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Equipment

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Content: Leica SP5 II • Olympus DSU • SEM • TEM • Prep Lab


picture of SP5 confocal microscopeLeica TCS SP5 II laser scanning confocal microscope

(user manualusing ImageJ with lif fileswhich confocal to use)

Confocal system

  • overview
    • with 4 spectral and 1 transmission detectors, 12-bit output
    • mostly software driven (LAS AF 2.6)
    • conventional scanner: 5 fps at 512×512 pixels, max at 8192×8192 pixels
    • resonant scanner: 28 fps at 512×512 pixels, max at 1024×1024 pixels
    • can do 4 simultaneous confocal scans and up to 16 between line or 32 between frame sequential scans
    • laser power can be modulated continuously
    • ROI scan, Mark & Find, tiling, FRAP & FRET Wizards
  • lasers (wavelength in nm)
    • diode: 405
    • Ar: 458, 476, 488, 514
    • DPSS: 561
    • HeNe: 633
  • beamsplitters (wavelength in nm)
    • RT 30/70: for reflective mode
    • substrate: plain glass
    • TD 488/561/633: 505-590, 570-615, 655-720 triple band pass
    • DD 458/514: 470-505, 530-570 double band pass
    • RSP 500: reflection shortpass 500
    • DD 488/561: 505-590, 570-615 double band pass
  • analyser/blocking filters (wavelength in nm)
    • 488/561/633
    • 458/514
    • polarizer

Microscope

Leica DMI6000: inverted, motorized XYZ stage with galvo insert (500 µm travel), infinity optics with epifluorescence (120 W mercury short-arc reflector lamp) and DIC, motorized objective and condenser turrets

  • objectives
    • PL S-APO 5×, NA 0.15, WD 12.1 mm (DIC not available)
    • HC plan apo CS 10×, NA 0.4, WD 2.2 mm (DIC not installed but available)
    • HC plan apo CS 20×, NA 0.7, WD 0.59 mm
    • HC plan apo CS 20× multi IMMersion, NA 0.7, WD 0.26 mm, correction collar for oil, glycerol, water, with or without coverglass
    • HC plan apo CS 40× oil, NA 1.25, WD 0.10 mm, aperture collar
    • HC plan apo CS 63× oil, NA 1.40, WD 0.10 mm, aperture collar
    • HC plan apo CS 63× water, NA 1.2, WD 0.22 mm, correction collar for cover glass thickness (0.14 mm to 0.18 mm)
  • filter cubes for epifluorescent attachment
    • A: EX 340–380, DM 400, BA LP485
    • GFP ET: EX 450–490, DM 495, BA 500–550
    • DsR: EX 541–552, DM 560, BA 568–640
    • I3 (not installed): EX 450–490, DM 510, BA LP515
    • N21 (not installed): EX 515–560, DM 580, BA LP590

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picture of DSU confocal microscopeOlympus DSU spinning disk confocal microscope

(user manualwhich confocal to use)

Illumination

  • Sutter Lambda XL: 300 nm to 700 nm (spectrum), stable Xenon plasma light source, built-in filter wheel for exciters
    Exciter Center Wavelength/Bandwidth (nm) Excitation Band (nm)
    DA 387/11 381.5 - 392.5
    FI 485/20 475 - 495
    TR 560/25 547.5 – 572.5
    Cy5 650/13 643.5 – 656.5
    GFP 472/30 455 - 485
    Texas Red 575/25 562.5 – 587.5
  • CoolLED pE-100 WH1 (white) for transmitted light

Rear light path for confocal, with DSU attachment, and widefield imaging

  • DSU with three disks of different slit widths and density
    • D1: low magnification, thick preps
    • D2: low magnification, thin preps (default)
    • D3: high magnification
  • Hamamatsu ImageEM Enhanced high dynamic range EM-CCD camera
    • 16-bit ADC, monochrome
    • 16 µm×16 µm cell
    • min. exposure time: 30.5 mS
    • transfer time: 32 mS at 512×512 pixels
    • readout noise: 1 electron max. at 1200 × EM gain (11 MHz)
  • filters for epifluorescence
    beamsplitters installed in the DSU filter turret
    Beamsplitter Reflection Band (nm) Transmission Band (nm)
    quad-band 381.5 - 392.5
    475 - 495
    547 - 572
    643 - 656
    420 - 460
    510 - 531
    589.5 - 623.5
    677 - 722
    GFP 350 - 488 502 - 950
    Texas Red 350 - 585 601 - 950
    emitters Installed in the DSU filter wheel (Sutter Lambda 10-3)
    Emitter Center Wavelength/Bandwidth (nm) Emission Band (nm)
    quad-band 440/40
    525/30
    607/36
    684/24
    420 - 460
    510 - 540
    589 – 625
    672 - 696
    GFP 520/35 502.5 – 537.5
    Texas Red 641/75 603.5 – 678.5

Front light path for widefield imageing

  • Hamamatsu Orca-flash2.8 CMOS camera
    • 12-bit ADC, monochrome
    • 3.63 µm×3.63 µm cell
    • min. exposure time: 20 µS
    • transfer time: 22 mS at 1990×1440 pixels
    • readout noise: 3 electrons at 8 × analog gain
  • epifluorescent beamsplitter and emitter
    • a quad-band set similar to the rear camera is used for the front camera
      • beamsplitter in front filter turret
      • emitters in front filter wheel (Sutter Lambda 10-3)
    • a triple-band set in the front filter turret for observation through the binocluar
      Beamsplitter Reflection Band (nm) Transmission Band (nm)
      triple-band 386 - 393
      466 - 490
      546 - 565
      414 - 450
      505 - 528
      584 - 645
      Emitter Center Wavelength/Bandwidth (nm) Emission Band (nm)
      triple-band 432/36
      516.5/23
      614.5/61
      414 – 450
      505 – 528
      584 – 645

Microscope

Olympus IX81: inverted stand, infinity optics with epifluorescence and DIC, motorized objective and condenser turrets

  • objectives
    • UPlanSApo 10×, NA 0.4, WD 3.1 mm
    • UPlanSApo 20×, NA 0.75, WD 0.65 mm
    • UPlanSApo 20× oil, NA 0.85, WD 0.20 mm
    • UPlanSApo 60× oil, NA 1.35, WD 0.15 mm
    • UApo N 340 20× water, NA 0.70, WD 0.35 mm
    • UApo N 340 40× water, NA 1.15, WD 0.25 mm, correction collar for cover glass thickness (0.13 mm to 0.25 mm)
    • UPlanSApo 60× water, NA 1.2, WD 0.28 mm, correction collar for cover glass thickness (0.13 mm to 0.21 mm)
  • ASI XYZ motorized stage with piezo insert (350 µm travel)
  • MetaMorph Premier (7.7) for hardware control and image acquistion

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picture of JSM-840A SEMJEOL JSM-840A scanning electron microscope

(user manualmagnification calibration)

Specification

  • tungsten filament
  • 0.2 kV to 25 kV, 10× to 300k×
  • secondary and backscattered electron detectors
  • uses 12.7 mm diameter pin type stub with a 3.2 mm diameter shank, or 12.5 mm and 32 mm diameter cylinder specimen mount

Accessories

  • JEOL DSG Plus digital scan imaging system: 320×240 to 2560×1920 pixel
  • Polaroid 4×5 and 35 mm roll film holders (seldom used)
  • GW specimen current amplifier

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picture of CM100 TEMPhilips CM100 transmission electron microscope

(user manualmagnification calibration)

Specification

  • tungsten filament
  • 40 kV to 100 kV, 18× to 450k×
  • resolution is currently about 4 nm
     
    islands of sputtered gold at 450k×

    Sample image: Islands of sputtered gold on carbon coated formvar taken with the Gatan 689 at 450k×, 100 kV, and emission 3. Scale bar is 10 nm.

Accessories

  • Gatan Model 689 digital slow scan camera: 14-bit, 128×128 to 1024×1024 pixels
  • camera for 3¼"×4" plate film (seldom used)

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Sample preparation lab

A059

  • Laboratory Safety Manual available on the EH&S website
  • MSDS are kept in A059 and available online through MyChem (accessable with UWNetID)
  • Denton DCP-1 critical point dryer: CO2, 1-1/8" diameter by 2-5/8" deep (28 mm by 66 mm) pressure chamber (user manual)
  • fume hood is in A079

A061

  • Technics Hummer V sputter coater with Au target (user manual)
  • LKB Type 7801B knife maker for 6 mm–7 mm thick glass (user manual)
  • Sorvall glass knife maker for 9 mm and 12 mm thick glass
  • RMC MT6000 ultramicrotome

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Date last changed Oct 5th, 2016 @ 15:26:05 PDT