Abstract

Multifocal multiphoton microscopy (MMM) enhances imaging speed by parallelization. It is not well understood why the imaging depth of MMM is significantly shorter than conventional single-focus multiphoton microscopy (SMM). In this report, we show that the need for spatially resolved detectors in MMM results in a system that is more sensitive to the scattering of emission photons with reduced imaging depth. For imaging depths down to twice the scattering mean free path length of emission photons (2×l em s), the emission point spread function (PSFem) is found to consist of a narrow, diffraction limited distribution from ballistic emission photons and a broad, relatively low amplitude distribution from scattered photons. Since the scattered photon distribution is approximately 100 times wider than that of the unscattered photons at 2×l em s, image contrast and depth are degraded without compromising resolution. To overcome the imaging depth limitation of MMM, we present a new design that replaces CCD cameras with multi-anode photomultiplier tubes (MAPMTs) allowing more efficient collection of scattered emission photons. We demonstrate that MAPMT-based MMM has imaging depth comparable to SMM with equivalent sensitivity by imaging tissue phantoms, ex vivo human skin specimens based on endogenous fluorophores, and green fluorescent protein (GFP) expressing neurons in mouse brain slices.

© 2007 Optical Society of America

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2007

W. Goebel, B. M. Kampa, and F. Helmchen, "Imaging cellular network dynamics in three dimensions using fast 3D laser scanning," Nat. Methods 473-79 (2007).
[CrossRef]

2006

2005

G. D. Reddy, and P. Saggau, "Fast three-dimensional laser scanning scheme using acousto-optic deflectors," J. Biomed. Opt. 10,064038 (2005).
[CrossRef]

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435,969-973 (2005).
[CrossRef] [PubMed]

W. C. Lee, H. Huang, G. Feng, J. R. Sanes, E. N. Brown, P. T. C. So, and E. Nedivi, "Dynamic Remodeling of Dendritic Arbors in GABAergic Interneurons of Adult Visual Cortex," PLoS Biol. 4e29 (2005).

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, "Single-photon counting multicolor multiphoton fluorescence microscope " J. Fluoresc. 15,41-51 (2005).
[CrossRef] [PubMed]

2004

B. R. Masters, P. T. C. So, C. Buehler, N. Barry, J. D. Sutin, W. W. Mantulin, and E. Gratton, "Mitigating thermal mechanical damage potential during two-photon dermal imaging," J. Biomed. Opt. 9,1265-1270 (2004).
[CrossRef] [PubMed]

R. D. Roorda, T. M. Hohl, R. Toledo-Crow, and G. Miesenbock, "Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes," J. Neurophysiol. 92,609-621 (2004).
[CrossRef] [PubMed]

T. Ragan, K. H. Kim, K. Bahlmann, and P. T. C. So, "Two-photon tissue cytometry," Methods Cell Biol 75,23-39 (2004).
[CrossRef] [PubMed]

G. C. Cianci, J. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc Res Tech 64,135-141 (2004).
[CrossRef] [PubMed]

2003

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

V. Iyer, B. E. Losavio, and P. Saggau, "Compensation of spatial and temporal dispersion for acousto-optic multiphoton laser-scanning microscopy," J. Biomed. Opt. 8,460-471 (2003).
[CrossRef] [PubMed]

K. Konig, and I. Riemann, "High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution," J. Biomed. Opt. 8,432-439 (2003).
[CrossRef] [PubMed]

C. Y. Dong, K. Koenig, and P. T. C. So, "Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium," J. Biomed. Opt. 8,450-459 (2003).
[CrossRef] [PubMed]

2002

E. Beaurepaire, and J. Mertz, "Epifluorescence collection in two-photon microscopy," Appl. Opt. 41,5376-5382 (2002).
[CrossRef] [PubMed]

B. Yu, K. H. Kim, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic Heterogeneity in Transdermal Transport revealed by High-Speed Two-Photon Microscopy: determination of representative skin sample sizes," J. Invest. Dermatol. 118,1085-1088 (2002).
[CrossRef] [PubMed]

T. P. Padera, B. R. Stoll, P. T. C. So, and R. K. Jain, "Conventional and High-Speed Intravital Multiphoton Laser Scanning Microscopy of Microvasculature, Lymphatics, and Leukocyte.Endothelial Interactions," Molecular Imaging 1,9-15 (2002).
[CrossRef]

J. Grutzendler, N. Kasthuri, and W.-B. Gan, "Long-term dendritic spine stability in the adult cortex," Nature 420,812-816 (2002).
[CrossRef] [PubMed]

2001

M. Oheim, E. Beaurepaire, E. Chaigneau, Jerome Mertz, and S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neuro. Method. 111,29-37 (2001).
[CrossRef]

2000

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc 201,368-376 (2000).
[CrossRef]

A. K. Dunn, V. P. Wallace, M. Coleno, M. W. Berns, and B. J. Tromberg, "Influence of optical properties on two-photon fluorescence imaging in turbid samples," Appl. Opt. 39,1194-1201 (2000).
[CrossRef]

D. N. Fittinghoff, P. W. Wiseman, and J. A. Squier, "Widefield multiphoton and temporally decorrelated multifocal multiphoton microscopy," Opt. Express 7,273-279 (2000).
[CrossRef] [PubMed]

G. Feng, R. H. Mellor, M. Bernstein, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28,41-51 (2000).
[CrossRef] [PubMed]

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

X. Gan, and M. Gu, "Spatial distribution of single-photon and two-photon fluorescence light in scattering media: Monte Carlo simulation," Appl. Opt. 39,1575-1579 (2000).
[CrossRef]

B. Lendvai, E. A. Stern, B. Chen, and K. Svoboda, "Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo," Nature 404,876-881 (2000).
[CrossRef] [PubMed]

1999

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

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, "Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with Cameleons," Biophy. J. 78,2412-2420 (1999).
[CrossRef]

J. Ying, F. Liu, and R. R. Alfano, "Spatial distribution of two-photon-excited fluorescence in scattering media," Appl. Opt. 38,224-229 (1999).
[CrossRef]

1998

1994

1990

W. Denk, J. H. Strickler, and W. W. Webb, "2-photon laser scanning fluorescence microscopy," Science 248,73-76 (1990).
[CrossRef] [PubMed]

Appl. Opt.

Biophy. J.

G. Y. Fan, H. Fujisaki, A. Miyawaki, R.-K. Tsay, R. Y. Tsien, and M. H. Ellisman, "Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with Cameleons," Biophy. J. 78,2412-2420 (1999).
[CrossRef]

J. Biomed. Opt.

V. Iyer, B. E. Losavio, and P. Saggau, "Compensation of spatial and temporal dispersion for acousto-optic multiphoton laser-scanning microscopy," J. Biomed. Opt. 8,460-471 (2003).
[CrossRef] [PubMed]

G. D. Reddy, and P. Saggau, "Fast three-dimensional laser scanning scheme using acousto-optic deflectors," J. Biomed. Opt. 10,064038 (2005).
[CrossRef]

K. Konig, and I. Riemann, "High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution," J. Biomed. Opt. 8,432-439 (2003).
[CrossRef] [PubMed]

C. Y. Dong, K. Koenig, and P. T. C. So, "Characterizing point spread functions of two-photon fluorescence microscopy in turbid medium," J. Biomed. Opt. 8,450-459 (2003).
[CrossRef] [PubMed]

B. R. Masters, P. T. C. So, C. Buehler, N. Barry, J. D. Sutin, W. W. Mantulin, and E. Gratton, "Mitigating thermal mechanical damage potential during two-photon dermal imaging," J. Biomed. Opt. 9,1265-1270 (2004).
[CrossRef] [PubMed]

J. Fluoresc.

C. Buehler, K. H. Kim, U. Greuter, N. Schlumpf, and P. T. C. So, "Single-photon counting multicolor multiphoton fluorescence microscope " J. Fluoresc. 15,41-51 (2005).
[CrossRef] [PubMed]

J. Invest. Dermatol.

B. Yu, K. H. Kim, P. T. C. So, D. Blankschtein, and R. Langer, "Topographic Heterogeneity in Transdermal Transport revealed by High-Speed Two-Photon Microscopy: determination of representative skin sample sizes," J. Invest. Dermatol. 118,1085-1088 (2002).
[CrossRef] [PubMed]

J. Microsc

T. Nielsen, M. Fricke, D. Hellweg, and P. Andresen, "High efficiency beam splitter for multifocal multiphoton microscopy," J. Microsc 201,368-376 (2000).
[CrossRef]

J. Neuro. Method.

M. Oheim, E. Beaurepaire, E. Chaigneau, Jerome Mertz, and S. Charpak, "Two-photon microscopy in brain tissue: parameters influencing the imaging depth," J. Neuro. Method. 111,29-37 (2001).
[CrossRef]

J. Neurophysiol.

R. D. Roorda, T. M. Hohl, R. Toledo-Crow, and G. Miesenbock, "Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes," J. Neurophysiol. 92,609-621 (2004).
[CrossRef] [PubMed]

J. Neurosci. Methods

R. Kurtz, M. Fricke, J. Kalb, P. Tinnefeld, and M. Sauer, "Application of multiline two-photon microscopy to functional in vivo imaging," J. Neurosci. Methods 151,276-286 (2006).
[CrossRef] [PubMed]

J. Opt. Soc. Am. A

J. Opt. Soc. Am. B

Methods Cell Biol

T. Ragan, K. H. Kim, K. Bahlmann, and P. T. C. So, "Two-photon tissue cytometry," Methods Cell Biol 75,23-39 (2004).
[CrossRef] [PubMed]

Microsc Res Tech

G. C. Cianci, J. Wu, and K. M. Berland, "Saturation modified point spread functions in two-photon microscopy," Microsc Res Tech 64,135-141 (2004).
[CrossRef] [PubMed]

Molecular Imaging

T. P. Padera, B. R. Stoll, P. T. C. So, and R. K. Jain, "Conventional and High-Speed Intravital Multiphoton Laser Scanning Microscopy of Microvasculature, Lymphatics, and Leukocyte.Endothelial Interactions," Molecular Imaging 1,9-15 (2002).
[CrossRef]

Nat. Biotechnol

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

Nat. Methods

W. Goebel, B. M. Kampa, and F. Helmchen, "Imaging cellular network dynamics in three dimensions using fast 3D laser scanning," Nat. Methods 473-79 (2007).
[CrossRef]

Nature

D. A. Sipkins, X. Wei, J. W. Wu, J. M. Runnels, D. Côté, T. K. Means, A. D. Luster, D. T. Scadden, and C. P. Lin, "In vivo imaging of specialized bone marrow endothelial microdomains for tumour engraftment," Nature 435,969-973 (2005).
[CrossRef] [PubMed]

J. Grutzendler, N. Kasthuri, and W.-B. Gan, "Long-term dendritic spine stability in the adult cortex," Nature 420,812-816 (2002).
[CrossRef] [PubMed]

B. Lendvai, E. A. Stern, B. Chen, and K. Svoboda, "Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo," Nature 404,876-881 (2000).
[CrossRef] [PubMed]

Neuron

G. Feng, R. H. Mellor, M. Bernstein, M. Wallace, J. M. Nerbonne, J. W. Lichtman, and J. R. Sanes, "Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP," Neuron 28,41-51 (2000).
[CrossRef] [PubMed]

Opt. Express

Opt. Lett.

Oxf.

A. H. Buist, M. Muller, J. Squier, and G. J. Brakenhoff, "Real time two-photon absorption microscopy using multi point excitation," J. Micros.-Oxf. 192,217-226 (1998).
[CrossRef]

PLoS Biol.

W. C. Lee, H. Huang, G. Feng, J. R. Sanes, E. N. Brown, P. T. C. So, and E. Nedivi, "Dynamic Remodeling of Dendritic Arbors in GABAergic Interneurons of Adult Visual Cortex," PLoS Biol. 4e29 (2005).

Science

W. Denk, J. H. Strickler, and W. W. Webb, "2-photon laser scanning fluorescence microscopy," Science 248,73-76 (1990).
[CrossRef] [PubMed]

Other

M. Gu, Principles of three-dimensional imaging in confocal microscopes (World Scientific Pub Co Inc, 1996).
[CrossRef]

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Figures (9)

Fig. 1.
Fig. 1.

An experimental setup for quantifying photon scattering effects on PSFem. Excitation beam path is very similar to conventional multiphoton microscopy. For measuring the PSFem FWHM variation, a CCD camera is used as a detector. For measuring the PSFem spatial distribution, a pinhole aperture of various sizes is positioned in the image plane and a PMT behind the aperture collects the transmitted emission light.

Fig. 2.
Fig. 2.

Variations in FWHM of PSFMMM tot as a function of imaging depth due to photon scattering. FWHMs in (a) transverse direction and (b) axial direction were measured from surface down to the imaging depth of 2×l em s (l em s=62.5 µm) at emission wavelength (515 nm). There was no significant change of PSFMMM tot FWHM in either direction.

Fig. 3.
Fig. 3.

Effects of emission photon scattering on PSFem peak intensity and shape. (a) Decay profiles of PSFem peak intensity and integrated intensity (over a 200 µm diameter circular area) as a function of normalized imaging depth down to 2.5×l em s. The lines denote best exponent fit of the data points. (b) Variations in the PSFem lateral profile (PSFem(r, ϕ) in cylindrical coordinates) with the imaging depth. The PSFem profile was measured up to 100 µm in radius from peak location. Data points are connected by lines for eye guidance.

Fig. 4.
Fig. 4.

A simulation of photon scattering effect on image contrast in MMM tissue imaging. An original image comprising 5×5 microspheres of 10 µm diameter equally spaced with 40 µm separation distance and the experimentally measured PSFtot was used in the simulation. (a) MMM image at the sample surface, (b) MMM image at imaging depth of 2.5×l em s, where increased background noise is observed in the center of the image. (c) The decay of SNR of the MMM image at its center location as a function of the imaging depth.

Fig. 5.
Fig. 5.

A schematic of the multifocal multiphoton microscope based on a MAPMT. Excitation beams are depicted in red/orange colors and emission beams are in green color. In this figure, only two beam-lets are ray-traced. The excitation beam is splitted into 8×8 beam-lets via a microlens array. Multiple excitation foci (8×8) scan the specimen. The emission beam-lets are collected either by a CCD camera or a MAPMT which has 8×8 pixels. L1, L2, and L3 are lenses. The transmission of light through the microlens array and high NA objective causes appreciable pulse dispersion which can be corrected by pre-chirping using a pair of prisms (not shown).

Fig. 6.
Fig. 6.

Quantifying signal decay with the increase of imaging depth. The intensity decay coefficient of a MAPMT-based MMM stands between that of a CCD-based MMM and that of a conventional SMM. A simple deconvolution algorithm (DC) is applied to MAPMT-based MMM. The decay coefficient of DC stands close to the that of SMM.

Fig. 7.
Fig. 7.

The effect of emission photon scattering on images acquired with MAPMT-based MMM. (a) An image of microspheres at 1.76×l em s deep from the surface in the tissue phantom sample. Ghost images appeared due to the scattered emission photons collected in neighboring pixels of the MAPMT. (b) Image after the deconvolution process. (c) Variation of PSFem due to emission photon scattering. The increase of intensity in the tail region was due to emission photon scattering. (d) Crosstalk in the MAPMT pixels. It was calculated by integrating the PSFem over the effective detection area of individual MAPMT pixels.

Fig. 8.
Fig. 8.

Human skin images acquired with MAPMT-based MMM. Epidermal layer of the skin is imaged. From left to right, imaging depth goes deeper from surface. Stratum corneum layer (a), stratum granular (b), and basal layer (c) are shown. Cells are visible based on autofluorescence. Image size: 360 µm×360 µm, input power: 7 mW per focus, objective: 20×, imaging speed: 2.5 frames/s with 320×320 pixels.

Fig. 9.
Fig. 9.

Images of GFP expressing neurons in the ex vivo mouse brain acquired with CCD-based MMM (a–c) and MAPMT-based MMM (d–f) at different depth locations (surface, 30 um, and 75 um deep). For images with MAPMT-based MMM, a deconvolution algorithm was applied to remove the effect of emission photon scattering (g–i). The objectiveused is a 20× water immersion with NA 0.95. The input laser power is 300 mW at 890 nm wavelength. The frame rate is 0.3 frames per second with a frame size of 320×320 pixels.

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