Fast four-dimensional fluorescence microscopy

Despite cellular architecture being inherently three dimensional (3D), almost all subcellular particle tracking studies are performed in 2D. The reason for this is due to the speed at which current microscope technologies are able to acquire 3D volumetric images. Conventional 3D imaging is performed by moving the sample in small increments through the focal plane of the microscope, and a 2D snapshot is taken at each step, creating a through-focal series known as a ‘z-stack’. The imaging speed is therefore limited by both the mechanical movement of the sample stage and the camera exposure time. Over the past decade, our lab has been developing methods which allow multiple focal planes (3 or 9) to be imaged simultaneously onto a single camera chip, using diffractive optical elements. This removes the need for mechanical movement of the sample, thus considerably improving the image acquisition times. However, because there is a wavelength dependence of the diffraction of light, chromatic aberrations are caused in the multi-focal images which drastically reduce the overall resolution. In this presentation, I will present our most recent work for compensating for these aberrations, and show how it enables high speed 4D (3D+t) microscopy.