very high (GaP 3.5) and closelymatches that of the studied sample. One of the critical is-sues related to this technique concerns the presence of theair gap between the surface of the SIL and the epitaxiallygrown sample [3].In this work we apply a solid immersion lens-based confo-cal fluorescence microscope to study pigment-protein com-plexes embedded in a polymer matrix. We investigatethe optical properties of a peridinin-chlorophyll-protein(PCP) light-harvesting complex. This is a water-solublelight-harvesting antenna from the dinoflagellate Amphi-dinium carterae [6]. Our experiments obtained for PCPcomplexes show that application of the SIL significantlyimproves the optical collection efficiency. We achieve anoptical resolution for the setup of about 600 nm, whichis almost half of the bare objective. Enhanced collectionefficiency results in much higher contrast in measured flu-orescence maps. The results provide solid evidence thatsuch an experimental setup can be applied for studyingindividual nanostructures.

2. Materials and methodsIn our experiment, we use PCP reconstituted with Chl asynthesized according to the protocol by Miller et al. [7].The structure of the reconstituted PCP has been deter-mined with better than < 1.5 Å resolution using X-raycrystallography [8] and is shown in Fig. 1a. Each pro-tein monomer of PCP surrounds a pigment cluster of fourperidinin molecules, one Chl a and one lipid molecule.The Chl a molecules in the PCP complex are separatedby less than 2 nm and peridinin molecules are in van derWaals contact with the Chl a molecules. The absorptionspectrum of the PCP complex (Fig. 1b) shows that peri-dinin is the primary absorbing pigment responsible foran absorption band ranging from 350 to 550 nm. On theother hand, the Chl a molecules absorb around 668 nm(Qy band) and 440 nm (Soret band). This light-harvestingcomplex is characterized with effcient energy transfer fromthe peridinins to the Chl a molecules with an efficiencyhigher than 90% [9]. The fluorescence of PCP originatesfrom the Qy transition of Chl a and is located at 673 nm(Fig. 1c) [9]. Relatively high quantum yield and stabil-ity of the PCP complexes make them suitable for single-molecule studies [9]. Large energy separation betweenperidinin absorption and Chl fluorescence together with

excellent matching of the peridinin absorption and typicalenergies of plasmon excitations in metallic nanoparticles,render PCP a model system for studying the plasmonicinteractions in light-harvesting biomolecules [10, 11].

Figure 1. (a) Structure of a homodimer of refolded PCP (RFPCP):four peridins (orange), one chlorophyll a (green) and onelipid molecule (blue). PDB entry 3IIS. (b) Absorption and(c) fluorescence spectrum of PCP complexes measuredat room temperature (after [9]). The excitation wavelengthfor the fluorescence was 485 nm.

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Bartosz Krajnik, Tim Schulte, Dawid Piątkowski, Nikodem Czechowski,Eckhard Hofmann, Sebastian Mackowski

In Fig. 2 we show the scheme of our home-built confocalfluorescence microscope based on the Olympus infinity-corrected microscope objective LMPlan 50x, characterizedwith a numerical aperture of 0.5 and a working distanceof 6 mm. The resulting laser spot size is about 1 µmfor the excitation laser of 485 nm. In order to improvethe collection efficiency and spatial resolution we use a3 mm diameter hemispherical solid immersion lens madeof S-LAH-71 glass with a refraction index n = 1.85. TheSIL is pressed against the sample by spring mount in or-der to reduce the air gap between the sample and thelens. The sample is placed on a XYZ piezoelectric stage(Physik Instrumente) with 1 nm nominal resolution of asingle step, which enables us to raster-scan the samplesurface in order to collect fluorescence maps. The maps

are formed by combining fluorescence intensity measure-ments with the motion of the XY translation stage. Forfluorescence excitation, we use one of four diode-pumpedsolid-state lasers with wavelengths of 405, 485, 532 and640 nm. Typical optical power of the laser sources is about7 mW, but in the case of actual measurements it needs tobe strongly reduced in order to prevent photobleachingof the molecules. We used excitation powers of about 40and 4 µW. Gaussian beams of the lasers are achieved byusing a spatial filter. The fluorescence is detected in aback-scattering geometry and focused on a confocal pin-hole (150 µm) in order to reduce stray light coming outof the focal plane. The emission of PCP complexes isextracted with HQ 650LP (Chroma) dichroic mirror andHQ 670/10 (Chroma) bandpass filter.

Figure 2. Experimental setup of the SIL-based confocal fluorescence microscopy.

Our experimental configuration allows for measuring flu-orescence intensity, spectra and lifetimes. The spectrum,dispersed using the Amici prism is measured with a CCDcamera (Andor iDus DV 420A-BV). The spectral reso-lution of the system is about 2 nm. Fluorescence in-tensity maps are collected with an avalanche photodi-ode (PerkinElmer SPCM-AQRH-14) with dark count rateof about 80 cps. Fluorescence lifetimes are measuredusing a time-correlated single photon counting module(Becker & Hickl) equipped with fast avalanche photodi-ode (idQuantique id100-50) triggered by a laser pulse.The time resolution of the TCSPC setup is about 30 ps.

First, in order to observe fluorescence maps for variousconcentrations of a fluorophore, a series of samples wasprepared. PCP with concentration of 0.49 mg/ml was dis-solved in a 2% water solution of PVA in a 1 to 10 ratio(stock solution). Next, we prepared 100-fold, 500-fold,and 2500-fold diluted PCP solutions. To make the sam-ples, 10 µl of each solution was dropped on a coverslipand spin-coated at 2500 rpm for 60 seconds. In this way,a thin, homogeneous layer was obtained. Embedding thePCP complexes in a polyvinyl alcohol (PVA) matrix hasbeen shown to improve the photostability of the PCP com-plexes [12] and should also minimize the impact of the air

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gap between the SIL surface and the sample.Next, we prepared a solution that was diluted by 12500times compared to the stock solution. The purpose was toobtain a sample with a concentration comparable to thesingle molecule limit for this system [13]. These highlydiluted samples were also spin-coated in a PVA matrixon glass coverslips. As a reference, samples containingonly the PVA water solution were prepared.

3. Results and discussionIn Fig. 3 we show fluorescence intensity maps obtainedwith the bare microscope objective for the 100-fold (3a),500-fold (3b), and 2500-fold (3c) diluted samples. The sizeof each map is 20×20 µm and the step size is 250 nm. ThePCP complexes were excited with a wavelength of 485 nmand the laser power at the objective was about 45 µW,while the integration time was 0.01 s. The fluorescencemaps are quite homogeneous, which suggests a uniformdistribution of the PCP complexes in a polymer matrix

for such concentrated solutions. We analyzed the mapsquantitatively by counting the occurrence of each fluores-cence intensity value within the map (histograms shown inFig. 3). The average fluorescence intensity decreases withdecreasing PCP concentration from about 8040 through2060 to 460 cps for the most diluted sample. The nor-malized widths of the histograms peaks are: 0.11, 0.27,and 0.47, respectively. The increase in the peak width isperhaps the result of reduced signal-to-noise ratio due toa decrease of the fluorophore concentration. As can beseen from Fig. 3, the histograms are not symmetrical andlow intensity tails are present. The origin of these tails isnot clear at this point: one possibility could be the pho-tobleaching of complexes during the experiment; on theother hand, such tails could also due to small variationsin PCP concentration across the sample. Nevertheless,the results shown in Fig. 3 indicate that the samples pre-pared in the PVA matrix indeed contain PCP complexes.This conclusion is important for carrying out experimentsfor highly diluted samples.

Figure 3. Upper row: Fluorescence intensity maps measured with the bare micorscope objective for PCP samples with concentrations diluted (a)100-fold (b) 500-fold, and (c) 2500-fold from the stock solution. Size of the maps is 20× 20 µm with a pixel size of 250× 250 nm. Bottomrow: Histograms displaying fluorescence intensities extracted from the respective maps.

In order to test the performance of the bare objective wehave measured fluorescence maps of a 12500-fold diluted

(4 · 10−6 mg/ml) PCP sample. Such a low concentrationis comparable to the single molecule limit described pre-

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viously for PCP in a PVA matrix [9]. The fluorescencemap of the sample is shown in Fig. 4a, and we compareit to the map obtained for our reference sample, the purePVA solution (Fig. 4b). The latter was prepared in exactlythe same way to facilitate straightforward comparison. Inorder to reduce the effect of photobleaching, particularly

important for low concentration samples, the laser powerwas reduced to 4 µW. At the same time the integrationtime was increased to 0.1 s. As in the case of the concen-trated samples (Fig. 3) we use histograms of fluorescenceintensity to quantify the observations.

Figure 4. Left column: (a) Fluorescence intensity maps measured with bare objective for highly diluted PCP sample (12500-fold diluted from thestock solution) compared to that of the reference PVA-only sample (b). Size of the maps is 20× 20 µm with pixel size of 250× 250 nm.Right column: Corresponding histograms of signal intensity obtained for the same two samples as in (a).

The reference sample (2% PVA water solution) intensityhistogram shown in Fig. 4b, is well defined by a Gaus-sian distribution with an average intensity of 1800 cps anda width of about 340 cps. In contrast, the PCP fluores-cence intensity map (Fig. 4a) shows many peaks with highfluorescence intensity, superimposed on a Gaussian back-ground distribution. The peaks correspond to PCP aggre-gates present in the sample, recognized as dark spots inthe image, a few pixels large (marked by the circles). Low

optical resolution makes estimation of the sizes of thesestructures extremely difficult.

The final step to demonstrate the advantages of using SILwas to repeat the experiment for the highly diluted PCPsample in a configuration where the microscope objec-tive is coupled with the SIL. A fluorescence map obtainedfor the highly diluted PCP sample (12500-fold from thestock solution) is shown in Fig. 5a together with an im-age collected for a reference sample comprised of PVA

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only (Fig. 5b). The excitation power of the laser remainedunchanged as compared to the data shown in Fig. 4.Since introduction of the SIL changes the overall numeri-cal aperture of the collection optics, the actual area mea-sured during the scan is smaller than the movement of theXY translation stage. From the calibration measurements(not shown) of back-scattered light from the semiconductorsample with 10 µm periodic squares, we have determinedthat the SIL reduces the image by a factor of 1.7 in a lineardimension. This value closely matches the refractive in-dex of the SIL material (1.85), indicating very good opticaldesign of the setup and in particular, negligible impact ofthe air gap between the sample and the PCP complexes.Consequently, the scan size of 20× 20 µm with step sizeof 250 nm translates into a map of 12×12 µm. We observethat distribution of the fluorescence intensity obtained forthe reference sample (Fig. 5b) has similar shape and av-erage intensity (about 1800 cps) as the results obtained

without the SIL (Fig. 4b). Although somewhat surpris-ing, this might indicate that we measure only stray lightnot associated with any unspecific emission from the ref-erence samples. Otherwise, the intensity should increaseby approximately 3-fold due to increased excitation powerdensity with the application of the SIL. In contrast, thefluorescence map measured for samples containing PCPcomplexes contains many extremely bright spots with av-erage intensities reaching 5000 cps. The intensities oftwo such spots A and B marked in Fig. 5a are 1700 and4220 cps, respectively. Moreover, the contrast betweenthe areas associated with emitting PCP complexes andregions where there is no fluorescing molecules is remark-ably high. Direct comparison between the fluorescencemaps obtained for highly diluted PCP samples measuredwith the SIL and with the objective only clearly point tosubstantial improvement of the data quality in the case ofthe former configuration.

Figure 5. Left column: (a) Fluorescence intensity maps measured with the objective coupled to the SIL for a highly diluted PCP sample (12500-fold diluted from the stock solution) compared to that of the reference PVA-only sample (b). Size of the maps is 12 × 12 µm with pixelsize of 150× 150 nm. Right column: Corresponding histograms of signal intensity obtained for the same two samples as in (a).

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In order to quantify the upper limit of the spatial resolu-tion in the SIL-based confocal microscope we show twoexemplary cross sections (Fig. 6) obtained for spots A andB marked in Fig. 5a. The time trajectory of the fluores-cence intensity measured for this spot features exponen-tial behavior, which suggests that the number of fluoresc-ing molecules is greater than ten [13]. For points A andB, we obtain widths of 640±10 and 770±20 nm, respec-tively. While the larger spot (B) is most probably due toan aggregate of PCP complexes, the emission attributedto the spot A, which is far less intense, could potentiallybe due to a smaller number of molecules. We note thatthese numbers do not reflect the actual size of the emitterbut are associated with the point spread function of theemitter which is in turn determined by the spatial resolu-tion of the collection optics. Therefore, the sizes obtainedin Fig. 6 provide an upper limit for our optical resolution.Future work will focus on studying the PCP complexeswithout the SIL-based confocal microscopy setup at thesingle molecule level. This should provide insight intothe photophysics of these complexes as well as allow fullcharacterization of the optical parameters of the setup.

Figure 6. Cross-sections along lines A and B shown in Fig. 5. Thenumbers correspond to the widths of the intensity profiles.

4. Conclusions

We demonstrate a configuration of fluorescence confo-cal microscope equipped with a solid immersion lens andits application for acquisition of high-resolution fluores-cence maps of light-harvesting complexes. Introductionof the SIL significantly increases the spatial resolution(∼ 600 nm) and light collection efficiency, allowing forimaging of molecule aggregates with low fluorescence in-tensity. The results presented in this work strongly sug-gest that this experimental setup is capable for studyingphenomena occurring in hybrid nanostructured systems ona single molecule level.

Acknowledgements

Financial support from the WELCOME program ‘Hybridnanostructures as a stepping-stone towards efficient artifi-cial photosynthesis’ awarded by the Foundation for PolishScience is gratefully acknowledged.

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sil-based confocal fluorescence microscope for investigating individual nanostructures

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