Figure 1.

(a) Detection path of the three-color multiplane fluorescence microscope. An adjustable slit aperture crops the light at the microscope’s side-port, thus defining the field of view. Lenses L₁ and L₂ comprise a 4f optics with magnification 1.33 that relays the fluorescence light to the sCMOS cameras 1 and 2. Between the lenses, the light sequentially passes two dichroic mirrors. The resulting three color channels of green (λ < 580 nm), orange (580 < λ < 624 nm), and red (λ > 624 nm) are further filtered with emission band-pass filters G (513/17 nm), O (593/40 nm), and R (692/40 nm), respectively. An off-axis tilt of the dichroic mirrors deflects the propagation direction of the green and deep red color away from the horizontal plane. Next, the multiplane prism generates eight images with uniformly increasing optical path length, corresponding to eight distinct focal planes inside the sample. (b) Raw eight-plane three-color images of a fixed fluorescently labeled COS-7 cell. Different focal planes are shown along the horizontal direction, different colors along the vertical direction. The different colors correspond to labeled vimentin, actin, and mitochondria with the corresponding peak emission wavelengths at 520, 593, and 692 nm, respectively. The numbers above the images show the axial order of the image planes (one is closest to sample surface, eight is farthest).