Shaping Light for Wider Fields of View

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How can we shape the illumination to reveal the most information?

Imagine capturing images of your living biological sample with excellent contrast, high x, y and z (axial) resolution and large field-of-view (FOV) with limited sample cellular damage from light exposure. Suddenly, probing inquiries into cellular development, morphology and behavior are within reach. Light-sheet microscopy is the powerful tool that enables this approach.

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Like many emerging technologies, there is a natural evolution the refines the details and in this case the detail is the light-sheet itself. Since the shape of the light-sheet defines the FOV and contributes to the axial resolution, how can changes to the beam shape illuminate the most information?

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Read the Paper (login may be required)
Vettenburg T, Dalgarno HIC, Nylk J, Coll-lladó C, Ferrier DEX, Cizmar T, Gunn-Moore FJ, Dholakia K. Light-sheet microscopy using an Airy beam. Nat Methods. 2014 May; 11(5):541-4. PMID 24705473.
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Light itself is one of the biggest problems facing microscopists—high levels are needed for good resolution and contrast, but too much leads to photo-bleaching and sample damage.

Light-sheet fluorescence microscopy was developed as a way to optically section the sample, providing fast, volumetric data while minimizing the negative effects of light. The goal—generate an extended, uniformly thin light sheet to ensure optimal excitation and isotropic resolution.

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Above: Illustration of the inverse relationship between axial confinement and field of view (FOV) of a light sheet. Illumination with a low numerical aperture (NA) results in a wide FOV, albeit at the cost of illuminating a relatively broad volume (A). At twice the numerical aperture, the FOV is reduced by a factor of four (B). Further reduction of the numerical aperture would lead to an impractically small field of view (C).

Not all light sheets are created equal.

There are several ways to shape light into a light sheet. Most current setups generate an apertured Gaussian beam, which illuminate the FOV with a light sheet that is wider than a biological cell. Such a light-sheet can be focused to sub-micron widths, due to diffraction, this is only possible for an impractically small FOV.

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Fortunately, not all light sheets have to be Gaussian. Other shapes, like Bessel beams have been proposed, but the outer rings of the beam contribute to background fluorescence which may lead to photo-bleaching or damage unless two-photon excitation is used.

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Beam waist: the narrowest portion of the beam, i.e., has the smallest cross-section.
Rayleigh range: the distance, in the direction of propagation, from the waist to the place where the cross section is doubled. For light sheets, a larger Rayleigh range translates into a wider field of view. Rayleigh range is only defined for Gaussian beam.
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Faced with these limitations of the illumination, the question asked by Tom Vettenburg, PhD, and the other team members in Dhoklia’s group was, “What about Airy beams?”

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Like a Bessell beam, an Airy beam has a propagation-invariant intensity profile, meaning the intensity is uniform along its length. Although highly asymmetric shape of the Airy beam may seem problematic, it avoids the low contrast caused by Bessell beam side lobes and the uneven Airy profile can be readily managed by standard deconvolution methods. Above: Depiction of the formation of a truncated Airy (A) light sheet (diagram not to scale). The light sheet propagates along the x-axis and is scanned through the sample in the z-dimension (B).

Light-sheet microscopy/biological samples setup

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Laser 1:
Coherent Verdi V6, 6 W, 532 nm
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Laser 2:
Spectra Physics argon-ion 2040E, 10 W, 488 nm
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Acousto-optical deflector (AOD):
Neos AOBD 45035-3
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Scan period:
50 µs
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Spatial light modulator (SLM):
Hamamatsu LCOS X10468-04
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Excitation and Detection objectives (MO1, MO2):
Nikon CFI Apo 40×/0.80 DIC, working distance = 3.5 mm
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Sample mount:
xyz piezo stage, Mad City Labs, Nano-LP200
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Power, back aperture:
30-300 μW
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70 W/cm2
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ORCA-Flash4.0, Hamamatsu
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Above: Maximum intensity projections of light-sheet microscopy volumetric imaging scans of neurons in the brain of a living zebrafish (daniorerio) embryo, five days post-fertilisation, visualised with a membrane tethered red fluorescent protein. A Gaussian light sheet (A) and Airy light sheet (B) scan the same sample volume slice-by-slice from top to bottom. The cell bodies of two neurons (large, faint, round structures) and the interconnecting axons and dendrites are clearly visible with Airy light-sheet illumination.

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Above: Maximum intensity projections of light-sheet microscopy volumetric imaging scans near the distal end of a juvenile amphioxus (Branchiostomalanceolatum) tail. Cell nuclei are visualised through staining with propidium iodide after fixation. A Gaussian light sheet (C) and Airy light sheet (D) scan the same sample volume slice-by-slice from top to bottom. With Airy light-sheet illumination, the nuclear structure is clearly defined across the whole image and different types of tissue can be differentiated based on this nuclear structure.

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What Vettenburg, et al.,1 realized is that, unlike those of the Bessel light-sheet, the side lobes of the Airy light sheet contribute positively to the contrast and axial resolution. As such, there is no need to remove these as long as an appropriate, efficient, deconvolution is applied. The other good news is that generating the Airy beam is relatively easy—existing experimental setups can be used with fairly straightforward modification. The exciting results speak for themselves.

Above: The light sheet formed by the Airy beam illuminates the sample from the top left. Fluorescence is collected from the right-hand objective and re-imaged onto the active surface of the Hamamatsu ORCA-Flash4.0 sCMOS camera.

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Above: Comparison of various scanned light sheet types without post-excitation fluorescence filtering

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Imagine a ten-fold field-of-view.

In a landmark study using a Gaussian light sheet, Misha Ahrens and Philipp Keller were able to visualize an intact, living zebrafish larva brain.10 With simple alterations to the light-sheet setup, imagine what processes researchers could follow with a FOV expanded ten-fold.

High-Speed Imaging of Amoeboid Movements Using Light-Sheet Microscopy

Daisuke Takao, Atsushi Taniguchi, Takaaki Takeda, Seiji Sonobe, Shigenori Nonaka PLoS One. 2012; 7(12): e50846. PMCID: PMC351548611


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