Tilted Two-Dimensional Array Multifocus Confocal Raman

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Tilted-Two-Dimensional-Array MultiFocus Confocal Raman Microspectroscopy Sohshi Yabumoto, and Hiro-o Hamaguchi Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 20, 2017

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Analytical Chemistry

Figure 6. Raman mapping image of polystyrene (PS) beads suspended in water. Microscopic image (a), Raman spectrum of PS and background luminescence spectrum reconstructed from the two most significant SVD components (b), the image of PS Raman scattering (c) and that of background luminescence. 183x146mm (300 x 300 DPI)

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Tilted-Two-Dimensional-Array Multi-Focus Confocal Raman Microspectroscopy Sohshi Yabumoto and Hiro-o Hamaguchi* Department of Applied Chemistry and Institute of Molecular Science, National Chiao Tung University, 1001 Ta Hsueh Rd., Hsinchu 30010, Taiwan ABSTRACT: A simple and efficient two-dimensional multi-focus confocal Raman microspectroscopy featuring the tilted-array technique is demonstrated. Raman scattering from a 4×4 square foci array passing through a 4×4 confocal pinhole array is tilted with a periscope. The tilted array of Raman scattering signals is dispersed by an imaging spectrograph onto a CCD detector, giving 16 independent Raman spectra formed as 16 bands with different heights on the sensor. Use of a state-of-the-art imaging spectrograph enables high-precision wavenumber duplicability of the 16 spectra. This high duplicability makes the simultaneously obtained spectra endurable for multivariate spectral analyses, which is demonstrated by a singular value decomposition analysis for Raman spectra of liquid indene. Although the present implementation attains only 16 measurement points, the number of points can be extended to larger than 100 without any technical leaps. Limit of parallelization depends on the interval of measurement points as well as the performance of the optical system. Criteria for finding the maximum feasible number are discussed.

INTRODUCTION Confocal Raman microspectroscopy is now well established as a powerful tool for investigating complex heterogeneous molecular systems in many research fields including bioscience,1,2 medicine,3–5 pharmaceutical science,6 food science,7,8 polymer science,9 stress analysis for semiconductors,10 and so on. It enables identification/characterization of molecules within a local microscopic volume of materials of virtually any sorts, nondestructively, low-invasively without any marking or pretreatments. It can effectively eliminate unwanted background light emitted from the substrate or the sample cell wall in defocus volume.11,12 An intrinsic difficulty is yet to be overcome, however, with respect to its efficiency. Because of the low cross section of Raman scattering, a long measurement time is usually required in Raman microspectroscopy. This low efficiency becomes particularly problematic in the Raman mapping experiment, in which hundreds to thousands of Raman spectra have to be collected to make an image. Considerably high excitation power is often employed in order to make Raman mapping time tolerably short. Such high-power excitation may well lead to undesirable effects on samples when the laser beam is tightly focused. In fact, a number of previous studies have shown that lower power of a few milliwatts or less is desperately required for photo-labile samples like living cells and organs.13–16 High speed is thus indispensably needed to make low excitation power compatible with short Raman mapping time. Speeding up of Raman mapping can be achieved by means of parallelization of the confocal optics. Bowden et al reported a line-scan system as the first demonstration of the onedimensional parallelization.17 This line-scanning technique, also known as slit-scanning, features a line illumination with excitation light and that enables the acquisition of multiple spectra within the line simultaneously.18–22 It is effective in shortening the mapping time considerably but intrinsically lacks in high depth resolution since the confocal effect is only one-dimensional.23 We developed a two-dimensional multifocus confocal Raman system using fiber image compression (FIC). Raman scattering signals aligned on a two-dimensional array pinholes were rearranged by using an optical fiber bundle into a one-dimensional array that fits to the entrance slit of the spectrograph.15 A 6×8-fold parallelization was achieved

first followed by an extension to 21×21-fold.24 The FIC technique has successfully shortened Raman mapping time while keeping the depth resolution as high as that of single point confocal Raman microspectroscopy. However, it requires a precision fiber bundle that is not readily available at the present time. Furthermore, use of a fiber bundle introduces extra challenges for the orientation matching between each fiber element and the signal beamlet. Kong et al developed another multi-focus confocal Raman microscope system employing galvo-scanner image compression (GIC).25,26 In this method, generation of multiple foci and the image compression are achieved temporally by multiplying the light path with galvo scanners. It has the advantage of having flexibility in the excitation pattern to customize the number of foci as well as their location without replacing any optics in the system. However, the parallelization is only as a result of time accumulation and samples have to be exposed to a focused laser beam of hundreds of milliwatts. Moreover, use of galvo scanners makes the apparatus rather complex and expensive. In this article, we demonstrate a new two-dimensional multi-focus confocal Raman microspectroscopic system without the use of a fiber bundle or galvo scanners. We shine the sample with 4×4 foci arranged in a square lattice pattern. Raman scattering from this foci array is tilted by a periscope, then dispersed and imaged by a spectrograph onto a charge-coupled device (CCD) detector. Raman spectra from the 4×4 (= 16) different sample points are imaged as 16 separate bands with different heights on the detector. A contemporary distortionfree imaging spectrograph enables two-dimensionally aligned signals to be dispersed and detected simultaneously without appreciable distortion. The present tilted-array technique makes parallel microspectroscopy much more simple and efficient. The multiplicity can in principle be increased to more than 100 without any technological leaps.

EXPERIMENTAL SECTION The tilted two-dimensional array method In tilted two-dimensional-array multi-focus confocal Raman microspectroscopy, sample is illuminated by excitation light with multiple foci arranged in a square lattice pattern. Raman scattering from this foci array is focused on a confocal pinhole array and then the square array is tilted by an angle θ (Figure


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Figure 1. Concept of tilted array technique. A Raman signal array with tilt angle θ (a) is input to spectrograph to form piled-up bands of spectra (b). For array with n points in row and with pitch of p, optimal tilt angle θ and interval of spectra p′ are derived as shown in (c). Tilt angle θ is adjusted by periscope (d). Here, L: lenses, M: mirror, and PM: periscope mirror pair.

1 (a)). The Raman scattering signals in the tilted square array are dispersed and imaged by a spectrograph onto a CCD detector. Within this transmitting path, the square pattern of signals is tilted but does not undergo any rearrangement unlike the cases of the FIC or GIC techniques. Because of the tilted configuration, Raman scattering signals from different foci do not overlap with one another upon dispersion; all the spectra are formed as separate bands with different heights on the CCD detector (figure 1 (b)). The optimal tilt angle θ depends on the number of points in a row of the array n. As shown in figure 1 (c), it can be derived as θ = arctan (1/n). In order to tilt the square array precisely, we use a periscopic mirror pair. By setting the angle between the incoming and outgoing beams as θ, the output image can be rotated around the optical axis by θ as shown in figure 1 (d). It makes the tilt angle adjustable independently of the excitation pattern, and consequently, we can optimize the tilt angle while maintaining the excitation pattern upright. The tilted two-dimensional array method works ideally when a distortion- and aberration-free imaging spectrograph is used. As the number of foci increases, the distance between the two adjacent spectra on the detector becomes smaller (figure 1 (c)). Aberrations, especially coma in the periphery, blur the image ending up with larger spread of spectra on the CCD detector to overlap with the adjacent. Aberrations also result in poorer spectral resolution, since blurring is equivalent to a wider slit width of the spectrograph. Radial distortion can also

degrade the performance of the tilted two-dimensional array system. It bends the spectra on the CCD detector to make it impossible to read them out by simple hardware binning. Thus, the imaging quality of the spectrograph crucially determines the performance of the system. Apparatus Figure 2 depicts the apparatus constructed in the present study. Excitation beamlets are generated with a commercial component manufactured by Tokyo Instruments, Inc.24 Briefly, beamlets with a given number and pitch are generated from a single laser beam with a diffractive optical element (DOE) and subsequently relayed with lenses to be introduced into the microscope. The specifications of the component are as follows: excitation wavelength 660 nm, excitation pattern 21×21 square lattice array, pitch 700 nm with a 100× objective lens. In the present study, the beamlets are thinned out to 4×4 arrays by a masking pinhole array so that the pitch of 3.5 µm with a 100× objective lens makes sufficient spaces that accommodate all the dispersed signals without overlapping. One might think that such a large pitch weakens the merit of the parallelization, especially when it comes to imaging. However, smaller clearance between the excitation spots also spoils the ability of depth sectioning of confocal microscopy drastically.24 Consequently, we consider that the pitch of 3.5 µm is a rather fair value for the size of target objects at the magnification of 100. The total excitation power at the sample is approximately 5.8 mW, and hence the average power per measurement point 0.36 mW. The microscope used in the present study is an inverted-type (Ti-S, Nikon). The excitation beamlets introduced to the microscope illuminate the sample through an objective lens, a Nikon’s CFI Plan Apo VC 100× Oil (NA1.4) or a CFI S Plan Fluor ELWD 40× (NA0.6), depending on the purpose of measurement. Note that the pitch of the excitation pattern depends on the magnification of the objective lenses, namely 3.5 µm with 100× and 8.75 µm with 40×. Raman back scattering from the sample is collected with the same objective lens and led to an analyzer through a long-pass edge filter that transmits

Figure 2. Diagram of present apparatus. Here, CPA: confocal pinhole array, DOE: diffractive optical element, M: mirrors, L: lenses, LEF: long-pass edge filter, MPA: mask pinhole array, OL: objective lens, PS: periscope, and TL: tube lenses. Asterisks stand for a part belonging to a commercial FIC-type multi-focus confocal Raman microspectroscopic system.



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the Raman-shifted signal light. The signal light is then focused on a pinhole array. The diameter of each pinhole is approximately 1.2 in Airy unit (1 Airy unit equals 1.22 λ / NA, where λ is wavelength and NA numerical aperture). The array is placed accurately so that each Raman signal beamlet goes through the corresponding confocal pinhole. The signal light is collimated and, subsequently, rotated around the optical axis by a periscope by 14.0 degree, which is optimal for the present case (n = 4), as mentioned above. The signal is finally focused on the entrance of an imaging spectrograph (IsoPlane SCT 320, Princeton Instruments), which employs a novel SchmidtCzerny-Turner configuration to minimize distortions and aberrations that occur in conventional spectrographs.27 In the present study, all measurements were conducted with a grating of 600 lines/mm. The entrance slit of the spectrograph is wide opened so as to accept the whole signal light. In the present set-up, the pinhole array, which is located distant from the spectrograph, in effect plays the role of the entrance slit in conventional spectrometers. The spectra are recorded on a TEcooled 1024×1024 electron multiplying CCD (EMCCD) detector (ProEM+:1024B, Princeton Instruments). Each spectrum from the different measurement points imaged on the CCD detector is digitized by vertical hardware binning of the most intense two rows of pixels in order to shorten the readout time to 50 ms and to avoid the contamination between the adjacent spectra as discussed in the following section. The microscope is equipped with a piezo scanning stage (PK3L150-100UA, Nano Control), so that a sample can be scanned with a precision of submicrometers. Spectra acquisition, sample scanning, and spectral and sensitivity calibrations are automated on a personal computer.

Figure 3. Raw CCD images of white light without dispersion (a) and with dispersion (b) by spectrograph. Each plot shown in right inset of image (b) is cross-section of image along line in corresponding color. (c) Point spread functions (PSF) of optical system derived from image (a). Bottom and right insets show its cross sections at center (solid) along with theoretical PSF at diffraction limit (dotted).

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RESULTS AND DISCUSSION The raw image of the confocal pinhole array obtained with the zeroth-order diffraction is shown in figure 3(a) together with the image obtained with the first-order in figure 3(b). The image was recorded by spatially homogeneous Köhler illumination of a white light lamp on the focal plane of the objective lens, and hence on the confocal plane at the pinhole array. As the image shows, aberrations and radial distortion are negligibly small over the present field of view. From the image in figure 3 (a), we can calculate the point spread function (PSF) of the optical system that transfers the image of a confocal pinhole array to the CCD detector as shown in figure 3(c). The diameter of the PSF’s center disk is approximately 5 pixels, which is comparable to that of the diffraction-limit Airy disk, indicating that the present optical system is close to the ideal. The diameter of the pinhole imaged on the CCD detector is calculated to be 5.0 pixels if there are no diffraction or aberrations. Therefore, the actual pinhole image, being the convolution of the PSF and the pinhole, spreads over about 10 pixels vertically and horizontally on the CCD detector. On the other hand, the interval of the spectra imaged on the CCD detector is as small as 6.3 pixels. Consequently, all the adjacent two spectra must overlap, resulting in contamination of the spectral data. Nevertheless, we found that such contamination is practically negligible when only the most intense two rows of each spectrum are binned to read out, although it substantially loses the signal counts. The displacement between the spectral axes of beamlet 1 and beamlet 16, which is the largest displacement between the beamlets on the CCD sensor, is 101 pixels. This displacement corresponds to 120 cm−1 in Raman shift. The actual measureable spectral range is approximately 1080 cm−1. Figure 4 (a) shows the original CCD readout that simultaneously records Raman spectra of liquid indene from 16 differ-

Figure 4. Raw readout of CCD detector that records Raman spectra of liquid indene sample from 16 different measurement points (a) and their calibrated and normalized spectra shown superimposed, in different colors (b). Each vertical bin in image (a) corresponds to a measurement point. Insets in graph (b) are zoom-up of Raman bands in boxes with broken line.


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Figure 5. Result of SVD analysis performed on Raman spectra of bulk indene: Singular values (a) and spectra (b) of five most significant components.

ent measurement points. Each row of the readout was obtained by vertical binning of two pixels on the CCD detector and corresponds to a particular measurement point. The raw Raman spectra were individually calibrated for the spectral and intensity axes to form Raman spectra that are ready to be analyzed as shown in figure 4 (b). The Raman intensities were normalized by their area intensities of the band at 1205 cm−1 in order to compensate intensity variations (~20 %) among the 16 beamlets from DOE. The spectral axes of the 16 raw Raman spectra have different CCD pixel numbers with specific shifts. They are consistently resampled upon calibration with at least twice as many points as those of the raw spectra. All the resultant 16 Raman spectra overlap almost perfectly with one another, indicating high spectroscopic duplicability among the different measurement points. For three bands at the leftmost, center and rightmost wavenumber regions in the spectra, peak positions and bandwidths were obtained with errors defined by 3σ (1σ equals standard deviation) as 1609.4 ± 0.1 cm−1 / 7.3 ± 0.1 cm−1, 1205.2 ± 0.1 cm−1 / 6.2 ± 0.1 cm−1, and 730.5 ± 0.2 cm−1 / 6.0 ± 0.2 cm−1, respectively. Such high spectroscopic duplicability even makes it possible to apply the spectra of the different measurement points to multivariate analyses, which are very sensitive to inconsistency in the spectral calibration and resolution. To evaluate such artifacts that may be caused in a multivariate analysis due to the spectroscopic inconsistency among the different measurement points, a singular value decomposition (SVD) analysis28 was performed for the indene spectra shown in figure 4 (b). Figure 5 (a) shows singular values of the most significant five components, and (b) their spectra. Component 0 is, by nature of the sample, the only physically meaningful component, and obviously represents the genuine Raman spectrum of indene. On the other hand, less significant components (1 to 4) feature first and second derivatives of indene peaks, which are ascribable to the arti-

facts due to the spectral-axis shift and the spectral-resolution inconsistency, respectively. The present analysis shows that the singular value of the largest artifact component (component 1) is only 1/166 as large as that of the physically meaningful component (component 0). Consequently, contribution from those artifacts to the spectra is of little account and, thus, may be safely ignored in most practical applications. Spectral resolution of the present system was also evaluated from line width of a neon lamp to be 5.6 ± 0.3 cm−1. It is not adjustable unlike conventional spectroscopes with a variable slit, since the slit function of the present apparatus is determined by fixed size of the confocal pinholes and the magnification of the relay lenses. Spatial resolution as defined by the full width at half maximum of the PSF was measured in water to be 0.3 µm for lateral and 0.7 µm for axial with the 100×/1.4 objective. In Figure 6 we show a Raman mapping image of 3-µm polystyrene (PS) beads suspended in water. The sample was scanned with the 4×4-array beamlets (10.5×10.5 µm2 with 3.5µm-pitch) at an interval of 0.35 µm to make 10×10 measurement points interposed between the pitches. Thus, the Raman data were collected from 40×40 (= 1600) points with the interval of 0.35 µm. The exposure time was 5 seconds and the overall measurement time 516 seconds. It would need a long collection time, ~8000 s (2.2 hr) at least, to make the same image with the single-beam conventional Raman microspectroscopic system. The obtained 1600 Raman data were analyzed by SVD. The following reconstruction by linear combinations of the two most significant SVD components gave the Raman spectrum of PS and the spectrum of background luminescence (figure 6 (b)), as well as their images (figure 6 (c), (d), respectively). Checkerboard-like-pattern artifact observed in the images is due to the variations among the laser intensities in the 16 excitation beamlets and the variations among the transmission efficiencies in the 16 signal beamlets. This result indicates that the 16 spectra from different points can be compared as they are in terms of Raman shifts but that careful

Figure 6. Raman mapping image of polystyrene (PS) beads suspended in water. Microscopic image (a), Raman spectrum of PS and background luminescence spectrum reconstructed from the two most significant SVD components (b), the image of PS Raman scattering (c) and that of background luminescence.



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normalization is required when they are compared in terms of intensity. Solutions for this problem may include (1) beam splitting optics that create uniform beamlets, (2) a higher precision pinhole array, (3) lower-distortion relay optics, and (4) larger intervals of the measurement points. In the present tilted-two-dimensional-array method, all the signal beamlets are transferred through free space to the spectrograph. They only undergo focusing and collimation with lenses, which is a natural extension of the conventional singlebeam technique. In contrast, the FIC technique requires the coupling of the signal beamlets to the fiber bundle. An advantage of the present approach lies in the fact that the coupling of the beamlets to a pinhole array is easier than that to the fiber bundle, which requires the orientation matching additionally. The intrinsic trade-offs of the present approach are two-fold. First, the density of the measurement points cannot be increased beyond the limit derived from the relation in figure 1 (c). However, this restriction is practically not important, since denser arrangement of measurement points increases the interference among beamlets and hence degrades the contrast of Raman images.24 Second, since the input of the signal beamlets to the spectrograph are horizontally displaced, certain number of CCD channels are not used making the measureable spectral range smaller. The actual measureable spectral range depends on the linear dispersion of the spectrograph and the magnification of the relay optics as well as the arrangement of the measurement points. The maximum measureable spectral range is accomplished with the minimum magnification of the relay optics that resolves the two neighboring spectral bands on CCD. We have demonstrated here multi-focus confocal Raman microspectroscopy with 16 measurement points. The method, however, can achieve more than 100 simultaneous measurements without any further technical leaps. The maximum number of simultaneous measurement points depends on the pitch of measurement point array and the PSF of optical system that images Raman signals on the CCD detector. It is also dependent on parameters such as numerical aperture and wavelength. In order to avoid the contamination of adjacent spectra on the CCD detector, the interval of the spectra p′ should be larger than the diameter of the pinhole image Φ on the CCD detector. As shown in figure 1 (c), the interval of the spectra p′ is expressed by = ⁄√ + 1, where p denotes the pitch of measurement point array and n the number of points in a row of the array. Therefore, from the relation ⁄√ + 1 ≥ , the allowed maximum number of measurement points in a row of the array, nmax can be derived as follows: max

= floor − 1 .

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Thus, the maximum number of measurement point in a row, nmax, can be finally rewritten as follows: max = floor

%1.22 "'Raman ⁄)*obj -

− 1 ,

where λRaman is the representing wavelength of Raman scattering, NAobj numerical aperture of the objective lens. The scaling factor, m, may ideally equal the diameter of pinhole in the Airy unit plus unity, but should be adjusted according to the performance of the optical system such as distortions and aberrations so that no spectral contamination among the different measurement points occurs. On the other hand, the maximum number of measurement points in a column is determined by sensor size or field-of-view size of the optical system, which can be ruled by any one of the objective lens, the relay optics, and the spectrograph. Thus, a pitch of 10 µm, an excitation wavelength of 660 nm and hence a representing Raman wavelength of 710 nm, an objective lens with numerical aperture of 1.4, and a scaling factor of 2.0 are assumed, for example, the maximum number of measurement points in a row of the array is calculated to be 8. If the field of view of the system accommodates 15 measurement points in a column of the array, Raman spectra from 120 different measurement points can be obtained simultaneously.

CONCLUSIONS We have demonstrated 16 multi-focus confocal Raman microspectroscopy based on the tilted two-dimensional array method. The method enables us to obtain simultaneously 16 Raman spectra from 16 different measurement points in the sample. The 16 spectra obtained from a homogeneous liquid sample show an excellent agreement with one another, indicating the high spectroscopic duplicability of the method. In other words, the simultaneously obtained 16 spectra from an inhomogeneous sample should carry spectroscopic information exactly the same as that of 16 Raman spectra measured point by point with single-focus confocal Raman microspectroscopy. The real 16 times parallelization of Raman microspectroscopic measurement has thus been achieved. It has also been applied to Raman imaging of polystyrene beads. A 1600-pixel Raman image was obtained in 8.5 minutes, while the conventional single beam microspectrometer may well need more than 2 hr to obtain the same. The potential of the present method with more than 100 times parallelization has been considered.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]

ACKNOWLEDGMENT

Since the image of a pinhole is expressed by a convolution of the pinhole with the PSF of the optical system, Φ may roughly approximate to the sum of the diameters of the pinhole and PSF’s center disk, pinhole + PSF , and they are typically as small as the Airy disk. Therefore, it may be convenient to express Φ as "Airy , where Airy is the diameter of the Airy disk and m a scaling factor. It is known that the diameter of the Airy disk can be written with the wavelength, λ, and the numerical aperture, NA, of the focusing optics as 1.22 λ / NA.

The authors acknowledge the support from the Ministry of Science and Technology of Taiwan (MOST105-2113-M-009-002 and MOST105-2745-M-009-001-ASP) and the Ministry of Education of Taiwan ("Aim for the Top University Plan" of National Chiao Tung University).

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Tilted Two-Dimensional Array Multifocus Confocal Raman

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