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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 5, Iss. 8 — Aug. 1, 2014
  • pp: 2526–2536
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Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device

Li-Chung Cheng, Chi-Hsiang Lien, Yong Da Sie, Yvonne Yuling Hu, Chun-Yu Lin, Fan-Ching Chien, Chris Xu, Chen Yuan Dong, and Shean-Jen Chen  »View Author Affiliations


Biomedical Optics Express, Vol. 5, Issue 8, pp. 2526-2536 (2014)
http://dx.doi.org/10.1364/BOE.5.002526


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Abstract

In this study, the light diffraction of temporal focusing multiphoton excitation microscopy (TFMPEM) and the excitation patterning of nonlinear structured-illumination microscopy (NSIM) can be simultaneously and accurately implemented via a single high-resolution digital micromirror device. The lateral and axial spatial resolutions of the TFMPEM are remarkably improved through the second-order NSIM and projected structured light, respectively. The experimental results demonstrate that the lateral and axial resolutions are enhanced from 397 nm to 168 nm (2.4-fold) and from 2.33 μm to 1.22 μm (1.9-fold), respectively, in full width at the half maximum. Furthermore, a three-dimensionally rendered image of a cytoskeleton cell featuring ~25 nm microtubules is improved, with other microtubules at a distance near the lateral resolution of 168 nm also able to be distinguished.

© 2014 Optical Society of America

1. Introduction

The multiphoton excitation (MPE) technique is widely applied in the biological imaging and microfabrication fields. With its superior axial sectioning capability and long excitation wavelength, MPE offers lower photobleaching and minimum invasiveness, and is therefore particularly suitable for imaging thick tissues and living animals. Further, with the two-photon absorption (TPA) confined to the focal volume, MPE provides an ideal solution for the fabrication of high-precision microstructures. However, one shortcoming of conventional MPE scanning is its low throughput due to point-by-point processing [1

1. B. R. Masters and P. T. C. So, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford, 2008).

,2

2. E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7(2), 93–101 (2013). [CrossRef] [PubMed]

]. Recent studies have shown that temporal focusing MPE microscopy (TFMPEM) can generate widefield and axially-resolved excitation on a plane-by-plane basis [3

3. D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express 13(5), 1468–1476 (2005). [CrossRef] [PubMed]

12

12. P. T. C. So, E. Y. S. Yew, and C. Rowlands, “High-Throughput Nonlinear Optical Microscopy,” Biophys. J. 105(12), 2641–2654 (2013). [CrossRef] [PubMed]

]. Different diffraction components such as a grating [3

3. D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express 13(5), 1468–1476 (2005). [CrossRef] [PubMed]

9

9. L.-C. Cheng, C.-Y. Chang, C.-Y. Lin, K.-C. Cho, W.-C. Yen, N.-S. Chang, C. Xu, C. Y. Dong, and S.-J. Chen, “Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning,” Opt. Express 20(8), 8939–8948 (2012). [CrossRef] [PubMed]

], an optical diffuser [10

10. J.-Y. Yu, C.-H. Kuo, D. B. Holland, Y. Chen, M. Ouyang, G. A. Blake, R. Zadoyan, and C.-L. Guo, “Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy,” J. Biomed. Opt. 16(11), 116009 (2011). [CrossRef] [PubMed]

], and a combination of two prisms and a grating [11

11. H. Dana and S. Shoham, “Remotely scanned multiphoton temporal focusing by axial grism scanning,” Opt. Lett. 37(14), 2913–2915 (2012). [CrossRef] [PubMed]

] can be utilized to diffract illuminating light frequencies for temporal focusing. The most common temporal focusing configuration uses a diffraction grating to separate frequencies into monochromatic waves at different spatial angles; then recombines them at the front focal plane of an objective lens. As such, only in the focal plane do the different frequencies overlap in phase and produce a short, high-peak power pulse, allowing MPE to simultaneously occur over the entire projection area.

Currently, the lateral and axial spatial resolutions of TFMPEM are restricted by the diffraction limitation and the confined excitation volume to within a few hundred nanometers and few microns, respectively. To improve the axial resolution near the diffraction limitation, using patterned illumination such as structured light and HiLo [13

13. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]

16

16. J. Mertz, “Optical sectioning microscopy with planar or structured illumination,” Nat. Methods 8(10), 811–819 (2011). [CrossRef] [PubMed]

] to eliminate the background noise and achieve optical sectioning is a reliable approach. Further, HiLo has also been employed to improve the axial resolution of TFMPEM [17

17. H. Choi, E. Y. S. Yew, B. Hallacoglu, S. Fantini, C. J. R. Sheppard, and P. T. C. So, “Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination,” Biomed. Opt. Express 4(7), 995–1005 (2013). [CrossRef] [PubMed]

]. However, there are many approaches [18

18. K. Weisshart, T. Dertinger, T. Kalkbrenner, I. Kleppe, and M. Kempe, “Super-resolution microscopy heads towards 3D dynamics,” Adv. Opt. Technol. 2, 211–231 (2013).

], to overcome the diffraction limitation and achieve superior lateral resolution, including photoactivation localization microscopy (PALM)/stochastic optical reconstruction microscopy (STORM) [5

5. A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008). [CrossRef] [PubMed]

,19

19. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006). [CrossRef] [PubMed]

21

21. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006). [CrossRef] [PubMed]

], super-resolution optical fluctuation imaging (SOFI) [22

22. T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106(52), 22287–22292 (2009). [CrossRef] [PubMed]

,23

23. S. Geissbuehler, C. Dellagiacoma, and T. Lasser, “Comparison between SOFI and STORM,” Biomed. Opt. Express 2(3), 408–420 (2011). [CrossRef] [PubMed]

], and (nonlinear) structured-illumination microscopy (SIM/NSIM) [24

24. M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000). [CrossRef] [PubMed]

28

28. K. Isobe, T. Takeda, K. Mochizuki, Q. Song, A. Suda, F. Kannari, H. Kawano, A. Kumagai, A. Miyawaki, and K. Midorikawa, “Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination,” Biomed. Opt. Express 4(11), 2396–2410 (2013). [CrossRef] [PubMed]

]. PALM and STORM activate sequentially and time-resolve the localization of photon switchable fluorophores to achieve a super-resolution image. However, they require anywhere from a thousand to tens of thousands of images to precisely determine a single molecule’s position. By contrast, SOFI is a technique that computes the temporal autocorrelation of the CCD pixels to determine the exact position of a single molecule. Lastly, SIM uses structured-illumination patterning, such as a sinusoidal pattern, to perform intensity modulation. With this technique, high spatial frequency information that is higher than the spatial cutoff frequency of the microscope is filtered out and modulated to a low frequency region detectable by the microscope. After remapping the high frequency information to its actual position on the frequency domain, a super-resolution image can be reconstructed. Theoretically, the lateral resolution can be enhanced 2-fold in SIM and at most m + 1-fold in NSIM, depending on the nonlinear mth order.

In this study, the lateral and axial spatial resolutions of TFMPEM are simultaneously improved through second-order NSIM and projected structured light, respectively. There are several methods to generate structured illumination, such as the conventional interference method [24

24. M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000). [CrossRef] [PubMed]

28

28. K. Isobe, T. Takeda, K. Mochizuki, Q. Song, A. Suda, F. Kannari, H. Kawano, A. Kumagai, A. Miyawaki, and K. Midorikawa, “Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination,” Biomed. Opt. Express 4(11), 2396–2410 (2013). [CrossRef] [PubMed]

]; however, this method for SIM requires a complex system setup that precisely shifts the phase delay of one of the interference beams and rotates the illumination pattern for multi-direction lateral resolution enhancement. Further, the initial phase of the illumination pattern must be found prior to the image reconstruction process. In digital light processing techniques, the use of a spatial light modulator (SLM) and digital micromirror device (DMD) have been proposed to generate arbitrary patterns in tandem with initial phase control, different phase shift, and rotation angle of illumination patterns for fast optical switching [29

29. P. Zhu, O. Fajardo, J. Shum, Y.-P. Zhang Schärer, and R. W. Friedrich, “High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device,” Nat. Protoc. 7(7), 1410–1425 (2012). [CrossRef] [PubMed]

], pattern excitation in optogenetics [6

6. E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010). [CrossRef] [PubMed]

], and high-throughput microfabrication [30

30. Y.-C. Li, L.-C. Cheng, C.-Y. Chang, C.-H. Lien, P. J. Campagnola, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned excitation,” Opt. Express 20(17), 19030–19038 (2012). [CrossRef] [PubMed]

]. Consequently, the second-order NSIM with projected structured light can be delivered via a DMD; and further, the conventional diffraction grating can be replaced by a DMD in TFMPEM [31

31. J. N. Yih, Y. Y. Hu, Y. D. Sie, L.-C. Cheng, C. H. Lien, and S.-J. Chen, “Temporal focusing-based multiphoton excitation microscopy via digital micromirror device,” Opt. Lett. 39(11), 3134–3137 (2014). [CrossRef] [PubMed]

]. Herein, the light diffraction of the TFMPEM and the excitation patterning of the second-order NSIM with projected structured light are simultaneously and accurately implemented via a single high-resolution DMD. Based on the concise TFMPEM configuration, the experimental results reveal that the lateral and axial resolutions are enhanced from 397 nm to 168 nm and from 2.33 μm to 1.22 μm, respectively, in full width at the half maximum (FWHM). Also, improvements to the three-dimensionally (3D) rendered image of a cytoskeleton cell with about 25 nm microtubules demonstrates the efficacy of the proposed TFMTEM setup.

2. Optical setup and principle

2.1. Temporal focusing-based multiphoton excitation microscopy via digital micromirror device

2.2. Second-order nonlinear structured-illumination microscopy

The equations from a linear SIM [25

25. M. G. Gustafsson, D. A. Agard, and J. W. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE 3919, 141–150 (2000). [CrossRef]

] can be easily extended into a higher order nonlinear expression. For nonlinear TPE, the detected fluorescence signal f(r) can be expressed as:
f(r)c(r)×I2(r)h(r),
(1)
where c(r) is the local concentration of fluorophores, I(r) is excitation intensity, and h(r) is the point spread function (PSF) of the fluorescence detection system. With a sinusoidal illumination pattern, generated by the DMD, the TPE intensity is:
I2(r)={I0[1+cos(kpr+ϕ)]}2,
(2)
where kp is the excitation spatial frequency of the sinusoidal pattern and ϕ is the pattern phase. By substituting Eq. (2) into Eq. (1) and converting it with a two-dimensional (2D) Fourier transform, the second-order representation can be given as:
F(k)=aH(k){32C(k)+C(k+kp)ejϕ+C(kkp)ejϕ+14C(k+2kp)ej2ϕ+14C(k2kp)ej2ϕ},
(3)
where a is a constant and 2kp is the nonlinear two-photon excitation spatial frequency. From Eq. (3), five different phase shifts of ϕ are needed to extract the five frequency components, namely C(k2kp), C(kkp), C(k), C(k+kp), and C(k+2kp). Herein, five phase shifts of 0, 2π/5, 4π/5, 6π/5, and 8π/5 are performed. After extracting the five components, each frequency component was remapped into its real position with appropriate weighting. To precisely remap the five frequency components in the spatial frequency domain, the image size was doubled by adding zeroes before taking the Fourier transform of the structured illumination images. With remapping and weighting all the frequency components, the data were converted to the space domain and an image with a radius of k0+2kp can be reconstructed in the frequency domain. Consequently, a superior resolution image with a lateral resolution enhancement of (k0+2kp)/k0 can be obtained. Based on the theoretical analysis, when kp equals k0, which is the cutoff spatial frequency of the microscope, a maximum 3-fold enhancement of the lateral resolution can be achieved. However, the excitation wavelength in TPE is longer than the emission wavelength, i.e. kp is never equal to k0. Under the current experimental conditions with the excitation wavelength of 750 nm and the emission wavelength of 570 nm, the maximum projected pattern frequency could approach 0.76k0, i.e. λemission/λexcitation×k0. Nevertheless, the modulation visibility at the higher spatial frequency becomes smaller. Furthermore, the precise structured illumination process only takes place in the focal plane; hence, the detected out-of-focus signals can be considered as background noise. According to [13

13. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]

], the background noise in c(r) was rejected. Furthermore, during the extraction process, the five frequency components were calculated by fitting a linear matrix model, enabling the background noise to be diminished via the least-squares fitting processing. At this moment, the axial resolution can be enhanced via the processing.

These processes enhance the lateral resolution in one direction, which in turn depends on the original structured illumination pattern direction. It should be noted that the linear SIM [25

25. M. G. Gustafsson, D. A. Agard, and J. W. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE 3919, 141–150 (2000). [CrossRef]

] requires different rotations of the structured pattern to enhance the 2D lateral resolution. Herein, four rotations were taken, namely 0°, 45°, 90°, and 135°, with each rotation undergoing five phase shifts. As such, a total of 20 sinusoidal illumination images were required for 2D super-resolution image reconstruction. The frame rate of the TFMPEM with structured illumination via the DMD can achieve 200 frames per second [9

9. L.-C. Cheng, C.-Y. Chang, C.-Y. Lin, K.-C. Cho, W.-C. Yen, N.-S. Chang, C. Xu, C. Y. Dong, and S.-J. Chen, “Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning,” Opt. Express 20(8), 8939–8948 (2012). [CrossRef] [PubMed]

], while the frame rate of the super-resolution TPE image based on the second-order NSIM with four rotations is lowered to 10 frames per second.

3. Experimental results and discussions

3.1. Improving the lateral and axial spatial resolutions

In the experiment, the sinusoidal patterns with five phase shifts, 0, 2π/5, 4π/5, 6π/5, and 8π/5, and four rotations, 0°, 45°, 90°, and 135°, are quickly generated via the DMD. To verify the structured TPE illumination, a R6G fluorescence thin film with a thickness less than 200 nm was utilized and excited with the sinusoidal patterns. The illumination patterns were controlled by both the ON-OFF pixel period and the gray level of the DMD. In this experiment, the ON-OFF period covers 16 pixels and sequentially changes the ON-OFF states to perform the phase shifts. 100 μm beads were measured for estimating the PSF of the system, which was then transferred to the spatial frequency domain to become k0. The current value of k0 is around 2.5 μm −1. To show the two peaks of kp and 2kp in the spatial frequency domain, the value of kp was adopted as 1.03 μm−1, which is slightly smaller than the 1.25 μm −1 half value of k0. The kp of 1.03 μm−1 and the 2kp of 2.06 μm−1 are both smaller than the cutoff frequency kp of 2.5 μm −1; hence, the two peaks can be observed in Fig. 2(b)
Fig. 2 (a) Sinusoidal illumination on the R6B fluorescent thin film. Blue circles: the detected fluorescent intensity; blue line: the fitting by [1+cos(kpr)]2. (b) Fourier transform of the detected structured pattern in Fig. 2(a), in which a nonlinear second-order peak caused by the intrinsic nonlinear TPE is indicated.
. Figure 2(a) shows the fluorescent intensity of the structured illumination on the R6G thin film. The blue circles are the normalization of the detected intensity on the EMCCD camera, while the dashed line is the fitting by [1+cos(kpr)]2. By converting the Fourier transform of the structured illumination image in Fig. 2(a), a nonlinear second-order peak caused by the intrinsic nonlinear TPE can be easily found, as shown in Fig. 2(b).

To verify the improvement of the lateral and axial spatial resolutions of the TFMPEM via the second-order NSIM, 100 nm fluorescent beads in water, acting as the specimen, were illuminated by the TFMPEM only and then by the TFMPEM with the second-order NSIM. The fluorescent bead solution was dropped and dried on a cover slide, and then sealed in glycerol for refractive index matching. Hereafter, to increase the enhancement of the lateral resolution, kp was increased to nearly 1.5 μm−1, i.e. 0.6k0; consequently, the enhancement based on (k0+2kp)/k0 can achieve roughly 2.2. The resulting images from the TFMPEM and the TFMPEM with NSIM are shown in Figs. 3(a)
Fig. 3 TPE images of 100 nm fluorescent beads by (a) TFMPEM only and (b) TFMPEM with second-order NSIM. (c) and (d) are the regions within red dashed circles in Figs. 3(a) and 3(b), respectively.
and 3(b), respectively. A region of interest highlighted by the red dashed circles in Figs. 3(a) and 3(b) is zoomed in, as respectively shown in Figs. 3(c) and 3(d). As can be seen in Fig. 3(c), the gathered beads in the TFMPEM image cannot be discriminated because the diffraction limit at the peak emission wavelength of 570 nm and the 1.2 NA is around 300 nm, which limits the lateral resolution of the widefield microscopy. Theoretically, the second-order NSIM can enhance the lateral resolution by a maximum of 2.52-fold with the 750 nm excitation wavelength in our system.

Ten beads were selected from Figs. 3(a) and 3(b) and fitted for a bead lateral profile according to Gaussian profile. Figure 4(a)
Fig. 4 (a) The 100nm fluorescent beads’ measured lateral profiles by TFMPEM alone (blue) and NSIM enhanced TFMPEM (red). The lateral resolution with TFMPEM only and NSIM enhanced TFMPEM are 397 nm and 168 nm, respectively, in FWHM. (b) The R6G thin film’s measured excitation volume axial profile by TFMPEM (black), and the 200 nm fluorescent beads’ measured axial profiles by only TFMPEM (blue) and NSIM enhanced TFMPEM (red). The excitation volume depth is 3.1 μm in FWHM, and the axial resolution with only TFMPEM and NSIM enhanced TFMPEM are 2.33 μm and 1.22 μm, respectively, in FWHM. The circles represent the measured intensities at different lateral and axial positions, while the dashed lines are the fitted curves.
shows the beads’ profiles and the lateral resolutions of the TFMPEM only and NSIM enhanced TFMPEM, which in FWHM are 397 nm and 168 nm, respectively. Therefore, the lateral resolution is enhanced 2.4-fold, which is in agreement with the theoretical (k0+2kp)/k0=2.2. In the experiment, we attempted to increase k0 to its maximum value. Based on the modulation transfer function theory, the modulation visibility decreases from 1 when the pattern frequency becomes higher; hence, the signal-to-noise ratio and the contrast of the structured TPE pattern on the specimen worsened, and as a consequence, the intensity modulation information was lost due to the induced distortion in the reconstructed image. Furthermore, to avoid photobleaching, the excited power on the sample was controlled to below 10 mW with an exposure time of 10 ms.

Nevertheless, SIM not only enhances the lateral resolution, but also provides optical sectioning capability [16

16. J. Mertz, “Optical sectioning microscopy with planar or structured illumination,” Nat. Methods 8(10), 811–819 (2011). [CrossRef] [PubMed]

,25

25. M. G. Gustafsson, D. A. Agard, and J. W. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE 3919, 141–150 (2000). [CrossRef]

]. The optical sectioning image without lateral resolution enhancement is reconstructed by structured illumination in one-photon widefield microscopy [13

13. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]

]. This requires three phase shift structured illumination images to restore a sectioning image, without considering high frequency components modulated in the low frequency region. The background noise in c(r) can be rejected. The patterns can be accurately projected in the focal plane region, ensuring the frequency component between the ±k0 region is restored by the structured illumination images. Herein, the five frequency components of the second-order NSIM were calculated by fitting a linear matrix model during the extraction process, enabling the background noise to be reduced via the least-squares fitting process. As such, when considering the high frequency component modulated into the ±k0 region to increase the lateral resolution, i.e. SIM, it can provide not only lateral resolution enhancement but also optical sectioning capability. In this study, the illumination sinusoidal pattern for the second-order NSIM can be clearly projected into the EMCCD in a confined region in depth, and depends on the NA of the objective lens and the excitation fluorescence wavelength [13

13. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]

]. Other fluorescence signals coming from the TPE volume of the TFMPEM, except for the confined region where the pattern can be imaged well, are rejected as background noise. The structured illumination has provided an optical sectioning ability even in widefield one-photon excitation microscopy [13

13. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]

,16

16. J. Mertz, “Optical sectioning microscopy with planar or structured illumination,” Nat. Methods 8(10), 811–819 (2011). [CrossRef] [PubMed]

,25

25. M. G. Gustafsson, D. A. Agard, and J. W. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE 3919, 141–150 (2000). [CrossRef]

]. Similar to the structured illumination approach, the TFMPEM with the NSIM can achieve a better axial resolution than conventional TFMPEM alone. To determine the current TPE depth volume of the TFMPEM only, an R6B fluorescent thin film was axially scanned and imaged, after which the TPE fluorescent signal was calculated at each axial position; hence, the fluorescent intensity profile as a function of axial position can be obtained. In this case, the excitation volume in depth based on this profile was 3.1 μm in FWHM, as shown in Fig. 4(b). To verify the axial resolution improvement, a specimen comprising 200 nm fluorescent beads sealed in glycerol was axially scanned and imaged by TFMPEM. Figure 4(b) shows that the axial resolution of the TFMPEM only via imaging the 200 nm fluorescent beads is about 2.33 μm in FWHM, i.e. only the axial information difference from 2.33 μm in depth can be distinguished. By contrast, for TFMPEM with the second-order NSIM, the axial resolution is enhanced to 1.22 μm in FWHM; hence the enhancement factor is 1.9-fold, as shown in Fig. 4(b). The axial resolution enhancement is limited by the excitation wavelength, the objective lens’ NA, and the period of the projected structured light. Similar to the formula in Ref [13

13. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]

], the axial resolution of the TFMPEM with the structured light near the cutoff spatial frequency could be improved to around 700 nanometers. However, in widefield TFMPEM, the diffraction laser profile in the back aperture of the objective lens is a line, and therefore does not cover the whole aperture. As a result, the full NA of the objective lens is not utilized, and so the optimal axial resolution enhancement cannot be achieved at this moment.

3.2. 3D image of the microtubules of cytoskeleton

4. Conclusions

The advantages of TPE, such as less photon damage and deeper sample penetration, can be maintained in TFMPEM. In this paper, it was demonstrated that the lateral and axial spatial resolutions of TFMPEM can be improved via the second-order NISM. The light diffraction of the temporal focusing and the sinusoidal nonlinear excitation patterns of the second-order NSIM can be simultaneously implemented via a concise configuration with only a high-resolution DMD. Since the DMD is placed on the image-conjugate plane of the objective lens’ focal plane, a sinusoidal MPE pattern with different rotations and phase shifts can be precisely projected on the focal plane. The lateral resolution is improved from 397 nm to 168 nm via the second-order NSIM, while the axial resolution is sharpened from 2.33 μm to 1.22 μm, for an enhancement factor of 1.9-fold, due the exact projected structured light and the rejection of background signals such as scattered light and out of focus signals. Furthermore, to demonstrate the superior lateral and axial resolutions of the TFMPEM with the second-order NSIM, microtubules of a cytoskeleton cell are observed as axially scanned 3D video. The diameter of the microtubules from the intensity profile is approximately equal to the lateral resolution of 168 nm; nevertheless, some close microtubules with a distance near the lateral resolution can be distinguished.

Acknowledgments

This work was supported by the National Science Council (NSC) in Taiwan with grant numbers NSC 101-2221-E-006-212-MY3 and NSC 101-2221-E-006-213-MY3.

References and links

1.

B. R. Masters and P. T. C. So, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford, 2008).

2.

E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics 7(2), 93–101 (2013). [CrossRef] [PubMed]

3.

D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express 13(5), 1468–1476 (2005). [CrossRef] [PubMed]

4.

G. Zhu, J. van Howe, M. Durst, W. Zipfel, and C. Xu, “Simultaneous spatial and temporal focusing of femtosecond pulses,” Opt. Express 13(6), 2153–2159 (2005). [CrossRef] [PubMed]

5.

A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105(51), 20221–20226 (2008). [CrossRef] [PubMed]

6.

E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7(10), 848–854 (2010). [CrossRef] [PubMed]

7.

D. Kim and P. T. C. So, “High-throughput three-dimensional lithographic microfabrication,” Opt. Lett. 35(10), 1602–1604 (2010). [CrossRef] [PubMed]

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O. D. Therrien, B. Aubé, S. Pagès, P. D. Koninck, and D. Côté, “Wide-field multiphoton imaging of cellular dynamics in thick tissue by temporal focusing and patterned illumination,” Biomed. Opt. Express 2(3), 696–704 (2011). [CrossRef] [PubMed]

9.

L.-C. Cheng, C.-Y. Chang, C.-Y. Lin, K.-C. Cho, W.-C. Yen, N.-S. Chang, C. Xu, C. Y. Dong, and S.-J. Chen, “Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning,” Opt. Express 20(8), 8939–8948 (2012). [CrossRef] [PubMed]

10.

J.-Y. Yu, C.-H. Kuo, D. B. Holland, Y. Chen, M. Ouyang, G. A. Blake, R. Zadoyan, and C.-L. Guo, “Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy,” J. Biomed. Opt. 16(11), 116009 (2011). [CrossRef] [PubMed]

11.

H. Dana and S. Shoham, “Remotely scanned multiphoton temporal focusing by axial grism scanning,” Opt. Lett. 37(14), 2913–2915 (2012). [CrossRef] [PubMed]

12.

P. T. C. So, E. Y. S. Yew, and C. Rowlands, “High-Throughput Nonlinear Optical Microscopy,” Biophys. J. 105(12), 2641–2654 (2013). [CrossRef] [PubMed]

13.

M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22(24), 1905–1907 (1997). [CrossRef] [PubMed]

14.

D. Lim, K. K. Chu, and J. Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett. 33(16), 1819–1821 (2008). [CrossRef] [PubMed]

15.

T. N. Ford, D. Lim, and J. Mertz, “Fast optically sectioned fluorescence HiLo endomicroscopy,” J. Biomed. Opt. 17(2), 021105 (2012). [CrossRef] [PubMed]

16.

J. Mertz, “Optical sectioning microscopy with planar or structured illumination,” Nat. Methods 8(10), 811–819 (2011). [CrossRef] [PubMed]

17.

H. Choi, E. Y. S. Yew, B. Hallacoglu, S. Fantini, C. J. R. Sheppard, and P. T. C. So, “Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination,” Biomed. Opt. Express 4(7), 995–1005 (2013). [CrossRef] [PubMed]

18.

K. Weisshart, T. Dertinger, T. Kalkbrenner, I. Kleppe, and M. Kempe, “Super-resolution microscopy heads towards 3D dynamics,” Adv. Opt. Technol. 2, 211–231 (2013).

19.

E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science 313(5793), 1642–1645 (2006). [CrossRef] [PubMed]

20.

S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91(11), 4258–4272 (2006). [CrossRef] [PubMed]

21.

M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3(10), 793–796 (2006). [CrossRef] [PubMed]

22.

T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106(52), 22287–22292 (2009). [CrossRef] [PubMed]

23.

S. Geissbuehler, C. Dellagiacoma, and T. Lasser, “Comparison between SOFI and STORM,” Biomed. Opt. Express 2(3), 408–420 (2011). [CrossRef] [PubMed]

24.

M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc. 198(2), 82–87 (2000). [CrossRef] [PubMed]

25.

M. G. Gustafsson, D. A. Agard, and J. W. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE 3919, 141–150 (2000). [CrossRef]

26.

M. G. Gustafsson, “Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102(37), 13081–13086 (2005). [CrossRef] [PubMed]

27.

M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94(12), 4957–4970 (2008). [CrossRef] [PubMed]

28.

K. Isobe, T. Takeda, K. Mochizuki, Q. Song, A. Suda, F. Kannari, H. Kawano, A. Kumagai, A. Miyawaki, and K. Midorikawa, “Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination,” Biomed. Opt. Express 4(11), 2396–2410 (2013). [CrossRef] [PubMed]

29.

P. Zhu, O. Fajardo, J. Shum, Y.-P. Zhang Schärer, and R. W. Friedrich, “High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device,” Nat. Protoc. 7(7), 1410–1425 (2012). [CrossRef] [PubMed]

30.

Y.-C. Li, L.-C. Cheng, C.-Y. Chang, C.-H. Lien, P. J. Campagnola, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned excitation,” Opt. Express 20(17), 19030–19038 (2012). [CrossRef] [PubMed]

31.

J. N. Yih, Y. Y. Hu, Y. D. Sie, L.-C. Cheng, C. H. Lien, and S.-J. Chen, “Temporal focusing-based multiphoton excitation microscopy via digital micromirror device,” Opt. Lett. 39(11), 3134–3137 (2014). [CrossRef] [PubMed]

32.

Y.-C. Li, L.-C. Cheng, C.-Y. Chang, C.-Y. Lin, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “High-throughput fabrication of gray-level bio-microstructures via temporal focusing excitation and laser pulse control,” J. Biomed. Opt. 18(7), 075004 (2013). [CrossRef]

OCIS Codes
(170.3880) Medical optics and biotechnology : Medical and biological imaging
(190.4180) Nonlinear optics : Multiphoton processes
(180.4315) Microscopy : Nonlinear microscopy

ToC Category:
Microscopy

History
Original Manuscript: May 13, 2014
Revised Manuscript: June 18, 2014
Manuscript Accepted: July 3, 2014
Published: July 8, 2014

Citation
Li-Chung Cheng, Chi-Hsiang Lien, Yong Da Sie, Yvonne Yuling Hu, Chun-Yu Lin, Fan-Ching Chien, Chris Xu, Chen Yuan Dong, and Shean-Jen Chen, "Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device," Biomed. Opt. Express 5, 2526-2536 (2014)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-5-8-2526


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References

  1. B. R. Masters and P. T. C. So, Handbook of Biomedical Nonlinear Optical Microscopy (Oxford, 2008).
  2. E. E. Hoover and J. A. Squier, “Advances in multiphoton microscopy technology,” Nat. Photonics7(2), 93–101 (2013). [CrossRef] [PubMed]
  3. D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express13(5), 1468–1476 (2005). [CrossRef] [PubMed]
  4. G. Zhu, J. van Howe, M. Durst, W. Zipfel, and C. Xu, “Simultaneous spatial and temporal focusing of femtosecond pulses,” Opt. Express13(6), 2153–2159 (2005). [CrossRef] [PubMed]
  5. A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A.105(51), 20221–20226 (2008). [CrossRef] [PubMed]
  6. E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods7(10), 848–854 (2010). [CrossRef] [PubMed]
  7. D. Kim and P. T. C. So, “High-throughput three-dimensional lithographic microfabrication,” Opt. Lett.35(10), 1602–1604 (2010). [CrossRef] [PubMed]
  8. O. D. Therrien, B. Aubé, S. Pagès, P. D. Koninck, and D. Côté, “Wide-field multiphoton imaging of cellular dynamics in thick tissue by temporal focusing and patterned illumination,” Biomed. Opt. Express2(3), 696–704 (2011). [CrossRef] [PubMed]
  9. L.-C. Cheng, C.-Y. Chang, C.-Y. Lin, K.-C. Cho, W.-C. Yen, N.-S. Chang, C. Xu, C. Y. Dong, and S.-J. Chen, “Spatiotemporal focusing-based widefield multiphoton microscopy for fast optical sectioning,” Opt. Express20(8), 8939–8948 (2012). [CrossRef] [PubMed]
  10. J.-Y. Yu, C.-H. Kuo, D. B. Holland, Y. Chen, M. Ouyang, G. A. Blake, R. Zadoyan, and C.-L. Guo, “Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy,” J. Biomed. Opt.16(11), 116009 (2011). [CrossRef] [PubMed]
  11. H. Dana and S. Shoham, “Remotely scanned multiphoton temporal focusing by axial grism scanning,” Opt. Lett.37(14), 2913–2915 (2012). [CrossRef] [PubMed]
  12. P. T. C. So, E. Y. S. Yew, and C. Rowlands, “High-Throughput Nonlinear Optical Microscopy,” Biophys. J.105(12), 2641–2654 (2013). [CrossRef] [PubMed]
  13. M. A. A. Neil, R. Juskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett.22(24), 1905–1907 (1997). [CrossRef] [PubMed]
  14. D. Lim, K. K. Chu, and J. Mertz, “Wide-field fluorescence sectioning with hybrid speckle and uniform-illumination microscopy,” Opt. Lett.33(16), 1819–1821 (2008). [CrossRef] [PubMed]
  15. T. N. Ford, D. Lim, and J. Mertz, “Fast optically sectioned fluorescence HiLo endomicroscopy,” J. Biomed. Opt.17(2), 021105 (2012). [CrossRef] [PubMed]
  16. J. Mertz, “Optical sectioning microscopy with planar or structured illumination,” Nat. Methods8(10), 811–819 (2011). [CrossRef] [PubMed]
  17. H. Choi, E. Y. S. Yew, B. Hallacoglu, S. Fantini, C. J. R. Sheppard, and P. T. C. So, “Improvement of axial resolution and contrast in temporally focused widefield two-photon microscopy with structured light illumination,” Biomed. Opt. Express4(7), 995–1005 (2013). [CrossRef] [PubMed]
  18. K. Weisshart, T. Dertinger, T. Kalkbrenner, I. Kleppe, and M. Kempe, “Super-resolution microscopy heads towards 3D dynamics,” Adv. Opt. Technol.2, 211–231 (2013).
  19. E. Betzig, G. H. Patterson, R. Sougrat, O. W. Lindwasser, S. Olenych, J. S. Bonifacino, M. W. Davidson, J. Lippincott-Schwartz, and H. F. Hess, “Imaging intracellular fluorescent proteins at nanometer resolution,” Science313(5793), 1642–1645 (2006). [CrossRef] [PubMed]
  20. S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J.91(11), 4258–4272 (2006). [CrossRef] [PubMed]
  21. M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods3(10), 793–796 (2006). [CrossRef] [PubMed]
  22. T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A.106(52), 22287–22292 (2009). [CrossRef] [PubMed]
  23. S. Geissbuehler, C. Dellagiacoma, and T. Lasser, “Comparison between SOFI and STORM,” Biomed. Opt. Express2(3), 408–420 (2011). [CrossRef] [PubMed]
  24. M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J. Microsc.198(2), 82–87 (2000). [CrossRef] [PubMed]
  25. M. G. Gustafsson, D. A. Agard, and J. W. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE3919, 141–150 (2000). [CrossRef]
  26. M. G. Gustafsson, “Nonlinear structured-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A.102(37), 13081–13086 (2005). [CrossRef] [PubMed]
  27. M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J.94(12), 4957–4970 (2008). [CrossRef] [PubMed]
  28. K. Isobe, T. Takeda, K. Mochizuki, Q. Song, A. Suda, F. Kannari, H. Kawano, A. Kumagai, A. Miyawaki, and K. Midorikawa, “Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination,” Biomed. Opt. Express4(11), 2396–2410 (2013). [CrossRef] [PubMed]
  29. P. Zhu, O. Fajardo, J. Shum, Y.-P. Zhang Schärer, and R. W. Friedrich, “High-resolution optical control of spatiotemporal neuronal activity patterns in zebrafish using a digital micromirror device,” Nat. Protoc.7(7), 1410–1425 (2012). [CrossRef] [PubMed]
  30. Y.-C. Li, L.-C. Cheng, C.-Y. Chang, C.-H. Lien, P. J. Campagnola, and S.-J. Chen, “Fast multiphoton microfabrication of freeform polymer microstructures by spatiotemporal focusing and patterned excitation,” Opt. Express20(17), 19030–19038 (2012). [CrossRef] [PubMed]
  31. J. N. Yih, Y. Y. Hu, Y. D. Sie, L.-C. Cheng, C. H. Lien, and S.-J. Chen, “Temporal focusing-based multiphoton excitation microscopy via digital micromirror device,” Opt. Lett.39(11), 3134–3137 (2014). [CrossRef] [PubMed]
  32. Y.-C. Li, L.-C. Cheng, C.-Y. Chang, C.-Y. Lin, N.-S. Chang, P. J. Campagnola, C. Y. Dong, and S.-J. Chen, “High-throughput fabrication of gray-level bio-microstructures via temporal focusing excitation and laser pulse control,” J. Biomed. Opt.18(7), 075004 (2013). [CrossRef]

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