Light sheet microscopy has been shown to be advantageous for the imaging of various specimens ranging from single cell nuclei to complete fixed or living animal embryos. Such specimens differ significantly in size and therefore require greatly differing light sheet geometries for illumination. In this paper we introduced a variable light sheet concept comprising a cylindrical illumination zoom lens system and different illumination objective lenses. This concept provides the necessary flexibility to the illumination geometry of a light sheet microscope, because it allows the observation of biological samples with different sizes using a single instrument. It has several advantages over alternative beam shaping methods.
An alternative method for beam shaping of light sheets is to utilize slit apertures. By using rectangular slit apertures in the beam path [11
] a significant amount of the illumination power is lost. Also, diffraction effects distort the excitation beam resulting in an uneven illumination profile and a reduced optical sectioning thickness. Diffractive effects are negligible for focal waists with FWHM values > 10 µm as it is the case in our setup for the light sheet width in y-direction.
A quite different approach to light sheet generation is digital scanned laser light-sheet fluorescence microscopy (DSLM) [20
]. Here, a laser beam is focused to a single line and rapidly scanned up and down during image acquisition thus generating a virtual light sheet. The advantages of this technique are the uniform illumination intensity over the complete field-of-view and a simple adjustment of the light sheet width through computer controlled scanning mirrors. But in DSLM only the light sheet width is scanned and can be adjusted, but the optical sectioning thickness stays constant. This technique is especially suited to observe large specimen and can also be used for structured illumination for contrast enhancement [5
],but the scan speed is to slow for applications like single molecule tracking, which require very high imaging rates [7
We could already adapt our setup to a large variety of specimen due to a light sheet thickness ranging from 2.4 µm to 6.6 µm. The corresponding optimal field-of-view ranged from 54 µm to 410 µm. Axial and lateral illumination widths could be further extended by using illumination objective lenses with lower numerical apertures. We demonstrated this by replacing the 0.28 NA illumination objective by an 0.11 NA objective lens achieving light sheet thicknesses from 8.3 µm to 36 µm with a corresponding field-of-view of 650 µm to 12.3 mm. Since the used illumination objective lenses were designed to operate in air, the glass and water interface introduced a certain amount of spherical aberration. Thus it was not possible to translate the six-fold increase in beam diameter achievable in the zoom lens system into a corresponding reduction of the light sheet thickness. To take advantage of the full zoom range, either a very low NA objective, for which the spherical aberrations would be even less significant, or an especially corrected objective, which considers the unique illumination geometry, must be used [22
]. In our current setup the width of the light sheet (y-direction) was adjustable in the range of 20 – 120 µm, which could easily be extended by lateral scanning or by choosing a focusing lens (C6) with a greater focal length, which would have to be traded against a greater system length for the illumination optics. Generally, reduction of the illumination field diminishes unwanted photon exposure and reduces light scattering.
Despite the encountered limitation we found the zoom lens concept to greatly expand the versatility of light sheet microscopes.