RESERVE THIS MICROSCOPE
The microscope is configured for simultaneous, two channel fast imaging with added capabilities for ROI photo-activation, FRAP, FRET and TIRF. This imaging system is specialized for fast imaging of live cells.
System specifications:
- Nikon TiE inverted microscope with Perfect Focus (PFS) mechanism
- Motorized XY Stage, allowing large images and multi-point time-lapse imaging
- Yokogawa CSU-X1 (1,800 rpm, 360 fps)/Borealis Upgrade/405/488/561/642 multichroic
- fast piezo Z stage (100 um travel, 15 ms step response)
- Nikon motorized TIRF illuminator
- WideField Imaging with large field of view (2560x2160 pixels / 16.6x14.0 mm)
- InVivo Scientific enclosure w/ environmental control (temperature and CO2)
- High Content Screening and Throughput using the JOBS/HCS module in Elements; allowed to apply automatic screening of multi-wells plates / Tracking of object over time using the xy stage / Phenotypic screening etc.
Software:
- NIS-Elements
NIS-Elements free viewer download page here:
http://www.nis-elements.com/resources-downloads.html
Objectives :
|
Name |
Magnification |
NA |
Immersion |
working distance |
|
PLAN APO |
4x |
0.20 |
air |
20 mm |
|
PLAN APO |
20x |
0.75 |
air |
1.00 mm |
|
PLAN APO |
40x |
1.15 |
water |
0.61 mm |
|
PLAN APO IR |
60x |
1.27 |
water |
0.17 mm |
|
PLAN APO |
60x |
1.40 |
oil |
0.13 mm |
|
PLAN APO-TIRF |
100x |
1.49 |
oil |
0.12 mm |
Confocal lasers:
- 405nm (100 mW)
- 488nm (50 mW)
- 561nm (50 mW)
- 642nm (100 mW)
Widefield illuminator:
- Lumencor SPECTRA III (LED)
- 390/22nm (500 mW)
- 440/20nm (500 mW)
- 475/28nm (500 mW)
- 510/25nm (400 mW)
- 555/28nm (500 mW)
- 575/25nm (500 mW)
- 637/12nm (500mW)
- 748/12nm (500mW)
fluorescence emission filter: |
||||
|
Description
|
||||
|
DAPI |
387/11 |
|||
|
CFP |
430/24 |
|||
|
Alexa488 |
490/20 |
|||
|
YFP |
500/20 |
|||
|
Alexa555 |
555/25 |
|||
|
CY5 |
645/30 |
|||
|
GFP/mCherry |
"GFP" 470/40, "mCherry" 573/35 |
|||
|
FURA2-C |
340/380 |
|||
|
Cy7 |
809/81 |
|||
Detection:
- Photometrics Prime94B sCMOS camera for the Spinning Disk
- Andor Neo sCMOS camera for the wide field and the TIRF
Funding:
This microscope was funded by jointly by the SOE and the Beckman Center (Dr. Alex Dunn and Jon Mulholland, PIs). Installation date: 09/2012
What to put in Material and Methods section (example for SDC):
Cells were cultivated in tissue culture dish with No. 1.5 cover glass bottom (FluoroDish) and mounted in an environmental chamber (in vivo scientific) for acquisition at 37°C and 5% CO2. DMEM media with 25mM Hepes (pH 7.2) and without phenol red was used during image acquisition, with a layer of mineral oil on top of the media to prevent evaporation. All image were collected with a Yokogawa spinning disk confocal on a Nikon Eclipse-TI inverted microscope (Nikon) equipped with a PLAN APO-TIRF 100X 1.49 N.A. oil immersion objective and the Perfect Focus System for continuous maintenance of focus. XX-EGFP fluorescence was excited with the 488nm line from a 50mW Cobolt Blues laser and collected with a quadruple band pass dichroic mirror (Semrock) and a 525/30 emission filter (Semrock). Images were acquired with a Photometrics Prime95B sCMOS camera controlled with NiS-Elements software. For timelapse experiments, images were collected every 1 min, using an exposure time of 500 ms and 2x2 binning, with illumination light shuttered between acquisitions. At each time point, 6 z-series optical sections were collected with a step size of 0.5 microns, using a piezo Z-axis stage (Mad City Labs). ZGamma, brightness, and contrast were adjusted on displayed images (identically for compared image sets) using NIS-Elements software.
