User:MicroscopyMeister/Multifocal plane microscopy

Multifocal plane microscopy is

Introduction
Fluorescence microscopy of live cells represents a major tool in the study of trafficking events. The current microscope design is well adapted to imaging 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 currently available 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.

To address the above limitations, a new imaging modality called multifocal plane microscopy (MUM) was developed by the Ward-Ober group that enabled 3D tracking of sub-cellular structures (e.g., vesicles) within a live cell environment [1,2]. In MUM, the sample is simultaneously imaged at distinct focal planes (Fig. 1), which enables the visualization of intracellular events that occur at different focal planes [1,2,3]. MUM has led to the discovery of new intracellular trafficking pathways, which otherwise would not have been possible with conventional microscopy techniques [3].

Recent technological advances have led to the advent of single molecule microscopy, which is an ultra sensitive optical microscopy technique that probes the behavior of individual molecules even within a live-cell environment. In biological applications, an important question arises as to whether single molecules can be tracked in 3D and how accurately can this be done. A major shortcoming of conventional optical microscopes is their depth discrimination capability [4]. As a result, it becomes highly problematic to accurately determine the z-location of a single molecule, especially when it is close to the plane of focus [4-5]. Thus this makes 3D single molecule tracking problematic with conventional micrscopes.

The identical problem arises in super-resolution microscopy approaches, such as PALM/STORM/FPALM/dSTORM etc., where also the 3D location of a single molecule needs to be estimated with the best possible accuracy. Here an important aspect of MUM arises in that it overcomes a severe problem of 3D location estimation in conventional microscopy, i.e. the depth discrimination problem (see below). This is in addition to MUM being able to image samples over a larger depth in three dimensions than conventional microscopy can do. Thus MUM supports 3D single molecule tracking [3,5-7] as well as 3D super-resolution [8-11] imaging over large depths.

Principle behind MUM


Figure 1 shows a schematic illustrating the principle behind MUM. 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 MUM setup shown in Fig. 1 is capable of imaging two distinct planes within the sample. 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. Presently, we have implemented a MUM setup that can image up to four distinct planes, the details of which are given below.

Implementation of MUM
MUM can be implemented in any standard optical microscope. Here, the details of the implementation in a Zeiss microscope are given (Figure 2); for additional details, please also see [1,3,6]. A Zeiss dual-video adaptor (Part # 1058640000) was first attached to the side port of a Zeiss Axiovert 200 microscope. Two Zeiss dual-video adaptors were 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 should be pointed out that there are many ways to implement MUM (e.g., see also [7,12]). The above implementation offers several advantages such as flexibility, ease of installation and maintenance, and adjustability for different configurations. Specifically, the above implementation has the advantage that it can be implemented with essentially off the shelf components and standard filters and dichroics can be used in the Zeiss dual-video adapter. For a number of applications it is important to be able to acquire images in different colors and at different exposure times. For example to visualize exocytosis in total internal reflection illumination mode, 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; see [1,3]. 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 in MUM


Modern microscopy techniques have generated significant interest in studying cellular processes at the single molecule level. Single molecule experiments overcome averaging effects and therefore provide information that is not accessible using conventional bulk studies. However, the 3D localization and tracking of single molecules poses several challenges. In addition to whether or not images of the single molecule can be captured while it undergoes potentially highly complex 3D dynamics, the question arises whether or not the 3D location of the single molecule can be determined and how accurately this can be done.

A major obstacle to high accuracy 3D location estimation is the poor depth discrimination of a standard microscope [4]. Even with a high numerical aperture objective, the image of a point source in a conventional microscope does not change appreciably if the point source is moved several hundred nanometers from its focus position (Fig. 3a). This makes it extraordinarily difficult to determine the axial, i.e., z position, of the point source with a conventional microscope.

More generally, quantitative single molecule microscopy for 3D samples poses the identical problem whether the application is localization\tracking or super-resolution imaging such as PALM/STORM/FPALM/dSTORM for 3D applications, i.e. the determination of the location of a single molecule in three dimensions. Here MUM offers several advantages [4-6,10]. In MUM, images of the point source are simultaneously acquired at different focus levels. These images give additional information that can be used to constrain the z position of the point source (Fig. 3b). This constraining information largely overcomes the depth discrimination problem near the focus [5, 6].

Figure 3c shows the 3D localization measure of z0 for a conventional microscope and for a two plane MUM setup with a focal plane spacing of 500 nm. The 3D localization measure provides a quantitative measure of how accurately the location of the point source can be determined (see Refs. 4-6 for details). A small numerical value of the 3D localization measure implies very high accuracy in determining the location, while a large numerical value of the 3D localization measure implies very poor accuracy in determining the location. For a conventional microscope when the point source is close to the plane of focus, e.g., z0 < 250 nm, the 3D localization measure predicts very poor accuracy in estimating the z position. For example, for z0 = 250 nm, the 3D localization measure predicts an accuracy of 31.79 nm and for z0 = 5 nm, the 3D localization measure predicts an accuracy of at least 150 nm, when 2000 photons are collected from the single molecule. Thus, in a conventional microscope, it is problematic to carry out 3D tracking when the point source is close to the plane of focus. On the other hand, for a two plane MUM setup the 3D localization measure predicts consistently better accuracy than a conventional microscope for a range of z-values, especially when the point source is close to the plane of focus. For example, for z-values in the range of 0–250 nm, the 3D localization measure of z0 predicts an accuracy of 20–25nm in determining the z position when 1000 photons are collected from the single molecule at each focal plane. An immediate implication of this result is that the z-location of the point source can be determined with relatively the same level of accuracy for a range of z-values, which is favorable for 3D single molecule tracking [5,6].

3D resolution and MUM
The resolution of an optical system is a measure of the system’s ability to distinguish two closely spaced point sources. Classical resolution criteria such as the Rayleigh’s criterion, although extensively used, are well known to be based on heuristic that are not well suited for modern imaging approaches. Recently, we introduced an information theoretic resolution measure for 3D optical microscopy [7-9]. Using this, we have quantified the resolving power of MUM and compared it to that of a conventional optical microscope. Our results show that in many practical conditions, MUM provides significantly improved 3D resolvability of closely spaced point sources when compared to a conventional optical microscope.

Dual objective multifocal plane microscopy


In single molecule/particle imaging applications, the number of photons detected from the fluorescent label plays a crucial role in the quantitative analysis of the acquired data. Currently, particle tracking experiments are typically carried out on either an inverted or an upright microscope, in which a single objective lens illuminates the sample and also collects the fluorescence signal from it. Note that although fluorescence emission from the sample occurs in all directions (i.e., above and below the sample), the use of a single objective lens in these microscope configurations results in collecting light from only one side of the sample (see Fig. 4a). Even if a high numerical aperture objective lens is used, not all photons emitted at one side of the sample can be collected due to the finite collection angle of the objective lens. Thus even under the best imaging conditions conventional microscopes collect only a fraction of the photons emitted from the sample.

To address this problem, a new microscope configuration known as dual objective MUM has been developed that uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position [10]; see Fig. 4b. The dMUM setup has higher photon collection efficiency when compared to a standard microscope [10]. Further, fluorescent point sources imaged in dMUM can be localized with better accuracy in 2D and 3D than when imaged through a standard microscope or MUM [10].