fast multicolor 3 d imaging using aberration - corrected multifocus microscopy

60 | VOL.10 NO.1 | JANUARY 2013 | nature methods fluorescence microscopy using beam splitters to image up to four focal planes on separate cameras3. This method provides excellent light efficiency and a large lateral field of view corresponding to the entire surface area of the camera chip. However, refocusing by translating the camera away from the nominal focal plane induces spherical aberration after a refocus of a few microns2. It should also be noted that in the current configuration, this approach requires one camera per focal plane. Here we report an aberration-corrected multifocus microscopy (MFM) method, which produces an instant focal stack of highresolution 2D images simultaneously displayed on a single camera (Fig. 1a–c). It is based on the use of a diffractive grating to form multiple focus-shifted images4,5 and on aberration-free refocusing2. Our system was designed with the following considerations: (i) sensitivity must be optimized to minimize photobleaching and phototoxicity and to enable high-speed imaging of weakly fluorescent samples such as single fluorophores; (ii) multiple focal planes must be acquired without aberrations, especially the dominating depth-induced spherical aberration, to avoid loss in resolution and contrast; (iii) the system must be corrected for the chromatic dispersion that arises when a diffractive element is used to image non-monochromatic light. Our multifocus microscope consists of three specially designed optical elements (Fig. 1d) appended to the camera port of a standard, high-resolution epifluorescence microscope. Two relay lenses are also used, to form a secondary pupil plane (Fourier plane) and the final image plane (Fig. 1a). The diffractive multifocus grating (MFG) is placed in the Fourier plane to form the multifocus image, and it is followed by the chromatic correction grating (CCG) and prism. The MFG performs two distinct functions. First, it splits the fluorescence light emitted from the sample into separate paths, thus forming an array of N × N images of the sample on the camera. Each image corresponds to a 2D diffractive order (mx, my) of the grating. Here we describe an MFG with nine focal planes, which are formed by the central 3 × 3 diffractive orders mx, my = 0, ±1. To minimize light loss, we use a phase-only grating of fused silica, with a grating function (Fig. 1d) designed to distribute the sample emission light evenly and efficiently among the nine focal planes. At the design wavelength (515 nm), the measured efficiency of our MFG (custom made by Creative Microsystems) is ~65% with even distribution between images, as illustrated in Supplementary Figure 1. This is close fast multicolor 3d imaging using aberration-corrected multifocus microscopy