Leica Stellaris with white light & MaiTai lasers
ACLSW 421  Badge Access Request Here

Leica Stellaris 8 Falcon laser scanning confocal microscope on inverted DMi8 CS stand with Super Z Galvo and resonant scanner, giving complete spectral freedom to choose probe wavelengths from 440 to 790 nm with a tunable third-generation white light laser, a 405 nm laser, four sensitive HyD detectors, fluorescence lifetime (FLIM) capability and imaging across the spectrum from 450 to 850 nm. Optics will accommodate an existing MaiTai DeepSee laser for second harmonics imaging. Objective lenses from 10X to 63X, an Okolab environmental chamber for use with live cells and tissues, and workstation and Stellaris modules for complex instrument operation, post-processing, and analysis included.

The Stellaris was purchased to fulfill a wide range of confocal based imaging needs. Of course, this is an exceptional instrument for standard confocal, but this microscope has unique features, such as time domain fluorescence lifetime (FLIM) to complement the lower resolution frequency domain Toggel FLIM system.

Why FLIM?

  • FRET sensors imaged quickly and with high spatial and temporal accuracy
  • Separate dyes with the same spectral characterists for additional multiplexing
  • Vital dyes may have different lifetimes when incorportaed into different organelles
  • Has promise to characterize or identify different cells or tissue types
                     Read more in Leica PDFs on Falcon and TauSense

Consider using the resonant scanner for faster image collection, especially when tiling large areas with Z series.

For excitation of fluorescence, any wavelengths may be chosen from 440 nm up to 1040 nm. From 440 to 790 nm, up to eight laser lines maybe chosen simultaneously and a MaiTai DeepSee may be used for a single wavelength in the range 760 to 1040 nm for second harmonics or 2 Photon using internal detectors with the pinhole open. This microscope does image bright 2P signal, but for most intravital imaging, we continue to recommend the Olympus FVMPE-RS. There is also a standard 405 nm diode laser.

Scanning is standard galvos or faster resonant. (This system does not allow for simultaneous use of galvos and resonant scanner, such as for highly localized photoactivation with galvos while scanning full field resonant.)


Signup to use microscope. Projects requiring unique functions of this microscope have priority. Please use other confocals for standard confocal work.


Instructions: 

At this time, there is no iLab Kiosk required to startup. However, it will likely be implemented. [Word doc]   [PDF]

 

 

A few of the specifications:

  • Antivibration table: Newport ST Series with autotune damping and I2000 series legs 1500 mm X 1800 mm X 203 mm (71 X 59 X 8 inches), 805 lbs not including the legs.

Lenses -- We chose these lenses for specfic reasons; click here for explanations.

  • 15506424 Obj. HC PL APO 10x/0.40 CS2 air
  • 11506517 Obj. HC PL APO 20x/0.75 CS2 air
  • 15506533 HC FLUOTAR L 16x/0.6 IMM CORR VISIR multi-immersion, usually set to glycerol
  • 15506375 Obj. HC FLUOTAR L 25x/0.95 W VISIR 0.17 water, a.k.a. Genteal
  • 15506425 Obj. HC PL APO 40x/1.10 W CORR CS2 water, a.k.a. Genteal
    15640019 Water Immersion Micro Dispenser
    for long term multifield imaging. This is for very long timelapse or for traversing full plates with full heat and CO2 such that water is continuously applied.
  • 15506350 Obj. HC PL APO 63x/1.40 CS2 oil
  • Not on turret, by request:
    15506361 Obj. HC PL APO 63x/1.20 W motCORR CS2

Four detectors for low light plus tramsmitted light detector.
Note that the detector in position S2 should be used for situations where the signal is bright.
The other detectors may be damaged by saturation; this one is safer.

  • Power HyD X SP pos 2 FLIM
  • Power HyD S SP Core Unit Pos 3 Si photocathode - use for reflection and second harmonics
  • Power HyD X SP pos 4 FLIM
  • Power HyD R SP pos 5 FLIM and reds out to 800+ nm
  • BF (brightfield) detector for transmitted light

 

Additional PARTS ORDER LIST:
10           158301100           1              STELLARIS 8
20           158301134           1              8k Tandem Scanner STELLARIS 8 DIVE/CRS
30           158204510           1              Fluorifier Disc Basis inkl. Analysator
40           158204512           1              Notch filter set VIS full
50           158301124           1              Scan optics VISIR with rotation for DMi8
60           158201310           1              SP Light Trap
110         158305400           1              Pulse trigger interface
120         158301140           1              STELLARIS 8 DMi8 Adapter
130         158301200           1              Laser Port 405/UV
140         158202140           1              Laser 405 DMOD
150         155933663           1              DMi8 CS Premium
180         158204202           1              Closed Loop Focus with AFC
200         158000677           1              Leica LED 3
210         158004141           1              Scanning stage inv. universal
230         15525226             1              XY advanced board for scanning stage
240         158004421           1              SuperZ Galvo Stage
250         158004424           1              Insert Universal Inverse SuperZ
260         15525309             1              Filter Cube DsRed
270         15525314             1              Filter Cube GFP
280         15525338             1              Filter cube LED 405
390         15555019             1              IC Condenser Prism K5
400         15555006             1              IC Prism A
410         15521524             1              IC Condenser Prism K10
420         15555018             1              IC Condenser Prism K4
430         15555056             1              IC Prism D1
440         15555017             1              IC Condenser Prism K3
450         15555046             1              IC Prism E
460         158206045           1              Okolab Laser Safety Box DMi8 4TUNE
470         158206043           1              Okolab Temp. Ctrl Laser Safety Box
480         158206037           1              Okolab Sample Chamber Inv Super Z
490         158206031           1              Okolab Insert multi well plate SuperZ
500         158206040           1              Okolab CO2, Hypoxia, Humidity Passive
505         158206042           1              Laser Safety Kit Okolab
510         158003151           2              High Brilliance Monitor flat
520         158203113           1              Workstation Expert
540         158203200           1              LAS X STELLARIS Control Software
550         158203204           1              LAS X Lightning Expert
560         158203206           1              LAS X Dye Finder
570         158203208           1              LAS X FRAP Zoomer
580         158203211           1              LAS X Assay Editor
590         158203214           1              LAS X Environmental Control
600         158003206           1              LAS X MicroLab
601         158203207           1              LAS X MicroLab
610         27100001            1              Aivia 3D
620         158305100           1              STELLARIS 8 FALCON
630         158305101           1              STELLARIS 8 FCS
690         158004512           1              MP Barrier Filter Set
700         158002641           1              Single beam routing with VBE
720         158002640           1              IR Laser attenuation AOM
730         158301212           1              IR multifunction port DIVE inverse
740         158005232           1              Tau excitation DIVE

Recommended alternative to water for using water immersion objectives.

 

General Confocal Best practices:

  • The pinhole is what makes the confocal a confocal. Set at 0.5 to 1AU (which means 1 Airy unit) and reset this each time you change lenses.
    If you are opening it for imaging fixed samples, you should go use a widefield fluorescence scope instead. If you open the pinhnole, when you write your methods, you won't be accurately describing your microscopy as "confocal".
    However, there are special cases for live cell imaging where opening the pinhole is ok, but needs to be noted.
  • Be careful of adjusting LUTs. Full range is usually best. However, this instrument has very sensitive detectors and may be used in photon counting mode where only a few counts at 8 bits is excellent signal.
  • Example of scan speed, averaging, filtering and noise click here.

Always save files as LIF.
Think of each LIF file as a container for one sample or experimental condition. Thisk of each LIF file as a folder containing only one condition.
When you change samples, start a new LIF file. Give it the name of the sample. This way, each image in the container does not need to be manually named. Or you can give shorter descriptive names.
LIF saves all metadata and you can reuse settings from individual images in the LIF containers. ImageJ/Fiji, Imaris, and other analysis software open LIF files.

 

Other Stuff

MaiTai laser control manually, for instance if LASX does not turn off the laser.

Notes from first day of training.   .rtf    .pdf

 


 

Staff only.


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