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Seeing Life in Four Dimensions

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THE QUESTION

How can we enable faster 3D imaging?

From understanding how molecules shift and reorient as cells divide to capturing the complex, coordinated events that occur within neural assemblies inside a living brain, how can we truly understand biological process unless we can watch them unfold at high-resolution in three dimensional space?

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Unlike the residents of Edwin A. Abbott’s Flatland,1 our lives are lived in three dimensions that are speeding forward through time.

Dealing with three spatial dimensions and the fourth dimension of time has often been a problem for cell biologists seeking to elucidate the complexities of molecular mechanisms occurring within a cell or within groups of cells. The time needed to capture sequential 2D slices can exceed the speed of biological processes, and maintaining high resolution can be difficult when moving a camera or a sample during image acquisition of a new focal plane.

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Above: Birefringence multifocus image of diatom.  (S. Abrahamsson)

Can we speed microscopy image acquisition to enable faster 3D imaging? This is the question that Sara Abrahamsson, a research fellow at Rockefeller University, has been fascinated with and has answered with a resounding yes. By pushing the boundaries of current microscopy techniques and leveraging optical physics innovations, Abrahamsson and her Ph.D. advisor, the late Dr. Mats Gustafsson, developed an elegant technique they call MultiFocus Microscopy (MFM).

Above: Movie of mRNA created with 9 plane MultiFocus Microscope. (S. Abrahamsson)

Their technique debuted in a 2013 publication which demonstrated capture of an instant focal stack of nine 2D images,2 and recent advances in camera technology have enabled the team to extend this approach to capturing an instant focal stack of up to twenty-five 2D images.3 The latest Abrahamsson paper also highlights the compatibility of their technique with polarization microscopy4,5—a technology pioneered by the Oldenbourg lab (Marine Biological Laboratory, MA) that can generate label-free high-contrast images of optically active samples—as an example of the generalizability of their approach.

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Read the Paper (login may be required)
Abrahamsson S, McQuilken M, Mehta SB, Verma A, Larsch J, Ilic R, Heintzmann R, Bargmann CI, Gladfelter AS, Oldenbourg R. MultiFocus Polarization Microscope (MFPolScope) for 3D polarization imaging of up to 25 focal planes simultaneously. Opt Express. 2015 Mar 23; 23(6):7734-54. PMID: 25837112.
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The System:

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Camera:
(2) Two ORCA-Flash4.0 cameras were used
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Microscope:
Inverted Olympus IX-83 microscope body
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Objectives:
Olympus 60x oil-immersion objective, NA=1.3 Standard tube lens
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Compensator:
Liquid crystal (LC) compensator, electronically controlled (polarizer)
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Software:
OpenPolScope (www.openpolscope.org) FIJI/ImageJ
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Grating:
Custom Diffractive Fourier optic - the MultiFocus Grating (MFG, see “The Solution” below)
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Fluorescence and transmission polarization imaging with 9 focal planes:
X-cite XLED1 (Lumen Dynamics), blue LED module
LC compensator
Green 515/30 nm emission filter (Semrock)
MultiFocus Grating for 9 focal planes (see “The Solution” below)
EMCCD camera (Andor iXon-888)
3D volume view, 33 × 33 × 2.5 μm3
250 nm focus step between image planes
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Transmission polarization imaging with 25 focal planes:
MultiFocus Grating for 25 focal planes (see “The Solution” below)
LC compensator
sCMOS camera (ORCA-Flash4.0, Hamamatsu)
3D volume of view, 55 × 55 × 38 μm3
1.5 μm focus step between image planes
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THE BARRIERS

Sequential image acquisition limits the time scales and resolutions that can be reliably imaged.

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While there have been a number of recent pioneering efforts focused on developing 3D microscopy that’s fast enough to observe biological processes happening on the time scale of seconds, these techniques—such as lightsheet microscopy PA1 —still require sequential image acquisition to capture the multiple focal planes that make up the imaged volume. This requirement places a limit on the time scales and resolutions that can be reliably imaged—the time needed to change from one focal plane to the next, the speed of image acquisition by the camera, and the stability of the system during the change of focal plane together impede fast and accurate 3D microscopy. These barriers are especially problematic for polarization microscopy, where the sample needs to be illuminated with light polarized in multiple orientations for each time point.

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Polarization microscopy: Label-free, information-rich

Polarization microscopy takes advantage of the optically-active nature of a number of biological structures, such as microtubules, spindle fibers, chromosomes, etc. Because they interact differently with polarized light depending on their orientation, polarization microscopy can be used to monitor changes in their orientation over time, offering submicroscopic information on structural changes. Further, polarized light can be used to generate high-contrast, label-free images at high resolutions. Above: Ed Uthman, MD/Wikimedia Commons CC BY-3.0

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Polarization microscopy: Birefringence

Birefrengence results from optically active materials that transmit polarized light at different speeds depending on whether the polarization is parallel to the axis of the molecular alignment or perpendicular. Light polarized parallel to the molecular axis moves faster than light polarized perpendicular to the molecular axis, resulting in a phase shift in the transmitted light, referred to as retardance. Above: Hans-Jürgen Schwarz/Wikimedia Commons CC BY-SA 3.0

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Polarization microscopy: Polarized Fluorescence

Both excitation and emission of a fluorescent molecule is polarization dependent, with the excitation efficiency dependent on the relative orientations of the fluorophore and the polarization orientation of the excitation light. Once the fluorophore becomes attached to a target molecule, its polarization becomes correlated with the target (depending on the rigidity of the linker), and can report on the orientation of the molecule it’s attached to.

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THE SOLUTION

Abrahamsson and colleagues solved the problem of lengthy data acquisition from sequential imaging by developing a method where multiple focal planes could be imaged at once.

With just one specially designed diffraction grating and two relay lenses, Abrahamsson and colleagues turned a standard, high-resolution epifluorescence microscope into a setup that could simultaneously acquire a stack of twenty-five focal planes.3 In the MFM approach, a diffraction grating is placed in the Fourier plane generated by the first relay lens and located behind the primary image. The design of the grating function—the shape of the pattern repeated across the grating—is calculated to diffract the incoming light in a way that results in the desired number of focal planes.

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A carefully calibrated geometrical distortion across the grating pattern refocuses the light and removes spherical aberrations. The result is an array of 2D images formed on the detector (the camera sensor), each of which corresponds to a single focal plane displaced from the others in the z-axis. For imaging a nucleus or even a single cell, nine focal planes provides good coverage in the third dimension. However for imaging larger structures such as organs or even whole organisms, increasing the number of focal planes is critical, with twenty-five focal planes sufficient for imaging whole C. elegans embryos. The expansion from nine focal planes in Abrahamson, et al’s, original paper2 to twenty-five in the most recent one was made possible by the wide field-of-view of the ORCA-Flash4.0.

The Multi-Focus Grating

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Above: The pattern on Abrahamsson’s multifocus grating.
Below: A multifocus grating before installation into the microscope.

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Above: extended volume 25-plane multifocus movie showing a developing C. elegans embryo inside an adult worm, recorded at one frame per second. Polarization imaging is used to obtain contrast, revealing chromatin and spindles rearranging during the first cell division into the two-cell stage. The size of each image tile (focal plane) is 55 μm Ā~ 55 μm laterally. The focus step between planes is 1.5 μm. Imaging depth is 38 μm covering the entire embryo.

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THE POSSIBILITIES

Rich data that provides new insights.

With straightforward integration into existing microscopy setups and easily interpretable data, the MFM approach can be combined with most microscopy techniques—including super-resolution microscopy—enhancing their capabilities into three dimensions and through time. Imagine the rich data sets that can be generated when you combine super resolution or PALM/STORM with MFM.

REFERENCES

  1. 01. Abbott, Edwin A. Flatland: A Romance of Many Dimensions. London, UK.: Seely & Co. 1884.
  2. 02. Abrahamsson, S, et al. Fast multicolor 3D imaging using aberration-corrected multifocus microscopy. Nat Methods. 2013 Jan; 10(1):60-3. PMCID: PMC4161287.
  3. 03. Abrahamsson, S, et al. MultiFocus Polarization Microscope (MFPolScope) for 3D polarization imaging of up to 25 focal planes simultaneously. Opt Express. 2015 Mar 23; 23(6):7734-54. PMID: 25837112.
  4. 04. R. Oldenbourg and G. Mei. New polarized light microscope with precision universal compensator. J. Microsc. 1995; 180(2), 140–147. www.openpolscope.org.
 
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