Advances in multiphoton microscopy technology

Journal name:
Nature Photonics
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Published online


Multiphoton microscopy has enabled unprecedented dynamic exploration in living organisms. A significant challenge in biological research is the dynamic imaging of features deep within living organisms, which permits the real-time analysis of cellular structure and function. To make progress in our understanding of biological machinery, optical microscopes must be capable of rapid, targeted access deep within samples at high resolution. In this Review, we discuss the basic architecture of a multiphoton microscope capable of such analysis and summarize the state-of-the-art technologies for the quantitative imaging of biological phenomena.

At a glance


  1. A typical multiphoton microscope fed by a near-IR laser.
    Figure 1: A typical multiphoton microscope fed by a near-IR laser.

    Typical multiphoton systems utilize near-IR (700–1,300 nm) light and use a raster scanning system to control the beam, either with 'close coupled' scan mirrors or with image-relayed scan mirrors (SMx and SMy, as shown here). In this epi-detection configuration, a dichroic (D) is used to separate two-photon excited fluorescence from the excitation light and direct this fluorescence to a PMT. L = lens.

  2. Multimodal image of a blood vessel in kidney tissue.
    Figure 2: Multimodal image of a blood vessel in kidney tissue.

    SHG (blue), TPEF (green) and coherent anti-Stokes Raman scattering (red). Image courtesy of Eric Potma, University of California, Irvine, USA.

  3. Illustrative fluorescence lifetime image with two similar fluorophores and comparison to TPEF imaging.
    Figure 3: Illustrative fluorescence lifetime image with two similar fluorophores and comparison to TPEF imaging.

    Fluorescence intensity and lifetime imaging of propidium iodide (PI)-labelled cells and Texas Red dextran (TR)-labelled vessels in a mouse model. a, TPEF image shows that the two dyes are indistinguishable. Scale bar (right) represents photon counts. b, Image is rescaled according to the measured fluorescent lifetime; the PI-label and the TR-label are now spatially distinct. Scale bar (right) is in nanoseconds. c, The images in a and b are combined, thus enabling facile detection of the two fluorophores. The arrowhead points to a PI-labelled cell, whereas the arrow points to a TR-labelled vessel. Figure reproduced with permission from ref. 35, © 2011 APS.

  4. Example of deep in vivo imaging through the use of longer excitation wavelengths.
    Figure 4: Example of deep in vivo imaging through the use of longer excitation wavelengths.

    1,280 nm light from an optical parametric oscillator is used to perform TPEF imaging of mouse vasculature labelled with Alexa680-Dextran. a, In vivo two-photon fluorescence images of cortical vasculature in mouse brain. 235 xy frames from 60 μm above the cortical surface to 1,110 μm below are taken at depth increments of 5 μm. The depth increments in the stack are 20 μm in the range of 1,110–1,490 μm and 30 μm in the range of 1,490–1,670 μm. 3D reconstruction is made in Image J software using the volume viewer plug-in. Expanded 3D stacks are shown for the deepest sections (>1,130 μm). b, Fluorescence intensity as a function of imaging depth for the stack shown in a. Fluorescence signal strength at a particular depth is represented by the average value of the brightest 1% of the pixels in the xy image at that depth. Scale bars are 50 μm for both a and b. Figure reproduced with permission from ref. 47, © 2011 SPIE.

  5. Simultaneous multilayer imaging achieved with remote focusing.
    Figure 5: Simultaneous multilayer imaging achieved with remote focusing.

    Four images of Drosophila melanogaster antennal lobe structure labelled with red fluorescent protein. The images are separated axially by 7 μm in depth and were all acquired simultaneously from a single-element detector. Figure reproduced with permission from ref. 105, © 2012 Wiley.


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  1. Center for Microintegrated Optics for Advanced Bioimaging and Control, Colorado School of Mines, 1523 Illinois Street, Golden, Colorado 80401, USA

    • Erich E. Hoover &
    • Jeff A. Squier
  2. Department of Physics, Colorado School of Mines, 1523 Illinois Street, Golden, Colorado 80401, USA

    • Erich E. Hoover &
    • Jeff A. Squier

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