A rainbow of colors – Imaging many colors in light sheet microscopy

With our new hyperspectral light sheet microscope we acquire the full emission spectrum in each pixel with nanometer resolution. Having the spectral information in hand, we resolved the signal of strongly overlapping dyes and fluorescent proteins and eliminated the autofluorescence signal in living zebrafish and drosophila embryos.

HFSP Career Development Award holder Jan Huisken and colleagues
authored on Mon, 21 December 2015

To understand the development of living organisms, one would ideally like to label all tissues in a single sample with differently colored markers and image their movements simultaneously. Until now, filter-based fluorescence microscopy was limited by the spectral overlap of these fluorophores and it was impossible to distinguish more than three or four colors within the visible spectrum. Therefore, we wanted to combine the power of spectral fluorophore detection with state-of-the-art light sheet microscopy.


Figure: Principle and benefits of hyperspectral light sheet microscopy. (a) For each line in the sample a full spectrum is recorded (yellow). The resulting x-lambda-images are then assembled into the well-known image representation. In this case only the sum is shown in the x-y-image. (b) In a zebrafish labeled with five colors the individual spectra can be recovered by spectral unmixing. Arrowheads indicate the wavelengths of the excitation lasers, which were suppressed in the spectrum by a notch filter. (c) Color representation of the zebrafish (area in the sketch is enlarged, scale bar 100µm).

In a light sheet microscope (or Selective Plane Illumination Microscope, SPIM), the sample is illuminated from the side with a thin sheet of light and the illuminated plane is imaged with the perpendicular detection arm. When compared with confocal microscopy, SPIM’s unique illumination scheme offers both increased acquisition speed and much lower phototoxicity, making it the technique of choice to image developing embryos over long periods of time. Yet, multiple colors have always been acquired using filter-based approaches, ultimately limiting the number of colors by the spectral overlap of fluorescent markers.

We have now designed a hyperspectral microscope based on line-scanning light sheet microscopy to acquire not only distinct colors, but the full emission spectrum with up to nanometer resolution. Since spectral information is available for each pixel in a three-dimensional volume, the spectra can be linearly unmixed to separate overlapping fluorophores. We resolved the signal of strongly overlapping GFP and YFP fluorescence and imaged up to five fluorophores in living zebrafish embryos. In strongly autofluorescent fruit fly embryos, we isolated the autofluorescence and recovered the desired faint fluorescence signal. Autofluorescence makes large structures such as the yolk or the outline of the sample visible, thus providing information on the morphological context of the sparse fluorescent label. This additional information comes free, as it requires neither extra labeling nor additional excitation.

Our hyperspectral SPIM provides the basis to simultaneously image dozens of fluorophores expressed in different tissues interacting during development. Instead of painstakingly assembling data sets acquired in several different individuals, all the necessary information is acquired in a single recording and in a single specimen. As new fluorescent markers are rapidly developed, the collection of samples expressing multiple fluorophores is constantly growing. Our hyperspectral imaging technique will serve as a powerful tool to provide a comprehensive atlas of embryonic development, in which different organs and different cellular components can be studied side by side.


Hyperspectral Light Sheet Microscopy. Wiebke Jahr, Benjamin Schmid, Christopher Schmied, Florian O. Fahrbach & Jan Huisken. Nature Communications 6:7990, doi: 10.1038/ncomms8990.

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