Content: Olympus DSU • Leica SP5 II • Radiance2000 • MRC-600 • SEM • TEM • Prep Lab

Olympus DSU spinning disk confocal microscope

(user manual, which confocal to use)

DSU attachment (suitable for live imaging)

• three disks with different slit widths and configuration
  • 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 at 512×512 pixels
  • readout noise: 1 electron max. at 1200 × EM gain (11 MHz)
• filters
  • beamsplitter and emitter from Semrock's "Pinkle" DA/FI/TR/Cy5-4X-A set (spectral data on Semrock's website)

    Beamsplitter	Reflection Band	Transmission Band
    Quad-band	381 - 392 nm 475 - 495 nm 547 - 572 nm 643 - 656 nm	420 - 460 nm 510 - 531 nm 589 - 623 nm 677 - 722 nm

    Emitter	Center Wavelength	Bandwidth
    Quad-band	440 nm 521 nm 607 nm 700 nm	40 nm 21 nm 34 nm 45 nm

  • Semrock FITC-3540B: EX 482/35 nm, DM 513, EM 536/40 nm

Illumination

• Sutter Lambda XL: 300 nm to 700 nm, highly stable Xenon plasma light source, built-in filter wheel with exciters from Semrock's "Pinkle" DA/FI/TR/Cy5-4X-A set (spectral data on Semrock's website)

  exciter	Center Wavelength	Bandwidth
  DA	387 nm	11 nm
  FI	485 nm	20 nm
  TR	560 nm	25 nm
  Cy5	650 nm	13 nm

• CoolLED pE-100 WH1 (white) for transmitted light

Setup for routine epifluorescent imageing

• Hamamatsu Orca-flash2.8 CMOS camera
  • 12-bit ADC, monochrome
  • 3.63 µm×3.63 µm cell
  • min. exposure time: 20 µS at 1990×1440 pixels
  • readout noise: 3 electrons at 8 × analog gain
• beamsplitter and emitter from Semrock's "Pinkle" DA/FI/TR/Cy5-4X-A set (see above or Semrock's website for spectral data)

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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Leica TCS SP5 II laser scanning confocal microscope

(user manual, using ImageJ with lif files, which 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 nm
  • Ar: 458, 476, 488, 514 nm
  • DPSS: 561 nm
  • HeNe: 633 nm
• beamsplitters
  • RT 30/70: for reflective mode
  • substrate: plain glass
  • TD 488/561/633: 505-590, 570-615, 655-720 nm triple band pass
  • DD 458/514: 470-505, 530-570 nm double band pass
  • RSP 500: reflection shortpass 500 nm
  • DD 488/561: 505-590, 570-615 nm double band pass
• analyser/blocking filters
  • 488/561/633
  • 458/514
  • polarizer

Microscope

Leica DMI6000: inverted, motorized XYZ stage with galvo insert (500 µm travel), infinity optics with epifluorescence and DIC, motorized objective and condenser turrets

• objectives
  • HC plan apo CS 10×, NA 0.4, WD 2.2 mm (DIC not 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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BIO-RAD Radiance2000 MP laser scanning confocal microscope

(user manual, using ImageJ, image quality, which confocal to use)

Confocal system

• overview
  • with 3 conventional and 1 transmission detectors
  • mostly software driven (LaserSharp 6.0 build 846)
  • can acquire up to 1280×1024 pixels
  • can do 3 simultaneous channels and up to 7 sequential channels
  • laser power can be modulated continuously
  • suitable for live imaging in 2-photon mode using the Mai Tai infrared laser and the Direct Detection System with 2 bi-alkali/multi-alkali detectors
• lasers
  • Kr/Ar: 488, 568 nm
  • red diode: 638 nm
  • Spectra-Physics Mai Tai: 780–920 nm
• dichroic mirrors
  • 440DCLPXR
  • 500DCLPXR
  • 560DCLPXR
  • 650DCLPXR
• blocking filters
  • BGG22
  • E625SP
  • E525/150
  • HQ575/150
• emission filters
  • HQ390/70
  • HQ450/80
  • D488/10
  • HQ500LP
  • HQ515/30
  • HQ528/50
  • E460LP
  • E570LP
  • HQ590/70
  • HQ600/50
  • E600LP
  • HQ660LP
• filter cubes for direct detectors
  • B/G: EmissionA HQ515/30, DM DC500LP, EmissionB empty
  • 495 DCXR: EmissionA HQ528/50, DM 495DCXR, EmissionB HQ450/80

Microscope

Nikon Eclipse E600 FN: upright, fixed stage, infinity optics with epifluorescence and DIC

• objectives
  • 4×, NA 0.10, WD 30
  • plan apo 10×, NA 0.45 DIC L, WD 4.0
  • plan apo 20×, NA 0.75 DIC L, WD 1.0
  • plan fluor 40×, NA 1.30 oil DIC H, WD 0.20
  • plan apo 60×, NA 1.40 oil DIC H, WD 0.22
  • plan fluor 20×, NA 0.50 water dipping
  • fluor 40×, NA 0.80 water dipping DIC M, WD 2.0
  • plan apo 60×, NA 1.2 water DIC H, WD 0.22
• filter cubes for epifluorescent attachment
  • UV-2E/C: EX 330–380, DM 400, BA 435–485
  • B-2E/C: EX 465–495, DM 505, BA 515–555
  • G-1B: EX 541–551, DM 565, BA 590

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BIO-RAD MRC-600 laser scanning confocal microscope

(user manual, using ImageJ, which confocal to use)

Confocal system

• overview
  • with 2 conventional detectors
  • mostly manual driven (CoMOS), including mirror alignments in the scanhead
  • can acquire up to 768×512 pixels
  • can do 2 simultaneous channels but at reduced resolution 384×512 pixels/channel
  • laser power is modulated via 4 ND filters (10 %, 3 %, 1 %, 0.1 %)
• laser
  • Kr/Ar: 488, 568, 647 nm
• filter blocks
  • BHS: DM 510LP, EM OG515LP
  • YHS: DM 585DRLP, EM 585EFLP
  • RHS: DM 660DRLP, EM 680EF32
  • K1: DM Z488/568rpc
  • K2: DM 560LP, EM HQ522/35m, EM 585EFLP

Microscope

Nikon Optiphot: upright, 160 mm tube length with eipfluorescence and phase contrast

• objectives
  • plan 4×, NA 0.13 DL
  • fluor 10×, NA 0.50
  • plan apo 20×, NA 0.75
  • plan apo 60×, NA 1.40 oil
• filter cubes for epifluorescent attachment
  • B-2A: EX 450–490, DM 510, BA 520
  • G-1B: EX 541–551, DM 580, BA 590

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JEOL JSM-840A scanning electron microscope

(user manual, magnification 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 mm diameter shank, or 12.5 mm and 35 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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Philips CM100 transmission electron microscope

(user manual, magnification calibration)

Specification

• tungsten filament
• 40 kV to 100 kV, 18× to 450k×
• resolution is currently about 4 nm
  

  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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Updated on 2012 February 09. Copyright© 2012 University of Washington, unless indicated otherwise.

Department of Biology • UW