Microfabricated devices for single objective single plane illumination microscopy (SoSPIM)

Abstract

Light sheet microscopy is a relatively new form of fluorescence microscopy that has been receiving a lot of attention recently. The strong points of the technique, such as high signal to noise ratio and its reduced photodamage of fluorescently labelled samples, come from its unique feature to illuminate only a thin plane in the sample that coincides with the focal plane of the detection lens. Typically this requires two closely positioned perpendicular objective lenses, one for detection and one for illumination. Apart from the fact that this special configuration of objective lenses is incompatible with standard microscope bodies, it is particularly problematic for high-resolution lenses which typically have a short working distance. To address these issues we developed sample holders with an integrated micromirror to perform single lens light sheet microscopy, also known as single objective single plane illumination microscopy (SoSPIM). The first design is based on a wet-etched silicon substrate, the second on a microfabricated polished polymer plug. We achieved an on-chip light sheet thickness of 2.3 μm (FWHM) at 638 nm with the polymer micromirror and of 1.7 μm (FWHM) at 638 nm with the silicon micromirror, comparable to reported light sheet thicknesses obtained on dedicated light sheet microscopes. A marked contrast improvement was obtained with both sample holders as compared to classic epi-fluorescence microscopy. In order to evaluate whether this technology could be made available on a larger scale, in a next step we evaluated the optical quality of inexpensive replicas from both types of master molds. We found that replicas from the polished polymer based mold have an optical quality close to that of the master component, while replicas from the silicon based mold were of slightly lower but still acceptable quality. The suitability of the replicated polymer based sample holder for single-lens light sheet microscopy was finally demonstrated by imaging breast cancer spheroids.

Light-sheet microscopy: a tutorial

“…Recently, new configurations have been developed where the same high-NA lens is used to both illuminate and image the specimen. This is the case of soSPIM [79][80][81], depicted in Fig. 5(c).…”

“…Finally, higher resolutions (NA > 1.1) can be obtained by using a conventional microscope with a single objective, which creates the LS and collects the fluorescence generated. Examples of these are the OPM technique [23,77,78] and the soSPIM [79][80][81][82] techniques that have been used for observing whole cells and cell aggregates in combination with superresolution (SR) localization microscopy.…”

“…Finally, there are applications that require mounting the sample on classical flat substrates (such as microscope cover slides or culture petri dishes). In such cases, novel LSFM schemes based on single lens [79,81] or 45°upright configuration implementations [28,73] can be used.…”

Microfluidic Based Optical Microscopes on Chip

“…So far, some miniaturized chips have fostered high and even super‐resolution light sheet microscopy. In particular, micro‐fabricated reflective components have been developed and tailored for light sheet illumination by Galland et al and Zagato et al : in these implementations a laser beam is reflected horizontally on the sample by a 45° mirror and forms the light sheet in the image plane of the detection objective of an upright or inverted microscope. The buffer solution can be further exchanged automatically by a microfluidic system coupled with the chip that incorporates the mirrors .…”

VIEW-MOD: A Versatile Illumination Engine With a Modular Optical Design for Fluorescence Microscopy

“…immersion objectives are almost precluded from LSFM applications due to the geometry configurations and the physical dimensions of the objectives. Both our research group and others have implemented LSFM in a single-objective system [22][23][24][25]; here we present how we can combine single-objective LSFM into a microscope design that allows for other FM techniques.…”