MICROSCOPY: 3-D microsurgery uses light-sheet fluorescence microscopy

In light-sheet microscopy, an illuminator encompasses the entire focal plane of a microscope objective lens and projects a microns-thick light sheet that illuminates only the specimens that lay within the objective’s focal depth (see www.laserfocusworld.com/articles/142833). Scientists at the European Molecular Biology Laboratory (EMBL; Heidelberg, Germany) are using this technique combined with fluorescence microscopy and plasma-induced laser-ablation methodologies to study in vivo biological functions and perform three-dimensional (3-D) laser microsurgery.1

A light-sheet or single-plane illumination microscope (SPIM) with fluorescence-imaging capability combined with a pulsed ablation laser in one instrument can perform imaging and microsurgery in three dimensions. A microtubule is easily targeted and cut (top; blue line). Two seconds after dissection (bottom), the cut region is clearly visible, with minimum impact on the surrounding medium. (Courtesy of European Molecular Biology Laboratory)
Click here to enlarge image

The researchers use the laboratory’s single-plane illumination microscope (SPIM)-an implementation of light-sheet-based fluorescence microscopy-combined with a pulsed-laser-based microsurgery setup in a single instrument to image and manipulate biological processes ranging in scale from microns to millimeters.2

In the optical setup, laser microsurgery is performed by a frequency-tripled Nd:YAG laser at 355 nm with 470 ps pulse duration and a pulse repetition rate up to 1 kHz. The laser is focused by a series of lenses with different numerical apertures to tailor the beam diameter to the size of the feature to be ablated or cut. The software-controlled ablation beam is coupled into the SPIM detection path using a dichroic mirror. The specimen can be translated along the z-axis for improved depth imaging during ablation or observation.

In one of several experiments, microtubules are targeted and dissected using the SPIM nanoscalpel system (see figure). Microtubules, confined in a glycerol solution, are illuminated at a fluorescence wavelength of 514 nm. One of the microtubules is cut using 50 pulses of the UV ablation laser with individual energies of 40 ± 5 nJ with a 1 kHz pulse repetition rate. The speed and tight focus of the ablation process minimizes ultraviolet photobleaching of the microtubules and reduces disturbances of the surrounding microtubules in solution.

In another experiment, the researchers performed 3-D microsurgery on a millimeter-scale caudal zebrafish fin. A section of fin was cut by 15 UV pulses with individual energies of 0.57 ± 0.03 µJ at a repetition rate of 800 Hz. Although 3-D fluorescence images show photobleaching in the vicinity of the laser cuts, transmission images demonstrate precise material removal, again with limited disturbance to surrounding tissue.

“The primary rationale for developing such a 3-D nanoscalpel was to be able to perform laser dissection deep into embryos but also in 3-D cell clusters,” said researcher Emmanuel G. Reynaud. “In fact, in the body, cells exist in complex 3-D arrangements. These arrangements are critical to the functions in the body and provide much more faithful replicates of cell behavior in vivo than is possible using two-dimensional substrata. Now, with this tool we can perform single-cell ablation anywhere in a cell cluster and follow the consequences, macrophage migration, tissue repair, and cell response in a more physiological and relevant environment.”

Gail Overton


1. C.J. Engelbrecht et al., Optics Express 15(10) 6420 (May 14, 2007).

2. K. Greger et al., Rev. Scientific Instruments 78, 023705 (2007).

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