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Equipment

Content: GE OMX SRLeica SP5 II, invertedLeica SP5 II, uprightOlympus DSUNikon EclipseWild M8Zeiss Axio ZoomVolumeScopeTEMPrep Lab

picture of the OMX SR microscope, click to see a larger version

GE DeltaVision OMX SR super-resolution microscope

(OMX docs—only available to registered users)

Overview

  • high speed 2D- and 3D-SIM, TIRF, SIM-TIRF, and widefield
  • dual 4-laser modules for 2 separate light paths: SI and Ring TIRF
  • LED transillumination
  • hardware UltimateFocus and software autofocus to compensate for axial drift
  • 3 sCMOS cameras: images are usually acquired at 512×512 pixels, 80 nm per pixel
  • environmental control for temperature, humidity, and gas
  • software: AcquireSR and softWoRx
  • please acknowledge grant proposal S10 OD021490 on any publications that have used this microscope

Specification

  • modules
    • Blaze structured illumination (SI) for 3D SI, 2D SI, and SI TIRF
    • Ring TIRF/Photokinetic (PK) for TIRF, bleaching (FRAP, FLIP), FRET, and photo activation
    • UltimateFocus to detect the coverglass surface in real time and maintaining the sample Z position to within 25 nm of the position set
  • 4-laser module (wavelength in nm)
    • diode: 405
    • diode: 488
    • diode: 568
    • diode: 640
  • optical filters
    polychroic filters: BGR Drawer
    channel laser line (nm) phase band (nm) Reflection Band (nm) Transmission Band (nm)
    DAPI 405 403–407 382–409 421–450
    AF488 488 487–488 462–492 505–549
    AF568 568 561–569 561–580 591.5–626.5
    Cy5 640 639–644 639–652 664–702
    emission filters
    channel Center Wavelength/Bandwidth (nm) blocking range (nm)
    DAPI 435.5/31 300–410, 461–710
    AF488 528/48 300–494, 562–710
    AF568 609/37 300–580.5, 637–710
    Cy5 683/40 300–653
  • camera: pco.edge 4.2 sCMOS
    • 6.5 µm×6.5 µm cell
    • readout noise: 1.5 electrons at 272.3 MHz
    • 16-bit ADC, monochrome
    • exposure time: 100 µS to 10 S
  • optics
    • PLAPON 60XOPSF: 60× oil, NA 1.42, WD 0.15 mm, tested for PSF
    • APON 60xOTIRF: 60× oil, NA 1.49, WD 0.1 mm, correction collar for coverglass thickness and temperature
  • environmental control
    • Cell Box: stage enclosure for temperature, humidity, O2 and CO2 control
    • objective heater
  • Capabilities of various imaging modes
      widefield widefield deconvolution 2D SIM 3D SIM
    1 for 488 nm excitation
    2 512×512 pixels, 1 mS exposure, no Z stepping, zero time delay
    lateral resolution1 320 nm 250 nm 120 nm 120 nm
    axial resolution1 600 nm 500 nm 600 nm 340 nm
    imaging speed2 ~400 fps ~400 fps + time for deconvolution 15 reconstructed fps 0.8 S/1 µm stack of 120 images
    application fast, live or fixed tissue imaging fast, live or fixed tissue imaging super resolution, live or fixed tissue imaging super resolution, live or fixed tissue imaging

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picture of the SP5 confocal microscope, click to see a larger version

Leica TCS SP5 II laser scanning confocal microscope on an inverted stand

(user manual, using ImageJ with lif files, which confocal to use)

Confocal system

  • scanner
    • with 4 spectral and 1 transmission detectors, 12-bit output
    • mostly software driven (LAS AF 2.7)
    • 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 (can be installed on demand, DIC not available)
    • HC plan apo CS 10×, NA 0.4, WD 2.2 mm (DIC not installed but available on demand)
    • 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 the SP5 confocal microscope

Leica TCS SP5 II laser scanning confocal microscope on an upright stand

(using ImageJ with lif files, which confocal to use)

Confocal system

  • scanner
    • with 3 spectral detectors, 12-bit output
    • mostly software driven (LAS AF 2.7)
    • conventional scanner: 5 fps at 512×512 pixels, max at 8192×8192 pixels
    • can do 3 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

Microscope

Leica DM6000B: upright, motorized XYZ stage with galvo insert (500 µm travel), infinity optics with epifluorescence (120 W mercury short-arc reflector lamp), motorized objective and condenser turrets

  • objectives
    • HC plan apo CS 10×, NA 0.4, WD 2.2 mm
    • 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
    • HCX apo L U-V-I 40× water dipping, NA 0.8, WD 3.3 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

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picture of the DSU confocal microscope, click to see a larger version

Olympus DSU spinning disk confocal microscope

(user manual, which 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
    TR-P 556/20 546–566
    Cy5 650/13 643.5–656.5
    GFP 472/30 455–485
    Texas Red 575/25 562.5–587.5
  • CoolLED pE-100 Wh2 (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) Edge Wavelength (nm) Transmission Band (nm)
    quad-band 381.5–392.5
    475–495
    547–572
    643–656
    410
    504
    582
    669
    420–460
    510–531
    589.5–623.5
    677–722
    GFP 350–488 495 502–950
    Texas Red 350–585 593 601–950
    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-flash3.8 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) Edge Wavelength (nm) Transmission Band (nm)
      triple-band 386–393
      466–490
      546–565
      403
      497
      574
      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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Other light microscopes

  • Nikon Eclipse E600FN: 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
    • Camera: color, 3 MP CMOS, 3.2 µm pixel, 2048×1536 pixels (user manual)
  • Wild M8 dissecting microscope
    • 10× ocular and plan 1× objective
    • Zoom Changer engravings indicate total magnifications available when used with a pair of 10× oculars: 6×, 9×, 12×, 18×, 25×, 40×, and 50×
    • additional oculars: 15× and 20×
    • Camera: color, 3 MP CMOS, 3.2 µm pixel, 2048×1536 pixels (user manual)
  • picture of the Axio Zoom, click to see a larger versionZeiss Axio Zoom.V16 microscope (user manual)
    • motorized zoom from 0.7× to 11.2×
    • objectives
      • PlanNeoFluar Z 1.0×, NA 0.25, WD 56 mm
      • PlanNeoFluar Z 2.3×, NA 0.57, WD 10.6 mm
    • transillumination base for bright field and dark field
    • Filter cubes for motorized epifluorescent attachment
      • blue: EX 365/100 DM 395 EM 445/50
      • green: EX 470/40 DM 495 EM 525/50
      • red: EX 545/25 DM 570 EM 605/70
    • Camera: Axiocam 506 with 1× and 0.63× adapter
      • monochrome, 6 MP, 4.54 µm pixel, 2752×2208 pixels
      • CCD Peltier-cooled to 18 °C

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picture of the Apreo VolumeScope, click to see a larger version

FEI Apreo VolumeScope, a serial blockface imaging scanning electron microscope

Specification

  • field emission gun assembly with Schottky emitter source
  • oil-free vacuum system
  • digital scanning engine to produce 16-bit images up to 6144×4096 pixels
  • Modular Automated Processing System (MAPS) and motorized stage to enable large volume coverage with automated multi-tile set runs
  • stage-mounted microtome
    • diamond knife
    • section thickness can be down to 1 nm, typical at 40 nm
    • cutting speed range is 0.1–1.2 mm/s, typical at 0.5–1 mm/s
    • sample block can be up to 600 µm×600 µm×600 µm, optimal size blockface is 200 µm×200 µm
    • electrostatic debris trap for the collection and immobilization of section debris prevents contamination of the blockface
  • in VolumeScope mode, resolution is
    • <3 nm at 1.18 kV, Hi-Vac (specimen chamber pressure at 10-4 Pa), with T1 detector and FilterRing installed
    • <7 nm at 2 kV, Lo-Vac (specimen chamber pressure at 50 Pa), with VS DBS detector
  • ThruSight, FEI’s multi-energy deconvolution method for virtual slicing with different accelerating voltages, can be used to obtain isotropic resolution up to 10×10×10 nm
  • typical images are 6144×4096 pixels with a resolution of 5 nm in XY and 40 nm in Z, the field of view is 31 µm×20 µm

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picture of the CM100 TEM, click to see a larger version

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

Accessories

  • Olympus Morada 11 megapixel digital camera: 14-bit, 9 µm×9 µm cell, can acquire images up to 2672×3615 pixels; typical image at high magnification has a pixel size of 0.55 nm and the field of view is 1.46 µm×1.98 µm
  • camera for 3¼"×4" plate film (seldom used)

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Sample preparation lab

A059

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

A061

  • EMS 105T ES sputter coater
  • LKB Type 7801B glass knife maker for 6 mm–7 mm thick glass (user manual)
  • Dupont Sorvall glass knife maker for 9 mm and 12 mm thick glass
  • RMC PT-XL ultramicrotome

A079

  • RMC MT6000 ultramicrotome
  • fume hood
  • VirTis BT6KEL-85 freeze dryer (user manual)

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Date last changed Aug 3rd, 2019 @ 20:26:01 PDT