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| Joël Beaudouin (left) and Jan Ellenberg (right) working on the LSM 510 system. A prophase cell labeled with histone-CFP and lamin-YFP can be seen on the computer screen. |
We use a custom built LSM510 (Carl Zeiss, Göttingen, Germany) confocal laser scanning microscope to image and photobleach living cells during cell division as well as in interphase.
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| Joël Beaudouin (left) and Jan Ellenberg (right) working on the LSM 510 system. A prophase cell labeled with histone-CFP and lamin-YFP can be seen on the computer screen. |
Microscope body and stage: Axiovert 100M microscope (Zeiss) equipped with a fast z-scanning stage (HRZ 200, to acquire rapid z-stacks) and an xy-scanning stage (for multiregion "tile" imaging and multilocation time-lapse imaging). In addition the microscope is equipped with a remote control to switch filter cubes for viewing, control focus and switch objectives (to operating the microscope without the software interface).
Objectives: PlanApochromat 63x DIC oil 1.4 NA (our workhorse for high resolution imaging of flat cells, i.e. fibroblasts). PlanApochromat 100x DIC oil 1.4 NA and alpha PlanFluar 100x 1.45 NA (best resolution of small objects such as nuclear pores in flat cells). C-Apochromat 40x and 63x water 1.2 NA (high resolution imaging of thick cells, i.e. starfish oocytes). Fluar 40x oil NA 1.3 (bright lens for single color fluorescence, beware of chromatic aberrations). PlanNeofluar 25x Immersion Corrected water, glycerol, oil 0.8 NA (for bleaching with large focal depth).
Heating: Cells are maintained at 37°C on the stage by an air stream incubator (ASI 400, Nevtek, Burnsville, VA) in conjunction with an objective heater (Bioptechs, Butler, PA).
Lasers: 80 mW Kr Ion laser (Coherent Lasers; 413 nm; strong for fast photobleaching). 100 mW Ar Ion laser (Spectra Physics; 458, 488, 514 nm; strong for fast photobleaching even with minor lines). 1 mW HeNe lasers (Spectra; 543, 633 nm, weak, good for imaging but useless for bleaching).
Scanhead: Custom designed dichroics and emission filters (Chroma Inc., Brattleboro, VT and Carl Zeiss) to discriminate CFP, GFP, YFP, DsRed in addition to traditional dyes in live cells with minimal crosstalk and maximum brightness at high speeds. Hand selected PMTs for better sensitivity.
Application examples:Gerlich, D., J. Beaudouin, M. Gebhard, J. Ellenberg, and R. Eils. 2001. 4-D Imaging and Quantitative Reconstruction to Analyze Complex Spatiotemporal Processes in Live Cells. Nature Cell Biol.3:852-855.
Daigle, N., J. Beaudouin, L. Hartnell, G. Imreh, E. Hallberg, J. Lippincott-Schwartz, and J. Ellenberg. 2001. Nuclear pore complexes form immobile networks and have a very low turnover in live mammalian cells. J Cell Biol. 154:71-84.
Belgareh, N., G. Rabut, S.W. Bai, M. van Overbeek, J. Beaudouin, N. Daigle, O. Zatespina, F. Pasteau, V. Labas, M. Fromont-Racine, J. Ellenberg, and V. Doye. 2001. An evolutionarily-conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells. J Cell Biol. 154:1147-1160.
Beaudouin, J., D. Gerlich, N. Daigle, R. Eils and J. Ellenberg. 2002. Nuclear Envelope Breakdown Proceeds by Microtubule-Induced Tearing of the Lamina. Cell. 108:83-96.
Gene Expression and Cell Biology/Biophysics Programmes, Location