Visualizing the development or response to stimuli of live, whole organisms at cellular resolution with fluorescence microscopy is critical in answering key biological questions. Over the last few years, selective plane illumination microscopy (SPIM) has emerged as an optical sectioning technique particularly well-suited for volumetric fluorescence imaging of whole organisms over an extended period of time with high temporal resolution. In SPIM, a sheet of excitation light passes through the specimen and is coincident with the focal plane of a detection objective. Scanning this light sheet in synch with the focal plane of the detection objective relative to the specimen provides volumetric imaging wherein portions of the specimen not in the focal plane receive no excitation light. Therefore, limited phototoxicity and photobleaching occur as compared to confocal or wide-field imaging methods.
In a common implementation of SPIM, the illumination and detection objectives are placed orthogonally on two sides of a liquid-filled chamber above a platform on which the chamber is placed. The specimen is held within a cylinder of gel that is vertically positioned within the chamber and that can be rotated and/or translated. Both objectives focus on the specimen either through glass windows on the sides of the chamber or with water-dipping objectives that enter the chamber. Other arrangements for SPIM enable the use of horizontally mounted specimens. In such cases, the illumination and detection optical axes are positioned above the platform and water-dipping objectives are immersed into the specimen chamber from above.
Although the above-described SPIM systems are compatible with some conventional specimen mounting protocols, they do not enable the use of commercially available multiwell plates or specimens held within microfluidic chambers. Furthermore, the benefits of using inverted microscopes, such as having easy access to the specimen from above and being able to quickly exchange specimens without adjusting the optics, are unavailable with these setups. It can therefore be appreciated that it would be desirable to have a SPIM system and method that enable the use of conventional specimen holders and possesses the benefits of inverted microscopy.