uniformity and fluorescence properties, these polymer layers may serve as a simple ..... ______. 0.0400 u. 0 2500 5000 750010000. FWHM. I_max. >- z_max.
SIPcharts Using Uniform Ultra-Thin And Thin Layers For Z-Response Measurements In Two-Photon Excitation Fluorescence Microscopy G. Vicidominia,c,f , J.M. Zwierb , P. Bianchinia,f , F. Cellaa , E. Ronzittia,f , S. Krola , T. Szellasd , G.F. Brakenhoffb and A. Diasproa,e,f . a LAMBS,
MicroSCoBiO, Department of Physics, University of Genoa, 16146, Genoa, Italy; Institute for Life Science, Section of Molecular Citology and Center of Advanced Microscopy, University of Amsterdam, 1098 SM, Amsterdam, The Netherlands; c Department of Computer Science, University of Genoa, 16146, Genoa, Italy; d Leica Microsystems CMS, D-68165, Mannheim, Germany; e CNR-Institute of Biophysics, 16149, Genoa, Italy; f IFOM The FIRC Institute for Molecular Oncology Foundation, 20139 Milan, Italy. b Swammerdam
ABSTRACT Layer-by-Layer or self-assembly techniques can be used to prepare fluorescent polymer samples on glass coverslips serving as benchmark for two-photon excitation microscopy from conventional to 4Pi set-up, or more in general for sectioning microscopy.1–3 Layers can be realized as ultra-thin ( 100 nm) or thin (approx. 100 nm) characteristics coupled to different fluorescent molecules to be used for different microscopy applications. As well, stacks hosting different fluorescent molecules can be also produce. Thanks to their controllable thickness, uniformity and fluorescence properties, these polymer layers may serve as a simple and applicable standard to directly measure the z–response of different scanning optical microscopes. In two-photon excitation microscopy z–sectioning plays a central role and uniformity of illumination is crucial due to the non-linear behaviour of emission. Since the main characteristics of a particular image formation situation can be efficiently summarized in a Sectioned Imaging property chart (SIPchart),3 we think that coupling this calibration sample with SIPchart is a very important step towards quantitative microscopy. In this work we use these polymer layers to measure the z–response of confocal, two-photon excitation and 4Pi laser scanning microscopes, selecting properly ultra-thin and thin layers. Due to their uniformity over a wide region, i.e. coverslip surface, it is possible to quantify the zresponse of the system over a full field of view area. These samples are also useful for monitoring photobleaching behavior as function of the illumination intensity. Ultrathin layers are also useful to supersede the conventional technique of calculating the derivative of the axial edges of a thick fluorescent layer. Polymer layers can be efficiently used for real time alignment of the microscope. Keywords: optical sectioning, z-response, ultra-thin uniform fluorescent layer, thin fluorescent layer, two-photon excitation microscopy, 4Pi microscopy, shift-variant system, optical alignment.
1. INTRODUCTION Although already known for a very long time,4 optical microscopy continues to gain importance, exploiting its unique capacity to enable high resolution in studies of biological systems from cells to tissues.5 In general, optical microscopy techniques offer a comparatively simple approach to study biological systems, combined with a suitable spatial resolution. As well, a continuous increase in radial and axial resolution has been achieved in the last twenty-five years: confocal microscope,6–8 multi-photon fluorescence excitation9, 10 and 4Pi microscope.11–13 The microscope resolution is known to be limited as a consequence of diffraction. For 3D (three-dimensional) optical sectioning the resolution performance along the z-direction (axial resolution) plays an important role. Further author information: (Send correspondence to G.V. or A.D.) V.G.: E-mail: vicidomini@fisica.unige.it, Telephone: +39 010 3536309 D.A.: E-mail: diaspro@fisica.unige.it, Telephone: +39 010 3536426 Multiphoton Microscopy in the Biomedical Sciences VII, edited by Ammasi Periasamy, Peter T. C. So, Proc. of SPIE Vol. 6442, 644224, (2007) · 1605-7422/07/$18 · doi: 10.1117/12.714250
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Unfortunately, the axial resolution is more sensitive to aberrations than the lateral component.14, 15 The 3D optical sectioning performance of the microscope is strongly related to the axial resolution of the system, which can be quantified by the so called 3D point spread function, i.e. P SFξ,η,ζ (x, y, z), that describes how the detected image of a point-like object at a given position (ξ, η, ζ) is blurred in space (x, y, z). Considering the optical microscope as a linear shift-invariant-system it can be fully described in terms of a unique PSF, i.e. P SF (x, y, z). The PSF of an optical microscope can be experimentally estimated as the response of the microscope to subresolution fluorescent objects. In physical terms, the effective PSF of a multi-photon confocal fluorescence microscope is given by: n → − → − (1) P SF (x, y, z) = A(x, y) ∗ | h det (x, y, z)|2 × | h ill (x, y, z)|2 , where: A(x, y) is the 2D function that describes the geometry of the detection pinhole; ∗ is the convolution product; hill denotes the field distribution of the illumination light in the focal region (thus |hill |2 is the intensity of the field) and hdet is the field distribution for the detection, which is similar to hill , but is calculated for the wavelength of fluorescence emission; with n=1,2,... for 1-, 2- and higher order multiphoton excitation. It is important to note that ideal confocal and multi-photon excitation microscopes PSFs can be obtain as limiting case of Eq. 1. For an ideal confocal microscope (ICM) the detection pinhole is infinitely small, thus A(x, y) can be modeled by an impulse function δ(x, y), moreover using one-photon excitation laser source (n=1) one obtain: → − → − (2) P SFICM (x, y, z) = | h det (x, y, z)|2 × | h ill (x, y, z)|2 . For a non-confocal multiphoton excitation microscope (MPEM) the detection pinhole is completely open, thus A(x, y) can be modeled by an infinitely large constant function and the convolution between A(x, y) and the → − | h det (x, y, z)|2 becomes a constant: n → − P SFMP EM (x, y, z) = | h ill (x, y, z)|2 . (3) However, the axial resolution of a sectioning fluorescence microscope is better quantified by its z–response to a subresolution fluorescent layer when scanned along the optical axis. The relationship between PSF and z– response under the shift-invariant assumption is given by the intensity distribution along the z-axis1 : I(z) = P SF (x, y, z)dxdy. (4) x
y
The full-width at half-maximum (FWHM) of the intensity profile along the axis serves as a reasonable measure for the axial resolution of the microscope. Due to the finite dimension of the fluorescent layers, it is not possible to measure exactly the z–response of a microscope system. But it is possible to measure the convolution of the z–response with the layer, according to the following relationship: Id (z) =
z
P SF (x, y, z)ld (z )dz ,
(5)
where ld is the function that describes uniform fluorescent layer of a given thickness d. As already demonstrated by Brakenhoff, et al.3 for a regular confocal microscope with a typical axial PSF width of around 600-700 nm (under high numerical aperture conditions) by using a layer thickness d of the order of 100nm (thin layer), convolution effects can be neglected. Same discussion can be made for two-photon excitation microscopy. On the other hand, for novel 3D imaging methods as 4Pi microscope, where the axial resolution decreases to around 100nm (a detailed description of 4Pi PSF can be found in Hell12 ), convolution effects must be taken into account. In order to neglect convolution effects for such systems, it is necessary to reduce the layer thickness d to few nanometers (ultra-thin layer). In the real case, the shift-invariant assumption is not true, thus the z-response of a sectioning fluorescence microscope is not identical over its imaging field. Eq. 2 shows that optimally the confocal PSF should be the product between diffraction-limited illumination and detection distribution function perfectly overlapping both
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Figure 1. Schematic of the multi-films deposition process using a coverslip and beakers. Steps 1, 3 and 5 represent the adsorption of a PEI, PAH-FITCH and PSS, respectively, and steps 2,4 and 6 are washing steps.
in the center as well as at the borders of the imaging field. However, optical aberrations (as chromatic aberration and off-axis abberation16 ) and alignments errors may damage this overlapping. This then will result in a different distribution of the confocal signal over the imaging field. In addition other parameter, as FWHM of the z-response, may show an irregular distribution over the field of the system. In MPEM the fluorescence signal is proportional to the quadratic or higher power of the intensity of the illumination (see Eq. 3). Well-focused, diffraction-limited distributions of the excitation result in the highest multiphoton yield. Multiphoton fluorescence signal is thus sensitive to off-axis aberration in the focusing of the excitation during the scanned acquisition. As in MPEM no detection pinhole is usually employed, the situation on the signal detection side will be less critical (in Eq. 3 detection term disappear). In 4Pi microscopy kept constant the phase difference between the two interfering wavefronts is very important. A change in the path of the two beams due to any kind of aberration radically alters the interference of the two beams and hence the structure of the 4Pi spot. This leads to greatly variations of the PSF in the imaging field. Brakenhoff et al.3 proposed a method to characterize the section properties of a 3D imaging system over all its imaging field. This method is based on the use of uniform fluorescent reference layers. Moreover, this methods developed a number of analysis criteria for the sectioning properties, and a simply but effective standard representation of these properties called Sectioning Imaging Property chart (SIPchart). The uniform thickness and uniform fluorescence properties are crucial for the success of the method proposed by Brakenhoff et al..3 Schrader et al. already used an ultra-thin (nanometre-scale) fluorescent layers for monitor the z-response of different sectioning system. Unfortunately these layers exhibited a limited uniformity over a small region, thus they were not aimed to provide characterization of the sectioning microscope over all its imaging field. In this work we propose a new fluorescent labeled polyelectrolyte (PE) ultra-thin (nanometre-scale) uniform layers for monitor the section properties of different fluorescence microscope. Ultra-thin dimension of the proposed layer it is particular important for monitoring 4Pi z–response. In particular we compare the SIPcharts obtained using the proposed ultra-thin layers and the thin (approx. 100 nm)layer developed by Zwier et al..17 We demonstrate as ultra-thin layers, despite their thickness and thus plausible low signal-to-noise ratio, are able for detecting small changes in the sectioning properties of a system. To compare SIPchart analysis based on thin and ultra-thin layer we used the same confocal system. This work is organized as follows. Section 2 shows materials and methods for PE layer preparation and an overview of the instrumentation used in this work. A characterization of the main properties of the proposed PE layer (thickness, uniformity over large area and fluorescence properties) is developed in section 3. Characterization of thin layers was already done byZwier et al..17 In section 4, the use of SIPchars with ultra-thin and thin layer for characterizing a confocal microscope and a TPE microscope are discussed. In section 5 we used ultra-thin layer for monitoring z–response of a 4Pi microscope system. Comments and discussion of the proposed PE layer conclude the work.
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2. MATERIALS AND METHODS 2.1. Preparation of ultra-thin and thin layers Polyethyleneimine (PEI: MW 25 kDa; Aldrich, Milan, Italy), poly(4-styrenesulfonate sodium) (PSS; MW 70 kDa; Aldrich, Milan, Italy), fluorescein labelled poly(allylamine hydrocloride) (PAH-FITC; compound was made using poly(allylamine hydrochloride), polymer base MW 15 kDa; Aldrich, Milan, Italy) were used to build the ultra-thin fluorescent layers. Multilayer films were prepared on 0.17 mm thickness glass coverslip (Forlab Carlo Erba, Milan, Italy) or 0.22 mm thickness circular quartz coverslip for 4Pi microscope. In both case the coverslips were cleaned for 30 min in NOCHROMIX solution (Godax Laboratories, Inc., Takoma Parc, MD, USA) and successively washed twice in Milli-Q grade pure water (Milli-Q-System, Millipore, Milan, Italy) under sonication (Transonic 130, ADAC Laboratories, Milpitas, CA, USA) for 15 min. The method used to prepare ultra-thin polyelectrolyte film is based on the Layer-by-Layer (LbL) technique.18 A pre-coating of the glass surface is mandatory since Lowman and Buratto19 found that films with an initial PEI layer produce a much smoother surface. Glass pre-coating was done by adsorbing a single layer of PEI on the coverslip. The coverslip was incubated into PEI solution for 15 min, then washed 3x by dipping in 0.15 M NaCl solution . PE layers were applied in the very same way. Only one type of film was utilized, namely: PEI/PSS/PAH-FITC/PSS (see Fig. 1). The coated glass coverslip was connected to a standard microscope slide (76x26mm). For 4Pi measurements, a drop of 87% glycerol (20l buffered with Milli-Q grade pure water) was placed between a coated and an uncoated quartz coverslip. Under a moderate pressure the glycerol was distributed and the coverslips were mounted on a 4Pi sample holder. More detail about ultra-thin layer fabbrication methods can be found in Vicidomini et al..2 Thin layers proposed by Zwier et al.17 are obtained by spin-coated of fluoresceine and polyvinyloalcohl solutions. More details regarding thin layers and their fabrication methods can be found in Zwier et al..17
2.2. Instrumentation The results presented in this work were obtained by using a Leica SP5 (Leica Microsystems, Mannheim, Germany) spectral microscope. The system mounts a HCX PL APO 63x 1.4 oil immersion objective (Leica Microsystems, Mannheim, Germany) resulting in a maximum imaging field of 245 x 245 µm. For confocal imaging we uses an Argon laser operating at λ=488 with an average power of approx. 40 µW, measured at the back focal plane of the objective. Airy 1 pinhole dimension was chosen. For two-photon excitation (TPE) imaging a Chameleon-XR (Coherent, Santa Clara, CA) laser source was coupled directly in the scanning head of the the system (pulse widths lie within the range of 100–140 fs full-width at half maximum at a repetition frequency of 80 MHz and 90 MHz, respectively, at the laser output window, excitation wavelengths from 705 to 980 nm, average output power of 1.5 W). TPE measurements were performed using an illumination of 800nm wavelength with an average power of around 200 µW, measured at the back focal plane of the objective. Pinhole completely open was chosen. Both confocal and TPE measurements were obtained using a pixel dwell time of 4.88 µs and, by means of spectral features of the system, fluorescent signals is detected within 520–550 and 560–590 nm (data not shown) spectral windows at the same time. 4Pi images were acquired by a Leica TCS 4Pi (Leica Microsystems, Mannheim, Germany). The 4Pi-unit was tightly mounted to the microscope turret to maintain all the capabilities of the scanning confocal unit in the microscope body. For two-photon excitation, the beam of a mode-locked Ti:Sapphire ultralaser Chameleon-Ultra (Coherent, Santa Clara, CA, USA) was coupled with the confocal microscope and directed towards the 4Pi-unit (Hanninen, et al. 1995). 4Pi measurements were performed using a glycerol immersion objective (HCX PL APO 100x/1.35 Glycerol 0.22/0.22 Pair, Leica Microsystems, Wetzlar, Germany), an illumination of 780nm wavelength with an average power of around 100W and fluorescence signal was detected in the 500-550 nm spectral range using an avalanche-photo-diode. To increase the signal-to-noise ratio, the imaging was performed with a pixel dwell time of 4.88 s, a scanning line average of 4 and a frame accumulation of 4.
2.3. SIPchart representation Various choices of parameters can be made to analyse the z-response of a 3D system. Section Imaging Propertycharts (SIPcharts) is a very powerful tool for 3D image characterization that needs simply a trough-focus data set of a fluorescent layer. Here we describe briefly the main imaging properties of sectioning involved in SIPcharts analysis. For a complete treatement we remand to Brakenhoff et al.3 :
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• Itotal , the total intensity of the z-response (the sum of Eq. 4 along z); • Imax , the maximum fluorescence intensity found along the z-response; • zmax , the axial position at which the value Imax is found; • F W HM , full width at the half maximum of the z-response; • Skew, the axial asymmetry of the z-response. It is defined as s = (a − b)/(a + b) , where a and b are the asymmetry factors of the z-response evaluated at the half maximum intensity level. As these parameters can be determined at each point in the imaging field of the scanning system it is a logical step to represent the data in the form of false-colours maps. These maps allow immediate access to the variation of the properties over the imaging field. Moreover, using the SIPcharts representation another three important insets are reported: • the intensity profile of the z-response measured in the centre and at four off-centre locations of the imaging field; • a resolution bar to gain an ’at glance’ impression of the experimental FWHM z-response versus the theoretical one expected (computed numerically Eqs. 1 and 4); • average and variation of the above parameters over the respective maps. All SIPcharts analysis reported in this work were evaluated on through-focus data sets of 64 x 64 points obtained from binning of original 3D stack images of 512 x 512 pixels. 3D stacks were acquired using the maximum scan fields of the systems and with a z-step of 100 nm. To be sure that SIPchart analysis were not hampered by bleaching effects we repeated each 3D axial acquisition two times. No relevant decrease of the best focus plane mean intensities appear using TPE and confocal imaging condition described above (see Subsection 2.2).
3. ULTRA-THIN FLUORESCENT PE LAYER PROPERTIES 3.1. Thickness To measure the z-response and the variations of the sectioned imaging property with acceptable resolution it is essential that the layers used are reasonably thin with respect to the dimensions of the axial PSF. On the other hand, a layer that is ’too thin’ will lead to lower signal-to-noise ration (SNR) in the fluorescence data. Brakenhoff et al.3 found that for a typical axial PSF width of around 700 nm a layer thickness of around 100 nm proved a good compromise. We demonstrate as also ultra-thin layers with a thickness of few nanometers can be used for monitoring sectioning imaging property both for confocal and TPE microscopes. Ultra-thin layer preparation as well as the concentrations used are in complete accordance with the protocol described by Etienne et al..20 There is a linear growth of the polyelectrolyte multilayer films, with an increment of approx. 40 ˚ A for a single layer.21 We occasionally checked for film thickness using atomic force microscope in scratching mode (data not shown).
3.2. Uniformity In order to use the proposed ultra-thin layer for SIPcharts analysis the most important requirement is the spatial uniformity. To demonstrate such a feature we followed the imaging analysis made by Zwier et al.17 but using confocal images in open pinhole configuration and not wide-field images: two fluorescence intensity images were acquired in two different positions of the layer (Fig. 2(g)). The former image was used as ”reference image” (Fig. 2(a)) and the latter as the ”object image” (Fig. 2(b)). Then we calculated the ratio between the ”reference image” and ”object image” (Fig. 2(c)). A narrow distribution of the intensity of this image with an average value of 1 indicates a uniformity of the sample (Fig. 2(f)). In our measurement, we get an average value of 0.901 and a FWHM of 0.143. Our values compared with the values obtained by Zwier et al.17 for the 100 nm layer (avg. 0.999 and FWHM 0.038) seem to indicate a lower uniformity. These differences can be explained by the lower amount of fluorescence molecules for the ultra-thin layer respect to the 100 nm layer that can cause a worse signal-to-noise ratio.
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Figure 2. .(a) ”reference” image; (b) ”object” image; (c) resulting ”calibrate” image; (d-f) corresponding histograms of pixel intensity values of these images; (h) the configuration of reference and object image.
3.3. Fluorescence property The emission spectra of the PAH-FITC layer were measured directly on the cover slip at an excitation wavelength of λ = 488 nm. Spectra shows an emission peak of FITC at around 530 nm, shifted to 10 nm with respect to the emission maximum for FITC described in literature (molecular probes). This is in agreement with a possible series of photo-reactions in the fluorophores, which are also responsible for changes in the bleaching rate.22 Other properties such as fluorescence linearity intensity in one-photon regime and bleaching behavior are discussed in Vicidomini et al..2
4. SIPCHARTS ANALYSIS This section demonstrates the sensitivity to track small changes of the sectioned imaging properties with ultrathin uniform fluorescence layers. In particular we want to demonstrate as ultra-thin and thin layers are able to characterize the very same distribution of the sectioned imaging properties both in confocal and TPE microscopy. Figures 3 and 4 show SIPchart analysis for SP5 confocal microscope system using respectively ultra-thin and thin layer. The comparison between SIPcharts based on ultra-thin and thin layer does not show particulary differences, especially for average and variation of all the properties mentioned in Subsection 2.3. Both for ultrathin and thin layer SIPcharts, areas with maximum intensity (Imax and Itot ) correspond well with those with better resolution (F W HM ). This suggests that both layers gives the same characterization. The differences between the pattern of the corresponding maps (in particular Imax ,Itot and F W HM maps) in the ultra-thin and thin SIPcharts can be explained by a possible different tilt of the fluorescent layer. This explains also the compliably different pattern of zmax maps.
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Figure 3. Sectioned Imaging Property chart for confocal microscope system using ultra-thin layer.
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Figure 4. Sectioned Imaging Property chart for confocal microscope system using thin layer.
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Due to the different thickness of the two layer, and thus the different amount of fluorescence intensity, the measurement with ultra-thin layer are obtained using an higher photomultiplier voltage amplification. A lower SNR of ultra-thin SIPchart maps in comparison with thin SIPchart maps is evident, but this do not hamper the results obtained.
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Figure 5 shows SIPchart analysis for TPE microscope system using thin layer. SIPchart shows a very high variation for Imax and Itot properties. This is in complete accordance with the theory: because the signal depends on the square of the excitation intensity (see Eq. 3), small variations of the position of the excitation diffraction spot can lead to rapidly decreases of the fluorescent signal. F W HM and Skew maps show a good uniformity of the TPE system in terms of resolution.
Figure 5. Sectioned Imaging Property chart for TPE microscope system using thin layer.
5. 4PI Z-RESPONSE MONITORING Ultra-thin fluorescent layers combined with z–scanning system were used to monitor the z-response of a 4Pi microscope operating under TPE regime. Unlike confocal analysis, the ultra-thin thickness of the layer became a very important condition for direct measurement of the z-response of the system. 4Pi microscope is known for a theoretical axial resolution of around 100nm. For this reason layers with a thickness of 100 nanometers can not be considered as sub-resolution objects. Ultra-thin fluorescent layers for direct measurements of the z-response of the 4Pi microscope were already used by Schrader, et al..1 Unfortunately, samples were not uniform over large regions. Thus monitoring the z-response of the system over the whole field of view was difficult. Figure 6(a) shows an axial scan over the maximum scanning field of view for the system (93.50 m). The intensity profiles in different positions within the scanning field of view (Fig. 6(b),(c) and (d)) show that it is possible, by using ultra-thin PE layer, to monitor z-response in all these positions. Figure 6(e) shows an axial scan for a smaller region of the scanning field of view (6.20 µm). Figure 6(g) is a typical intensity profile of a 4Pi z-response with a sharp maximum and two pronounced lobes. The results found with PE layer are in complete accordance with the results obtained by Schrader, et al.1 : the FWHM of the sharp maximum (Fig. 6(g)) is 117 nm and after fast linear one-step deconvolution 96nm.23 These values can be improved by a rigorous alignment of the system.
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Figure 6. Figure describes z-response of 4Pi system with the typical interference pattern. (a) 4Pi xz image of the whole imaging field of view. Spatial resolution was 93.54 µm 3.65 µm. (b)-(d) Intensity profile through the xz image in three different positions, respectively in positions 1,2 and 3 (all the profiles are lateral averaged across 8 pixel = 365nm). (e) Physically zoomed image of the area inside the white box of (a) (scale bar = 1µm). (f) same image of (e) after fast linear one-step deconvolution (scale bar = 1µm). (g) Intensity profile of the uncorrected image (e, black line) and corrected image (f, gray line) along position 4 (profiles are lateral averaged across 8 pixel = 97nm)
6. CONCLUSION Brakenhoff et al.3 presented a method for characterizing the sectioning properties in confocal and multiphoton microscopy by means of a thin (100 nm thickness) fluorescent polyvinyloalcohl layer. The present work has shown that also ultra-thin2 (few nanometer thickness) fluorescent polyelectrolyte layers could be an optimum candidate for monitoring the variations of z-response over the imaging field of confocal and multi-photon microscopes. As well, our data further enforce the use of SIPcharts representation as an optimum tool for microscope characterization. Moreover, because of its few nanometer thickness ultra-thin layer can be used for monitoring z-response of novel 3D imaging methods as 4Pi microscope, where the axial resolution decreases to around 100nm. Because the polyelectrolyte can be covalently labeled with different dyes and the easy to use LbL technique is useful to create multilayer films; the development of a unique sample containing different ultra-thin layers of different excitation and emission spectral properties is under development. This new sample might be used to monitor the chromatic aberrations of the optical system: short distances between fluorescent polyelectrolyte layers guarantee that no spherical aberration effects induced by mismatch in refractive index can hamper results.
Acknowledgment We are greatly indebted to Rolf Borlinghaus, Martin Hoppe and Paolo Sapuppo of Leica Microsystems for 4Pi measurements. This work was partially granted by MIUR, IFOM (Milan) and Compagnia di San Paolo (Turin).
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