User:Papain exclio/Multifocal plane microscopy

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.

Multifocal plane microscopy (MUM) is a microscope imaging modality that overcomes these problems and allows for 3D sub-cellular trafficking studies within a live cell environment. 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. Prabhat, P., Ram, S., Ward, E. S. and Ober, R. J. (2004) “Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimension”, IEEE Transactions on Nanobioscience, 3, 237-242. (First report of 3D subcellular tracking with MUM in live cells)

2. Prabhat, P., Ram, S., Ward, E. S. and Ober, R. J. (2006) “Simultaneous imaging of several focal planes in fluorescence microscopy for the study of cellular dynamics in 3D”, Proceedings of the SPIE, 6090, 115-121.

3. Prabhat, P., Gan, Z., Chao, J., Ram, S., Vaccaro, C., Gibbons, S., Ober, R. J. and Ward, E. S. (2007) “Elucidation of intracellular recycling pathways leading to exocytosis of the Fc receptor, FcRn, by using multifocal plane microscopy”, Proceedings of the National Academy of Sciences, USA, 104, 5889-5894. (First report of 3D single molecule tracking of QD labeled IgGs in live cells)

4. Ram, S., Ward, E. S., and Ober, R. J. “How accurately can a single molecule be localized in three dimensions using a fluorescence microscopy?” Proceedings of the SPIE, 5699: 426-435, 2005. (Reports the quantitative analysis of the poor depth discrimination capability of conventional optical microscopes) 5. Ram, S., Chao, J., Prabhat, P., Ward, E. S., and Ober, R. J. “A novel approach to determining the three-dimensional location of microscopic objects with applications to 3D particle tracking”, Proceedings of the SPIE, 6443: 6443-0C, 2007 (First report of the improved depth discrimination capability of MUM)

6. Ram, S., Prabhat, P., Chao, J., Ward, E. S., and Ober, R. J. (2008) “High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells”, Biophysical Journal, 95, 6025-6043. (Reports the implementation and validation of The MUM localization algorithm MUMLA, and a detailed analysis of depth discrimination problem.) (The paper also reports 3D single molecule tracking of QD labeled IgGs undergoing endocytosis in live cells.)

7. Chao, J., Ram, S., Abraham, A., Ward, E. S., and Ober, R. J (2008). “Resolution in 3D in multifocal plane microscopy”. Proceedings of the SPIE, 6861: 68610Q, 2008. (First report discussing the 3D resolving power of MUM)

8. Chao, J., Ram, S., Ward, E. S., and Ober R. J. “3D resolution measure for multifocal plane microscopy”, Proceedings of the 2008 IEEE International Symposium on Biomedical Imaging: From Nano to Macro, 1339-1342. (Report contains additional results on 3D resolution of MUM)

9. Chao, J., Ram, S., Abraham, A., Ward, E. S., and Ober, R. J. (2009) “A resolution measure for three - dimensional microscopy”, Optics Communications, 282: 1751-1761. (Report contains a detailed analysis regarding quantifying the 3D resolution of optical microscopes)

10. Ram, S., Prabhat, P., Ward, E. S., and Ober, R. J. “Improved single particle localization accuracy with dual objective multifocal plane microscopy”, Optics Express, 17, 6881-6898, 2009. (First report of dual objective MUM imaging configuration and its applications to high accuracy 2D/3D single molecule localization)