Leica SP5 upright Confocal, multi-photon, FLIM

RESERVE THIS MICROSCOPE

The microscope is coupled with a Spectra Physics MaiTai, DeepSee ultrafast pulsed laser system for multi-photon excitation deep within tissue. It is fully enclosed for heating and environmental control with media perfusion capabilities and CO2 control. For high resolution, deep tissue imaging the SP5 is also equipped with the Leica HCX APO L 20x NA 1.0 water immersion objective offering a large field with high resolution. Additionally, the system can scan at 8000Hz for ultra-fast scanning (and greatly reduced photobleaching) via its selectable resonant scanner. The SP5 is equipped with a prism spectrometer detector which allows selection of emission detection bandwidth as well as for spectral scanning of emission profiles. The SP5 is also equipped with an optional 6-position motorized objective turret that holds a standard set of air and oil objectives for confocal imaging of samples. These features make the Leica SP5 MP/confocal a highly sensitive, flexible 2-photon imaging system for thick, live tissue as well as routine confocal imaging of fixed samples. It is not recommended for imaging tissue culture in multiwell plates or chambered coverslips due to the upright configuration that requires 'dipping' the lens into the media for live cell imaging (see Leica SP8 in SOM Beckman CSIF or Zeiss LS780 2-photon and Nikon spinning disk confocal in SOE Shriram CSIF for imaging cells in multi well plates).

SP5 FLIM:The pulsed ultra-fast MaiTai laser allows SP5 confocal to be used for fluorescence lifetime measures. FLIM measurements are done using the Becker Hinkl Inc., Simple-Tau 150 system (http://www.beckerhickl.com/simpletau152.htm) with a single ultra-fast Time Correlated Single Photon Counting TCSPC channel. These electronics measure the time between the laser pulse and the arrival of photons at the detector; this constitutes the basis of the FLIM measurement. FLIM is the best way to do FRET measurements as only the donor lifetimes need to be measured. (NOTE:use of the FLIM hardware requires coordination with CSIF staff, two channel FLIM can be done in consultation with CSIF staff).

System specifications:

DM6000 upright microscope platform with motorized x,y stage with z-glavo stage

Objectives :

lasers:

1p Lasers: HeNe 633nm/10mW, HeNe 594nm/2.5mW, HeNe 543/1.0 mW, Ar 458nm/5mW, 476nm/5mW, 488nm/20mW, 514nm/20mW

2P Laser: Spectra Physics 15W MaiTai DeepSee Tuning range 690-1040 nm

Leica tandem scanner system: variable speed galvo/resonant scanner single device X2Y-Scanner design for high frame rate. Variable Scanner 1 - 1400 Hz Line frequency max: 2800 Hz 5 Images/second, 512 x 512 ~54 Images/second, 512 x 16 Max. Image resolution 8192 x 8192 Line frequency up to 2800 Lines/second. Resonant scanner, 8000 Hz X2Y-Scanner for high frame rate: 28 Images/second, 512 x 512 ~290 Images/second, 512 x 16 Max. Image resolution 1024 x 1024 Line frequency up to 16000 Lines/second Very big scanfield with 12,52 mm (SFZ) diagonal in the Intermediate image plan Hardware zoom, stepless 1,7x - 64x

Detection:

4 channel descanned/confocal detection: 2 descanned Hybrid-GaAsP detectors for increased light sensitivity, 2 standard fluorescent photomultiplier tubes (PMT)

3 channel non-descannced detection (NDD): 2 reflected, 1 transmitted/SHG for multi-photon imaging 

1 Dodt contrast, transmitted light detector

FLIM hardware and filters:

Becker and Hinkl Simple Tau 150 with HPM-100-40 high speed hybrid detector

mTFP-Venus FRET cube:

mTFP filter 475/28 BP ( donor emission: 461-489nm) , dichroic is BS506 and the Venus filter is a560/25 BP (acceptor emission 557-572)

Standard Cube:

510/42 BP (emission 489-531), dichroic is BS555 , 607/36 BP (emission 589-625).

Funding:This microscope was funded by a NIH Shared Instrumentation grant (Dr. Stuart Kim, PI). Installation date: 02/2010.

Required Acknowledgement:The following acknowledgement is required when publishing data obtained with this instrument:"The project described was supported, in part, by Award Number S10RR02557401 from the National Center for Research Resources (NCRR). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or the National Institutes of Health.