Inclined selective plane illumination microscopy adaptor for conventional microscopes

Abstract

Driven by the biological sciences, there is an increased need for imaging modalities capable of live cell imaging with high spatial and temporal resolution. To achieve this goal in a comprehensive manner, three-dimensional acquisitions are necessary. Ideal features of a modern microscope system should include high imaging speed, high contrast ratio, low photo-bleaching and photo-toxicity, good resolution in a 3D context, and mosaic acquisition for large samples. Given the importance of collecting data in live sample further increases the technical challenges required to solve these issues. This work presents a practical version of a microscopy method, Selective Plane Illumination Microscopy re-introduced by Huisken et al. (Science2004,305,1007-1009). This method is gaining importance in the biomedical field, but its use is limited by difficulties associated with unconventional microscope design which employs two objectives and a particular kind of sample preparation needed to insert the sample between the objectives. Based on the selective plane illumination principle but with a design similar to the Total Internal Reflection Fluorescence microscope, Dunsby (Dunsby, Opt Express 2008,16,20306-20316) demonstrated the oblique plane microscope (OPM) using a single objective which uses conventional sample preparation protocols. However, the Dunsby instrument was not intended to be part of a commercial microscope. In this work, we describe a system with the advantages of OPM and that can be used as an adaptor to commonly used microscopes, such as IX-71 Olympus, simplifying the construction of the OPM and increasing performance of a conventional microscope. We named our design inclined selective plane illumination microscope (iSPIM).

Figure 1

Setup of iSPIM. Panel A shows the overview of the system. Laser beam is inserted into a 10x beam expander and reflected by a mirror (M) both mounted on a vertically moving component (panel B). Cylindrical lens (CL) and achromatic doublet (L1) are mounted on the “injection arm” base (panel C). The dichroic mirror (D) reflects the beam vertically into the back aperture of the objective (Obj1) (panel D). The emission light collected through Obj1 is reflected out of the body microscope into a tube containing two achromatic doublet lenses (L2). The virtual image is formed after the second objective (Obj2) and its light is collected perpendicularly with Objective three (Obj3), mounted on an objective wheel (panel E). The resulting image is focused on the camera through tube lens (L3).

Figure 2

geometrical model for excitation angle. The scheme represents an objective lens with acceptance angle θ. The light sheet, in green, has an half angle of the excitation Φ_ex. The sample in the center (black circle), has a half angle of emission Φ_em.

Figure 3

images of live MMT H2B-EGFP acquired with iSPIM with a step size of 0.5μm. The fluorescence signal derives from the nucleus. Renderings are in color, whereas reference section slices are in grayscale. Scale bars are 10μm. A–B) 98 frames stack; C) 280 frames stack; D) 361 frames stack.

Figure 4

comparison of images of live MMT H2B-EGFP acquired with confocal (A, C) and iSPIM (B, D). Scale bars are 20 μm. A and B represent the x-z view. The lateral resolution observed is comparable to confocal. C and D show the view of x-y plane. In these two panels the advantage of gray levels camera-based acquisition is noticeable. The confocal acquisition is performed using settings comparable to those used for the iSPIM system and considering that the zooming function of a confocal microscope is not applicable to a SPIM camera based system.

Figure 5

Mosaic of 9012 frames 512 × 512 pixels acquired with iSPIM. Scale bar is 100μm. Due to size issues, the dataset has been downsampled a factor of 2 to allow visualization. 4a) vertical view; 4b) inclined view with reference plane resulting from stitched frames. The fluorescence image corresponds to single cell nuclei

Figure 6

outline of volumetric acquisition through iSPIM. The image represents an objective (in black) with inclined light sheet excitation (in green) exiting the lens. The specimen (in red) placed over the objective is thus sectioned by the excitation light yielding a real volume subtended by the inclined light sheet, that is a 3D parallelogram.