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Optics Express

Optics Express

  • Editor: C. Martijn de Sterke
  • Vol. 18, Iss. 8 — Apr. 12, 2010
  • pp: 8491–8498
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A simple scanless two-photon fluorescence microscope using selective plane illumination

Jonathan Palero, Susana I.C.O. Santos, David Artigas, and Pablo Loza-Alvarez  »View Author Affiliations


Optics Express, Vol. 18, Issue 8, pp. 8491-8498 (2010)
http://dx.doi.org/10.1364/OE.18.008491


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Abstract

We demonstrate a simple scanless two-photon (2p) excited fluorescence microscope based on selective plane illumination microscopy (SPIM). Optical sectioning capability is presented and depth-resolved imaging of cameleon protein in C. elegans pharyngeal muscle is implemented.

© 2010 OSA

The past two decades saw the emergence of two-photon excited fluorescence (TPEF) laser-scanning microscopy as a powerful imaging tool for thick tissue imaging. Owing to its inherent optical sectioning capability, deep penetration of the excitation light in tissues and its capability to excite multiple fluorophores, TPEF laser-scanning microscopy has been used in a wide range of imaging applications. The demand for two-photon imaging of fast biological events prompted the development of scanning techniques that pushed two-photon imaging at video rates including resonant scanning [1

1. Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca(2+) imaging,” Cell Calcium 30(6), 383–393 (2001). [CrossRef] [PubMed]

,2

2. M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002). [CrossRef] [PubMed]

], line-scanning [3

3. G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996). [CrossRef] [PubMed]

], multifocal scanning [4

4. A. Egner and S. W. Hell, “Time multiplexing and parallelization in multifocal multiphoton microscopy,” J. Opt. Soc. Am. A 17(7), 1192–1201 (2000). [CrossRef]

], polygonal mirror scanning [5

5. K. H. Kim, C. Buehler, and P. T. So, “High-speed, two-photon scanning microscope,” Appl. Opt. 38(28), 6004–6009 (1999). [CrossRef]

], and diffractive optical element-based (DOE) scanning [6

6. B. O. Watson, V. Nikolenko, and R. Yuste, “Two-photon imaging with diffractive optical elements,” Front Neural Circuits 3, 6 (2009). [PubMed]

,7

7. L. Sacconi, E. Froner, R. Antolini, M. R. Taghizadeh, A. Choudhury, and F. S. Pavone, “Multiphoton multifocal microscopy exploiting a diffractive optical element,” Opt. Lett. 28(20), 1918–1920 (2003). [CrossRef] [PubMed]

].

In recent years, novel TPEF imaging techniques that do not involve mechanical scanning of the excitation light have emerged. Temporal focusing of 10 fs infrared laser pulses achieved depth resolved images of DAPI-labeled Drosophila embryos, albeit with out-of-focus fluorescence background [8

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

]. A subsequent study has demonstrated that combining spatial modulation of the excitation light with the temporal focusing technique resulted in better axial confinement, with an axial resolution practically similar to that of two-photon line-scanning microscope [9

9. E. Papagiakoumou, V. de Sars, V. Emiliani, and D. Oron, “Temporal focusing with spatially modulated excitation,” Opt. Express 17(7), 5391–5401 (2009). [CrossRef] [PubMed]

]. Excitation multiplexing by using a spatio-temporal light modulator (SLM) also enabled scanless two-photon imaging and simultaneous photostimulation of neurons [10

10. V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, “SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators,” Front Neural Circuits 2, 5 (2008). [CrossRef]

]. Recently, lateral wavelength division scanning for TPEF was demonstrated whereby different wavelengths are focused on to the sample at different lateral positions by tuning the excitation wavelength [11

11. K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006). [CrossRef] [PubMed]

] and this has been extended to axial wavelength division scanning using a Fresnel lens which has a large chromatic aberration [12

12. Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two photon imaging,” in Lasers and Electro-Optics, 2008 and 2008 Conference on Quantum Electronics and Laser Science. CLEO/QELS 2008. Conference on(2008), pp. 1–2.

]. Axial scanning has also been achieved by simultaneous spatial and temporal focusing which was controlled by adjusting the group velocity dispersion [13

13. M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing for axial scanning,” Opt. Express 14(25), 12243–12254 (2006). [CrossRef] [PubMed]

].

The practical setup for 2p-SPIM is shown in Fig. 1. For the excitation source, a typical Kerr lens mode-locked Ti:sapphire laser (MIRA 900f, Coherent, France), with pulses of 160 fs with a repetition rate of 76 MHz was operated at a central wavelength of 860 nm. After attenuation of the excitation beam using a variable neutral density filter wheel, its diameter was increased using a pair of spherical lenses in a telescope configuration. The beam was allowed to pass through a square aperture (4 mm × 4 mm) and through the first cylindrical lens (f = 200mm) that determines the height (δy) of the plane illumination. After passing through a mirror, the beam is focused horizontally to the sample specimen by a second cylindrical lens (f = 20mm) resulting in an effective excitation NA of 0.1. Samples were embedded in ~1.5% agar gel and immersed in a water-filled chamber made of cover slip windows.

For the C. elegans imaging experiments, adult hermaphrodite worms were immobilized using sodium azide-NaN3 (50 mM). To obtain optical section at different depths (z-direction), we attached the specimen to a computer-controlled linear translation stage (M-505.6DG, Physik Instrumente GmbH & Co. KG, Karlsruhe, Germany). An air objective lens (20 × /NA0.40/WD3.90, Nikon) oriented perpendicular to the excitation axis was used to collect the fluorescence from the sample. To block scattered laser excitation light an emission filter (BG39, Schott) was placed before the high-sensitivity imaging detector. Images were recorded on a back-illuminated EMCCD (iXonEM + 897, Andor Technology plc., Belfast, Northern Ireland) with a full frame image size of 512 × 512 pixels and a pixel size of 16 µm.

In order to estimate the achievable resolution of 2p-SPIM, we have calculated the two-photon illumination point-spread function (PSFill) in the transversal (δz) and axial (δx) directions (see Fig. 1, inset) [20

20. W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003). [CrossRef] [PubMed]

]. Here, the transversal and the axial PSFill determine the thickness and length of the two-photon plane illumination, respectively. Note here that the transversal PSFill plays a role in determining the axial resolution of the image (i.e., relative to the detection axis, z) while the axial PSFill determines the length of the image field-of-view. In theory, the length of the two-photon illumination length is expected to be by a square root of two shorter than the one-photon illumination length (at the same illumination thickness). Notice that the lateral imaging resolution is determined by the objective lens and the pixel size of the CCD but it is independent of the illumination numerical aperture. Shown in Fig. 2
Fig. 2 Calculated FWHM of TPEF axial and transversal PSF as a function of the illumination numerical aperture of cylindrical lens 2 (see Fig. 1) for two-photon excitation wavelength of 860 nm.
are the results of the calculations for the axial and transversal PSFill measured at the full-width at half-maximum (FWHM) as a function of the illumination numerical aperture of the illumination system (NAill) at the two-photon excitation wavelength of 860 nm in air (refractive index is 1.0). Based on Fig. 2, a two-photon PSFill with length of ~100 µm and thickness ~3 µm can be achieved using NAill = 0.1. Longer PSFill (>100µm) can be achieved by using lower NAill (<0.1) albeit accompanied by an increase in PSFill thickness. On the other hand, higher NAill (>0.1) results in narrower PSFill but with shorter PSFill length. For example, a two-photon illumination of ~1 µm can be achieved using PSFill ≈0.3 but with a short illumination plane of ~10 µm. The vertical extent of the PSFill (δy) can be conveniently varied by the first cylindrical lens oriented horizontally with a long focal length as shown in the experimental setup (see Fig. 1). Alternatively, the height of the aperture can be varied at the expense of losing excitation intensity.

To gain knowledge on the vertical and horizontal dimensions of the two-photon excitation profile in 2p-SPIM, we recorded an XY image of the fluorescence of Coumarin 540 (Excition, Dayton, OH, USA) in methanol as shown in Fig. 3, A
Fig. 3 A: Pseudocolor 2p-SPIM fluorescence image of Coumarin 540 dye in methanol. Depicted also are the horizontal (B) and vertical (C) PSF obtained from cross-sectional intensity profiles (green lines). We measured the two-photon illumination length (δx) and height (δy) as 268 µm and 21.9 µm, respectively. D: Transversal PSF of the two-photon plane illumination obtained from z-section intensity profile of a typical XZ pseudocolor image (D, inset) of a 0.028 µm fluorescent bead excited. An average thickness (δz) of 3.4 ± 0.2 µm (n = 5) were measured. Image dimensions: (A) 590 µm × 150 µm (X × Y), (D, inset) 4 µm × 16 µm (X × Z).
. Here, we adjusted the first cylindrical lens (see Fig. 1) so that we obtain the minimum height in the fluorescence image. We measured the horizontal extent (δx) of the two-photon illumination to be 268±21 µm (see Fig. 3, B) which is more than two-fold larger than the computed axial PSFill length of 143 µm for NAill = 0.1 (see Fig. 2, red line). This discrepancy may be due to the combined effect of optical aberration introduced by the cylindrical lens and the refractive index mismatch between the different media (i.e. air, water and agar gel). Moreover, an experimental error in the value of the numerical aperture may also be attributed to this large discrepancy. We calculate using the relation between the axial PSF and the NA, that 268±21 µm corresponds to an experimental numerical aperture NA = 0.07±0.01. On the other hand, the vertical extent (δy) measured 21.9±1.0 µm (see Fig. 3, C) which is in good agreement with the minimum achievable height of the light sheet (20.4 µm) considering the focal length of cylindrical lens 1 to be 200 mm corresponding to a numerical aperture (in the horizontal direction) of 0.01.

To demonstrate the optical sectioning capability of the constructed 2p-SPIM experimental setup, images of fluorescent polymer microspheres (Firefli Fluorescent Green, size: 0.028 µm, Duke Scientific Corporation, Palo Alto, CA, USA) embedded in 2% agar gel were acquired for different relative depths (1 µm steps) of the sample. Shown in Fig. 3, D is a typical z-profile of a microsphere depicting a Gaussian fit with FHWM of 3.4 µm and an XZ image perpendicular to the optical section (inset, image dimension: 4 µm × 16 µm) of the microsphere. The gray level is presented in pseudocolor. This clearly indicates optical sectioning capability of the 2p-SPIM setup with an average two-photon plane illumination thickness of 3.4±0.5 µm obtained from five measurements of microspheres. Although this value is in good agreement with the calculated PSFill thickness of 3.2 µm for NAill = 0.1, the obtained two-photon plane illumination thickness in fact corresponds to a numerical aperture NA = 0.09±0.01. This is close to the value (NA = 0.07±0.01) obtained when we calculated for the corresponding experimental numerical aperture to two-photon illumination length.

We then proceeded to test the sectioning capabilities of 2p-SPIM in a typical imaging experiment involving a living model organism, the C. elegans. To do that we acquired depth-resolved images of C. elegans pharyngeal muscle expressing the fluorescent protein “cameleon”. This protein is normally used as calcium indicator in imaging calcium transients in intact C. elegans [21

21. R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron 26(3), 583–594 (2000). [CrossRef] [PubMed]

]. It is composed of four domains, cyan fluorescent protein (CFP), calmodulin, M13 (a calmodulin binding domain), and yellow fluorescent protein (YFP). In our proof-of-principle experiment, the worms were deeply anesthetized and no calcium transients were expected. Hence, the observed fluorescence signal was attributed only to two-photon excitation of CFP.

Figure 4
Fig. 4 Upper: 2p-SPIM fluorescence image section of cameleon-expressing in C. elegans pharyngeal muscles. Lower: Overlay of the 2p-SPIM fluorescence (cyan) and brightfield image of anterior of C. elegans depicting colocalization between the fluorescence signal and the pharynx. Also indicated are the distinct structures in the fluorescence image associated with the known anatomy of C. elegans. BC: bucal cavity, PC: procorpus, MC: metacarpus, IS: isthmus, TB: terminal bulb. Scale bars: 50 µm.
shows the observed 2p-SPIM fluorescence signal (Fig. 4, upper) and its overlay on a brightfield image of the anterior body section of C. elegans (Fig. 4, lower). In the fluorescence imaging, we used an average laser excitation power of ~40mW and an acquisition frame rate of 5Hz (image exposure time, 200ms). At this excitation power levels, no evidence of photobleaching could be observed. The overlay image clearly shows that the fluorescence signal originates from the pharynx. Moreover, the fluorescence image illustrates identifiable anatomical structures of the pharynx, including the spherical terminal bulb, the elongated isthmus, the metacarpus and the procorpus (see Fig. 4).

The capability of 2p-SPIM to record optical sections, as demonstrated earlier (Fig. 3) allowed us to reconstruct a three-dimensional image of the cameleon-expressing pharynx in the soil worm by acquiring images at different specimen depth. Shown in Fig. 5
Fig. 5 A to J: 2p-SPIM fluorescence optical sections of cameleon in C. elegans pharyngeal muscle at 4 µm depth intervals clearly depicting the optical sectioning capability of 2p-SPIM. K: Movie showing the reconstructed three-dimensional structure of the C. elegans pharynx (Media 1). Scale bars: 50 µm.
are 2p-SPIM optical sections of the specimen at 4 µm step intervals (Fig. 5, A to J), and the three-dimensional reconstruction of the pharynx (Fig. 5, K and Media 1).

Whereas this report demonstrated a practical 2p-SPIM setup for the first time, demonstration of its advantages over other imaging techniques is yet to be carried out. In the future, our experiments will be focused on showing the capabilities of 2p-SPIM in terms of fast imaging rates, large specimen imaging, and long-term imaging. It is our view that the potential advantages of 2p-SPIM lies primarily in its use of long-wavelength excitation light. Near infrared (NIR) light scatters less inside turbid media than visible or UV light implying deeper penetration of the excitation light inside tissues and large specimens can potentially be imaged by 2p-SPIM. This exemplifies a possible future application of 2p-SPIM in large-specimen imaging. Moreover, the use of near infrared as excitation light source in 2p-SPIM has its advantages in terms of wavelength-dependent phototoxicity issues. It has been demonstrated that long wavelength illumination has less detrimental effects than visible- or UV-light in mammalian embryos [27

27. J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17(8), 763–767 (1999). [CrossRef] [PubMed]

]. This sensitivity to short-wavelength light, mainly attributed to radical oxygen species (ROS) production has also been reported in the development of mammalian zygotes [28

28. M. Takenaka, T. Horiuchi, and R. Yanagimachi, “Effects of light on development of mammalian zygotes,” Proc. Natl. Acad. Sci. U.S.A. 104(36), 14289–14293 (2007). [CrossRef] [PubMed]

]. Thus, it is likely that imaging studies on long-term biological processes such as mammalian embryogenesis will benefit from the low phototoxicity of NIR-excited 2p-SPIM.

Acknowledgements

We thank Dr. J. Swoger and Prof. J. Sharpe from the Centre for Genomic Regulation (Barcelona, Spain) for the interesting and encouraging discussions on SPIM; Dr W. Schafer from the MRC Laboratory of Molecular Biology (Cambridge, UK) for providing the C. elegans strain and C. Alonso for the help in their preparation; Dr. Hendrik Fuß of Molecular Machines & Industries GmbH (Munich, Germany) for the helpful discussion on the setup and for his assistance in obtaining the imaging detector; Iberlaser, s.a. (Madrid, Spain) for lending us the EM-CCD detector. This work is supported by the Generalitat de Catalunya grant 2009-SGR-159, the Spanish government grant TEC2009-09698, and the EU project STELUM (FP7-PEOPLE-2007-3-1-IAPP, 217997). This research has been partially supported by Fundació Cellex Barcelona.

References and links

1.

Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca(2+) imaging,” Cell Calcium 30(6), 383–393 (2001). [CrossRef] [PubMed]

2.

M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002). [CrossRef] [PubMed]

3.

G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996). [CrossRef] [PubMed]

4.

A. Egner and S. W. Hell, “Time multiplexing and parallelization in multifocal multiphoton microscopy,” J. Opt. Soc. Am. A 17(7), 1192–1201 (2000). [CrossRef]

5.

K. H. Kim, C. Buehler, and P. T. So, “High-speed, two-photon scanning microscope,” Appl. Opt. 38(28), 6004–6009 (1999). [CrossRef]

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B. O. Watson, V. Nikolenko, and R. Yuste, “Two-photon imaging with diffractive optical elements,” Front Neural Circuits 3, 6 (2009). [PubMed]

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L. Sacconi, E. Froner, R. Antolini, M. R. Taghizadeh, A. Choudhury, and F. S. Pavone, “Multiphoton multifocal microscopy exploiting a diffractive optical element,” Opt. Lett. 28(20), 1918–1920 (2003). [CrossRef] [PubMed]

8.

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

9.

E. Papagiakoumou, V. de Sars, V. Emiliani, and D. Oron, “Temporal focusing with spatially modulated excitation,” Opt. Express 17(7), 5391–5401 (2009). [CrossRef] [PubMed]

10.

V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, “SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators,” Front Neural Circuits 2, 5 (2008). [CrossRef]

11.

K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006). [CrossRef] [PubMed]

12.

Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two photon imaging,” in Lasers and Electro-Optics, 2008 and 2008 Conference on Quantum Electronics and Laser Science. CLEO/QELS 2008. Conference on(2008), pp. 1–2.

13.

M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing for axial scanning,” Opt. Express 14(25), 12243–12254 (2006). [CrossRef] [PubMed]

14.

P. J. Keller and E. H. Stelzer, “Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008). [CrossRef]

15.

J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004). [CrossRef] [PubMed]

16.

P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007). [PubMed]

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P. J. Scherz, J. Huisken, P. Sahai-Hernandez, and D. Y. Stainier, “High-speed imaging of developing heart valves reveals interplay of morphogenesis and function,” Development 135(6), 1179–1187 (2008). [CrossRef] [PubMed]

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E. H. K. Stelzer and S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111(5-6), 536–547 (1994). [CrossRef]

20.

W. R. Zipfel, R. M. Williams, and W. W. Webb, “Nonlinear magic: multiphoton microscopy in the biosciences,” Nat. Biotechnol. 21(11), 1369–1377 (2003). [CrossRef] [PubMed]

21.

R. Kerr, V. Lev-Ram, G. Baird, P. Vincent, R. Y. Tsien, and W. R. Schafer, “Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans,” Neuron 26(3), 583–594 (2000). [CrossRef] [PubMed]

22.

H. J. Koester, D. Baur, R. Uhl, and S. W. Hell, “Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage,” Biophys. J. 77(4), 2226–2236 (1999). [CrossRef] [PubMed]

23.

K. König, T. W. Becker, P. Fischer, I. Riemann, and K. J. Halbhuber, “Pulse-length dependence of cellular response to intense near-infrared laser pulses in multiphoton microscopes,” Opt. Lett. 24(2), 113–115 (1999). [CrossRef]

24.

A. Hopt and E. Neher, “Highly nonlinear photodamage in two-photon fluorescence microscopy,” Biophys. J. 80(4), 2029–2036 (2001). [CrossRef] [PubMed]

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A. Schönle and S. W. Hell, “Heating by absorption in the focus of an objective lens,” Opt. Lett. 23(5), 325–327 (1998). [CrossRef]

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Y. Liu, D. K. Cheng, G. J. Sonek, M. W. Berns, C. F. Chapman, and B. J. Tromberg, “Evidence for localized cell heating induced by infrared optical tweezers,” Biophys. J. 68(5), 2137–2144 (1995). [CrossRef] [PubMed]

27.

J. M. Squirrell, D. L. Wokosin, J. G. White, and B. D. Bavister, “Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability,” Nat. Biotechnol. 17(8), 763–767 (1999). [CrossRef] [PubMed]

28.

M. Takenaka, T. Horiuchi, and R. Yanagimachi, “Effects of light on development of mammalian zygotes,” Proc. Natl. Acad. Sci. U.S.A. 104(36), 14289–14293 (2007). [CrossRef] [PubMed]

OCIS Codes
(170.0110) Medical optics and biotechnology : Imaging systems
(170.0180) Medical optics and biotechnology : Microscopy
(170.2520) Medical optics and biotechnology : Fluorescence microscopy
(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: February 26, 2010
Revised Manuscript: March 24, 2010
Manuscript Accepted: March 31, 2010
Published: April 7, 2010

Virtual Issues
Vol. 5, Iss. 8 Virtual Journal for Biomedical Optics

Citation
Jonathan Palero, Susana I.C.O. Santos, David Artigas, and Pablo Loza-Alvarez, "A simple scanless two-photon fluorescence microscope using selective plane illumination," Opt. Express 18, 8491-8498 (2010)
http://www.opticsinfobase.org/oe/abstract.cfm?URI=oe-18-8-8491


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References

  1. Q. T. Nguyen, N. Callamaras, C. Hsieh, and I. Parker, “Construction of a two-photon microscope for video-rate Ca(2+) imaging,” Cell Calcium 30(6), 383–393 (2001). [CrossRef] [PubMed]
  2. M. J. Miller, S. H. Wei, I. Parker, and M. D. Cahalan, “Two-photon imaging of lymphocyte motility and antigen response in intact lymph node,” Science 296(5574), 1869–1873 (2002). [CrossRef] [PubMed]
  3. G. J. Brakenhoff, J. Squier, T. Norris, A. C. Bliton, M. H. Wade, and B. Athey, “Real-time two-photon confocal microscopy using a femtosecond, amplified Ti:sapphire system,” J. Microsc. 181(3), 253–259 (1996). [CrossRef] [PubMed]
  4. A. Egner and S. W. Hell, “Time multiplexing and parallelization in multifocal multiphoton microscopy,” J. Opt. Soc. Am. A 17(7), 1192–1201 (2000). [CrossRef]
  5. K. H. Kim, C. Buehler, and P. T. So, “High-speed, two-photon scanning microscope,” Appl. Opt. 38(28), 6004–6009 (1999). [CrossRef]
  6. B. O. Watson, V. Nikolenko, and R. Yuste, “Two-photon imaging with diffractive optical elements,” Front Neural Circuits 3, 6 (2009). [PubMed]
  7. L. Sacconi, E. Froner, R. Antolini, M. R. Taghizadeh, A. Choudhury, and F. S. Pavone, “Multiphoton multifocal microscopy exploiting a diffractive optical element,” Opt. Lett. 28(20), 1918–1920 (2003). [CrossRef] [PubMed]
  8. D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express 13(5), 1468–1476 (2005). [CrossRef] [PubMed]
  9. E. Papagiakoumou, V. de Sars, V. Emiliani, and D. Oron, “Temporal focusing with spatially modulated excitation,” Opt. Express 17(7), 5391–5401 (2009). [CrossRef] [PubMed]
  10. V. Nikolenko, B. O. Watson, R. Araya, A. Woodruff, D. S. Peterka, and R. Yuste, “SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators,” Front Neural Circuits 2, 5 (2008). [CrossRef]
  11. K. Shi, S. Yin, and Z. Liu, “Wavelength division scanning for two-photon excitation fluorescence imaging,” J. Microsc. 223(2), 83–87 (2006). [CrossRef] [PubMed]
  12. Q. Xu, K. Shi, S. Yin, and Z. Liu, “Chromatic two photon imaging,” in Lasers and Electro-Optics, 2008 and 2008 Conference on Quantum Electronics and Laser Science. CLEO/QELS 2008. Conference on(2008), pp. 1–2.
  13. M. E. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing for axial scanning,” Opt. Express 14(25), 12243–12254 (2006). [CrossRef] [PubMed]
  14. P. J. Keller and E. H. Stelzer, “Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy,” Curr. Opin. Neurobiol. 18(6), 624–632 (2008). [CrossRef]
  15. J. Huisken, J. Swoger, F. Del Bene, J. Wittbrodt, and E. H. Stelzer, “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305(5686), 1007–1009 (2004). [CrossRef] [PubMed]
  16. P. J. Verveer, J. Swoger, F. Pampaloni, K. Greger, M. Marcello, and E. H. Stelzer, “High-resolution three-dimensional imaging of large specimens with light sheet-based microscopy,” Nat. Methods 4(4), 311–313 (2007). [PubMed]
  17. P. J. Scherz, J. Huisken, P. Sahai-Hernandez, and D. Y. Stainier, “High-speed imaging of developing heart valves reveals interplay of morphogenesis and function,” Development 135(6), 1179–1187 (2008). [CrossRef] [PubMed]
  18. T. F. Holekamp, D. Turaga, and T. E. Holy, “Fast three-dimensional fluorescence imaging of activity in neural populations by objective-coupled planar illumination microscopy,” Neuron 57(5), 661–672 (2008). [CrossRef] [PubMed]
  19. E. H. K. Stelzer and S. Lindek, “Fundamental reduction of the observation volume in far-field light microscopy by detection orthogonal to the illumination axis: confocal theta microscopy,” Opt. Commun. 111(5-6), 536–547 (1994). [CrossRef]
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