RESERVE THIS MICROSCOPE
SP8 WLL Confocal:
Confocal imaging using a super continuum, white light (WLL) pulsed laser source that allows users to specify any excitation wavelength between 470nm and 670nm. Together with an AOBS (acoustical optical beam splitter) up to 8 discrete laser lines can be selected simultaneously with a 1 nm precision. Because the WLL system is a pulsed laser it can be used in conjunction with hybrid detectors to provide gated suppression (removal) of autofluorescence and reflected light. The SP8's spectral detection system uses a prism spectrometer based emission separation (not filters) that allows user selection of emission detection bandwidth as well as spectral scanning of emission profiles. This allows users to precisely select any emission bandpass they require. Additionally, the system can scan at 8000Hz for ultra-fast scanning (and greatly reduced photobleaching) via its selectable resonant scanner. It is fully enclosed for heating and environmental control with media perfusion capabilities and CO2 control. The system is ideal for imaging live cells, tissue in culture dishes and chambered coverglass (#1.5).
SP8 gSTED:
Stimulated Emission Depletion (gSTED) is a super resolution method. The combination of pulsed WLL and hybrid detectors has allow the development of gated or gSTED. gSTED has improved resolution (< 50 nm resolution in x,y) and contrast over continuous wave (cw) STED using significantly less laser intensity than cwSTED allowing the application of STED super resolution in both fixed and living samples. The SP8-gSTED uses a 591 nm depletion laser. More info on sample prep and STED fluorophore options can be found on our super resolution page.
SP8 FLIM:
The use of a pulsed WLL source allows SP8 confocal to be used for fluorescence lifetime measures. FLIM measurements are done using the Becker Hinkl Inc., Simple-Tau 150 system that consist of an 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. Photon detection is with an external HPM-100-40 high speed hybrid PMT/ GaAsP detectors. FLIM is the best way to do FRET measurements as only the donor lifetimes need to be measured. (NOTE: FLIM hardware is shared with the upright 2P-SP5, therefore setup and use of the FLIM hardware requires coordination with CSIF staff.)
System general specifications:
DMI 6000 inverted microscope platform with motorized x,y stage with z-glavo stage
Objectives :
|
Magnification |
N. A. |
Working Distance (um) |
|
10X HC PL Apo, air |
0.40 |
2200 |
|
20X HC PL APO IMM CORR CS2, H2O/Glycerol/oil |
0.75 |
680 |
|
40X HC PL APO, CS2, oil |
1.30 |
240 |
|
40X HC PL APO W CORR CS2 water |
1.10 |
650 |
|
63X HC PL APO, CS2, oil |
1.40 |
140 |
|
63X HC PL APO W CORR CS2 water |
1.20 |
300 |
|
100X HC PL APO, CS2, oil STED obj |
1.40 |
100 |
Lasers:
WLL2 Laser; avg. power 1.5 mW: 470nm to 670 nm, 78 Mhz;
with integrated pulse picker: 78, 39, 19.5, 9.75 MHz
405nm/AOTF laser 50mW
Argon laser (FRAP, bleach), 65 mW: 458, 476, 488, 496, 514 nm
STED (depletion) laser 592nm
Leica tandem galvo/resonant scanner system:
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, 8 kHz 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 1,3x - 48x
Detection:
5 channel confocal detection: 3 Hybrid-GaAsP detectors for increased light sensitivity, 2 standard fluorescent photomultiplier tubes (PMT)
1 transmitted light detector
Software:
Leica AF: control software, Dye Finder, MicroLab, FRAP Zoomer, FRAP, FRAP-XT, FRET-AB, FRET-SE;
Huygens Professional: deconvolution software.
Funding: This microscope was funded by a NIH S10 Shared Instrumentation Grant (Dr. Margaret Fuller, PI) with significant contribution from Stanford's Beckman Center. Installation date: 11/2013
Required Acknowledgement: The project described was supported, in apart, by Award Number 1S10OD010580 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.
