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Marsico
Hall Microscopes Folk
(MHMF.ORG) |
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CFC
Confocal Proposal
Leica Stellaris 5 WLL - Is an inverted confocal microscope platform for
fixed and live cell/tissue imaging with environmental control using a large
environmental box
User Information
Page - http://microscopy.unc.edu/resources/leicastellaris5
Specifications:
- Inverted body
- Software controlled motorised x-y stage
- Fast Z-galvo for slides, 35 mm petri - x-z mode - microtitre plates
- Standard focus for microtitre plates - encoded focus drive
- Automatic focus - 850 nm total internal reflection based
- Laser excitation
- Diode 405nm
-
White light laser
with output at all wavelengths from 485nm (blue) to 685 nm (far red) with
band selections carried
out with an AOTF (acoustical tunable optical filter)
- 5x HyD S detectors (hybrid photo multiplier tubes
HyD S)
- 410 to 850 nm with spectral discrimination and fluorescence
lifetime gating (485 to 685 nm) -
- Sensitivity 410 - 850 nm pde* 60%
480, 40% 610 nm, 30% 700nm, 20% 800 nm and flat
- HyD S normally use analogue mode with PMT voltage (gain) control
or Photon counting mode or lifetime discrimination
mode (TauSense) [not FLIM]
- AOBS electronic dichroic
-
Whole
stage and microscope upper body incubator -
Okolab enclosure
- This system accommodates the fast z-galvo for glass bottom
petri dishes, microchamber slides, standard 1" x 3" slides
- Temperature control from room+3C to 45C
- CO2 control 0 - 10% - uses 100% CO2 supply and mixes with air
- Can do controlled anoxia (0 to 10% O2)
- Active humidity control
- Samples on slides, 35 mm glass bottom dishes, multichamber coverslips, uTitre
plates 12, 24, 96 well with metal heat conducting plate
- LAS X software
- Live data modes - FRET, FLIP, time lapse, mark and scan, etc.
Manual setup with parameters saved, not wizards. FRAP at 405 nm.
FRAP at other wavelengths may be possible
- Lightning Expert software - Deconvolution and noise reduction and other
features
- Colocalization
- Mark and find tile scan
- Dye unmixing
- Tau gating - removes reflections, can reduce autofluorescence
- Objectives:
- 5x 0.15NA wd ~17 mm?
- 10x 0.4NA, wd ~3.6 mm
- 20x 0.75NA wd 660 um
- 40x 1.25NA glycerol wd 350um DIC
- 63x 1.4NA oil wd 140um DIC
- spare position
- Typical Scanning Task:
- Multichannel - DAPI, FITC, CY2 to CY7, Alexa 405, Alexa 488 to Alexa
680, Opal 520 to 690, dye groups references -
CYs, Opals, Alexas - Of peak excitation of Alexa 700 to 790 may be
possible
- Z-series, 3-D, timelapse, line scans
- Fast x-z scanning, e.g. for ASL imaging. rapid high accuracy
z-series
- FRAP (405 nm)/FRET/FLIP
- Slides, 35 mm petri, 4 or 8 or etc. multi-chamber slides and
coverslips
- Time gated detection - can remove reflections or autofluorescence,
notch filters
- Monochrome Transmitted light, DIC, fluorescence, reflection
- ASL from slides or petri dishes at 37C, 5% CO2, active humidity
control
- Tile scanning / stitching / montaging
- Perfusion based multimode, e.g. Saber technique, should be possible with minor
additions to the system.
Does not have:
- White light laser is sufficiently intense enough for FRAP - and can use
wide excitation bandwidth in order to bleach more strongly. Strong photo
conversion/activation/bleaching can be done with 405 nm. Additional standard
laser(s) and merge module can be filed retrofitted.
- No resonance scanner - can retrofit ~$55K. Don't think a high speed
resonance scanner is really needed,
but may be good for very brightly labeled CBF
- Excitation for a larger range of colors 440 nm to 790 nm is
available on the next model up, the Stellaris
8, and adds ~$220K. Pricey! Will 690
to 790 nm excitation be used? Can not retrofit. $27.5K/yr
maintenance contract.
- No CFP - cyan fluorescence protein excitation at 440 nm is not included -
can retrofit a single wavelength c.w. laser ~$8K or get the ~$220K extra Stellaris
8
- No wizards for FRET/FRAP/FLIP/timelapse - setup manually
- Annual service & software maintenance contract is $26K/yr
for Stellaris 5 -
Needed and best to prepay upfront with initial purchase P.O. and get 20% discount.
Think can prepay in 1st too and get discount. 1st year maintenance is
included with purchase.
- 40x long working distance to image through 1 mm plastic (have an old one
available but only good for 440 to 700 nm i.e. ~DAPI to CY5)
- 20x long working distance to image through 1 mm plastic (have an old one
available but only good for ~440 to ~700 nm i.e. ~DAPI to CY5)
- 16x 0.5 multi immersion lens wd 150 um (have an old one available
but only good for ~440 to ~700 nm i.e. ~DAPI to CY5)
- Can't do Fura-2 or Quinn-1 - best done with widefield anyway
- No demonstration system which we can visit. The Stellaris model 5 &
8 are a 2nd generation white light laser system. Release date was May 2020.
An installed system is at Atlanta in a locked down covid-19 clinical research division.
The previous model, SP8, is no longer
available and has a more limited excitation and detection ranges.
- Stellaris 5 can not do proper FLIM and can not be upgraded for proper
time resolved FLIM. FLIM is interesting, useful, but difficult. A Stellaris 8 is
required for FLIM. I do not think its
worth while given the small necessity for it. There is an Leica SP8 FLIM system
in the Neuro Science Microscopy Core if it is ever needed.
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* = Photon Detection Efficiency (A measurement analogous the Quantum
Efficiency applied to photomultiplier tubes which includes geometric corrections
and, amazingly, Cherenkov radiation)
Super continuum laser
