Multifocal plane microscopy (MUM), also known as multiplane microscopy or multifocus microscopy, is a form of light microscopy that allows the tracking of the 3D dynamics in live cells at high temporal and spatial resolution by simultaneously imaging different focal planes within the specimen. In this methodology, the light collected from the sample by an infinity-corrected objective lens is split into two paths. In each path the split light is focused onto a detector which is placed at a specific calibrated distance from the tube lens. In this way, each detector images a distinct plane within the sample. The first developed MUM setup was capable of imaging two distinct planes within the sample. However, the setup can be modified to image more than two planes by further splitting the light in each light path and focusing it onto detectors placed at specific calibrated distances. It has later been improved for imaging up to four distinct planes. To image a greater number of focal planes, simpler techniques based on image splitting optics have been developed. One example is by using a customized image splitting prism, which is capable of capturing up to 8 focal planes using only two cameras. Better yet, standard off-the-shelf partial beamsplitters can be used to construct a so-called z-splitter prism that allows simultaneous imaging of 9 individual focal planes using a single camera. Another technique called multifocus microscopy (MFM) uses diffractive Fourier optics to image up to 25 focal planes.
Introduction Fluorescence microscopy of live cells represents a major tool in the study of trafficking events. The conventional microscope design is well adapted to image fast cellular dynamics in two dimensions, i.e., in the plane of focus. However, cells are three-dimensional objects and intracellular trafficking pathways are typically not constrained to one focal plane. If the dynamics are not constrained to one focal plane, the conventional single plane microscopy technology is inadequate for detailed studies of fast intracellular dynamics in three dimensions. Classical approaches based on changing the focal plane are often not effective in such situations since the focusing devices are relatively slow in comparison to many of the intracellular dynamics. In addition, the focal plane may frequently be at the wrong place at the wrong time, thereby missing important aspects of the dynamic events.
Implementation MUM can be implemented in any standard light microscope. An example implementation in a Zeiss microscope is as follows. A Zeiss dual-video adaptor is first attached to the side port of a Zeiss Axiovert 200 microscope. Two Zeiss dual-video adaptors are then concatenated by attaching each of them to the output ports of the first Zeiss video adaptor. To each of the concatenated video adaptor, a high resolution CCD camera is attached by using C-mount/spacer rings and a custom-machined camera coupling adaptor. The spacing between the output port of the video adaptor and the camera is different for each camera, which results in the cameras imaging distinct focal planes. It is worth mentioning that there are many ways to implement MUM. The mentioned implementation offers several advantages such as flexibility, ease of installation and maintenance, and adjustability for different configurations. Additionally, for a number of applications it is important to be able to acquire images in different colors at different exposure times. For example, to visualize exocytosis in TIRFM, very fast acquisition is necessary. However, to image a fluorescently labeled stationary organelle in the cell, low excitation is necessary to avoid photobleaching and as a result the acquisition has to be relatively slow. In this regard, the above implementation offers great flexibility, since different cameras can be used to acquire images in different channels.
3D super-resolution imaging and single molecule tracking using MUM
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