Content OMX: GE SIM • SP5: Leica confocal, inverted • SPU: Leica confocal, upright • A1R: Nikon confocal • DSU: Olympus spinning disk and widefield • YX1: Yokogawa spinning disk and widefield • Nikon Eclipse • Wild M8 • Zeiss Axio Zoom • AVS: FEI VolumeScope • TEM: Philips CM100 • Prep Lab

OMX: GE DeltaVision OMX SR super-resolution microscope, LSB B204

(OMX docs—only available to registered users)

Overview

• high speed 2D- and 3D-SIM, TIRF, SIM-TIRF, and widefield (compare between modes)
• 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, per Z slice, zero time delay
  lateral resolution1	320 nm	250 nm	120 nm	120 nm
  axial resolution1	600 nm	500 nm	600 nm	340 nm
  imaging time2	~3 mS	~3 mS, needs post processing: deconvolution	<45 mS per slice (9 images per slice)	<100 mS per slice (15 images per slice)
  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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SP5: Leica TCS SP5 II laser scanning confocal microscope on an inverted stand, PAA A033

(user manual, using ImageJ with lif files)

Confocal system

• scanner
  • with 4 spectral detectors for fluorescence and 1 detector for transmitted light, 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: 505–540, 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: 505–545, 580–740 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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SPU: Leica TCS SP5 II laser scanning confocal microscope on an upright stand, LSB 322

(using ImageJ with lif files)

Confocal system

• scanner
  • with 3 spectral detectors for fluorescence, 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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A1R: Nikon A1R HD25 laser scanning confocal microscope, LSB B202A

(user manual, extended log)

confocal system

• scanner
  • 2 multialkaline and 2 GaAsP detectors for fluorescence, and 1 multialkaline detector for transmitted light, 12-bit output
  • mostly software driven (NIS Elements 5.30)
  • galvano scanner: 0.7 fps at 512×512 pixels, max at 4096×4096 pixels
  • resonant scanner: 15 fps at 1024×1024 pixels max
  • simultaneous and sequential scans
  • laser power can be modulated continuously
  • Mark & Find, tiling, FRAP &
• LU-N4 laser unit (wavelength in nm): 405, 488, 561, 640
• beamsplitters (wavelength in nm)
  • 1st dichroic mirror: 405/488/561/640
  • BS 20/80: for reflective mode
• Detector configuration
  • Ch1: DM 495 LP EM 447/57 (419–476)
  • Ch2, GaAsP: DM 560 LP EM 524/42 (503–545)
  • Ch3, GaAsP: DM 652 EM 600/45 (578–623)
  • Ch4: EM 650 LP

Microscope

Nikon Ti2-E: inverted stand with Perfect Focus System, infinity optics, epifluorescence, and DIC; motorized components include objective and filter cube turrets, condenser, and XY stage

• objectives
  • Plan Apochromat Lambda 10×, NA 0.45, WD 4.0 mm
  • Plan Apochromat Lambda 20×, NA 0.75, WD 1.0 mm
  • Plan Apochromat Lambda 60× oil, NA 1.4, WD 0.13 mm
  • Plan Apochromat 60× water, NA 1.2, WD 0.22 mm, correction collar for cover glass thickness (0.15 mm to 0.18 mm)
  • Apochromat Lambda S LWD 20× water, NA 0.95, WD 0.90–0.99 mm, correction collar for cover glass thickness (0.11 mm to 0.23 mm)^{loaded on demand only}
  • Apochromat Lambda S LWD 40× water, NA 1.15, WD 0.59–0.61 mm, correction collar for cover glass thickness (0.15 mm to 0.19 mm)^{loaded on demand only}
• X-Cite 110LED epifluorescence light source: 375–660 nm
• P736 PInano Z piezo stage with 200 µm travel, universal adapter for slides and dishes, and adapter for well plate; can accept the Tokai Hit stage top incubator (see the description below in the YX1 section)
• filter cube turret
  • EX 340–380 DM 400, EM 435–485
  • EX 465–495, DM 505, EM 515–555
  • EX 528–553, DM 565, EM 590–650

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DSU: Olympus DSU spinning disk confocal microscope, PAA A065

(user manual)

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-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)	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 4×, NA 0.16, WD 13 mm
  • 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, BFP1
  • 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), BFP1
  • 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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YX1: Yokogawa CSU-X1 spinning disk confocal microscope, LSB B212

(user manual, extended log)

Spinning disk, left camera port

• CSU-X1 unit
  • Microlens-enhanced Nipkow disk
  • 1500–10000 rpm, continuously adjustable
  • 3-position dichroic filter wheel with a quad band filter: 429–491, 516–552, 583–636, 672–748
  • 6-position emission filter wheel with 4 single band and a quad band filters
    • 455/50 (430–480)
    • 525/36 (507–543)
    • 605/52 (579–631)
    • 705/72 (669–741)
    • 428–466, 509–531, 578–616, 671–738
  • image size is about 7.8 mm×10.7 mm (710×970 pixels as projected on to the camera, usable ROI is 650×928 pixels)
• Nikon LUN-F XL laser launch with 4 solid state lasers: 405, 488, 561, 640 nm
• Photometrics Prime95B back illuminated scientific CMOS camera
  • 95% QE at 550 nm
  • 16-bit digital output, monochrome
  • 11 µm×11 µm cell
  • readout time: 24 mS (41 fps) at 16-bit, full frame
  • 13.2 mm×13.2 mm sensor area, 1200×1200 pixel
• Gataca Live-SR super resolution module
  • motorized magnification switcher with standard (1×) and SR position (1.7×)
  • Live-SR deconvolution algorithm can be applied to live images or in post-processing
  • can be used with low or high magnification objective lenses
  • resolution up to 120 nm in XY

Widefield imaging^IPD, right camera port

The components marked ^{IPD or Winkler} are owned by the Insititue of Protein Design or Winkler Lab. They are made available through a collaboration between the Department of Biology and the Department of Biochemistry, or the Winkler Lab.

• Lumencor Celesta light engine with 7 laser lines: 408, 445, 473, 518, 545, 635, 750 nm
• Hamamatsu ORCA-Fusion scientific CMOS camera
  • 80% QE at 550 nm
  • 16-bit digital output, monochrome
  • 6.5 µm×6.5 µm cell
  • readout time: 11.22 mS (89 fps) at 16-bit, full frame
  • 14.98 mm×14.98 mm sensor area, 2304×2304 pixels
• Finger Lakes Instrumentation HS-625 high speed emission filter wheel with 5 bandpass filters:
  • 514/30 (499–529)
  • 565/24 (553–577)
  • 624/40 (604–644)
  • 676/29 (662–691)
  • 732/68 (698–766)

Microscope

Nikon Ti2-E: inverted stand with Perfect Focus System, infinity optics, epifluorescence, motorized stage and motorized objective and filter cube turrets

• objectives
  • Plan Apochromat Lambda 4×, NA 0.2, WD 20.0 mm^IPD
  • Plan Apochromat Lambda 10×, NA 0.45, WD 4.0 mm
  • Plan Apochromat Lambda 20×, NA 0.75, WD 1.0 mm
  • Plan Apochromat Lambda 40×, NA 0.95, WD 0.17–0.25 mm, correction collar for cover glass thickness (0.11 mm to 0.23 mm)^IPD
  • S Plan Fluor ELWD ADM 40×, NA 0.60, WD 3.6–2.8 mm, correction collar for cover glass thickness (0 mm to 2 mm), phase contrast PH2^Winkler
  • Plan Apochromat Lambda 60× oil, NA 1.4, WD 0.13 mm
  • Apochromat Lambda S LWD 20× water, NA 0.95, WD 0.90–0.99 mm, correction collar for cover glass thickness (0.11 mm to 0.23 mm)^{loaded on demand only}
  • Apochromat Lambda S LWD 40× water, NA 1.15, WD 0.59–0.61 mm, correction collar for cover glass thickness (0.15 mm to 0.19 mm)^{loaded on demand only}
• X-Cite 110LED epifluorescence light source: 375–660 nm
• P736 PInano Z piezo stage with 200 µm travel, universal adapter for slides and dishes, and adapter for well plate
• upper filter cube turret^IPD:
  • pentaband filter cube
    • dichroic: 422–467, 482–533, 558–623, 651–733, 764–865
    • emission: 424–462, 486–531, 562–620, 657–731, 769–865
  • CFP: DM 458, EM 483/32 (467–499)
  • CFP/YFP: DM 458, EM 542/27 (529–556)
  • YFP: DM 526, EM 544/24 (532–556)
• lower filter cube turret
  • EX 340–380 DM 400, EM 435–485
  • EX 465–495, DM 505, EM 515–555
  • EX 528–553, DM 565, EM 590–650
• Tokai Hit stage top incubator
  • TIZWX incubation system for dishes and well plate
  • STXF controller with analog flowmeter and STX–CO2O2 for hypoxia condition
  • setting range
    • CO₂: 5.0% ~ 20.0%
    • O₂: 0.1% ~ 18.0%
    • temperature: 30 °C ~ 40 °C
• National Instrument DAQ

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Other light microscopes

• Eclipse: Nikon Eclipse E600FN with an upright stand, fixed stage, infinity optics, epifluorescence and DIC, PAA A061
  • 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 M, 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 0.21
    • 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
    • blue: EX ~350, DM 400, BA ~430
    • green, B-2A: EX 450–490, DM 505, BA 515–555
    • red: EX 541–551, DM 565, BA 580
  • Camera: color, 3 MP CMOS, 3.2 µm pixel, 2048×1536 pixels (user manual)
• M8: Wild M8 dissecting microscope, PAA A061
  • 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)
• Axiozoom: Zeiss Axio Zoom.V16 microscope, PAA A053 (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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AVS: FEI Apreo VolumeScope, a serial blockface imaging scanning electron microscope, MOL G44L

The Apreo VolumeScope is owned by the Department of Biostructure and is made available through a collaboration between the Department of Biology and Department of Biostucture.

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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TEM: Philips CM100 transmission electron microscope, PAA A071

(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

PAA 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)
• supplies for SEM sample preparation

PAA A061

• EMS Quorum 105T ES sample preparation system for sputter coating, carbon evaporation, etc.
• 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

PAA A079

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

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