Multifocal plane microscopy

[5] In each path the split light is focused onto a detector which is placed at a specific calibrated distance from the tube lens.

[8] 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.

[9][10] Another technique called multifocus microscopy (MFM) uses diffractive Fourier optics to image up to 25 focal planes.

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.

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.

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.

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.

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 particle tracking.

In single 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.