Confocals

Nikon Ti2E AXR NSPARC laser scanning with resonance and super-resolution (Porter B049)

Lasers & Filters

Lasers

Launch: LUD-S4Ìý

  • Excitation at: 405, 488, 561, 640 (nm)

Emission filters & molecules imaged (All filters are Chroma)

Confocal scanning:Ìý

  • Emission filter: 430-475 (nm); DAPI, Hoechst, DyLight-405, Alexa405, etc.
  • Emission filter: 499-551 (nm); Alexa488, FITC, EGFP, Opal520, etc.
  • Emission filter: 571-625 (nm); Alexa555, 568, 594, TxRed, mRFP, mCherry, TdTomato, Opal570, etc.
  • Emission filter: 663-738 (nm); Alexa647, Cy5, Opal620, etc.
  • DIC imaging (laser scanning confocal only, 10, 20, 40, 60x)

NSPARC scanning:Ìý

  • Emission filter: 430-475 (nm); DAPI, Hoechst, DyLight-405, Alexa405, etc.
  • Emission filter: 502-546 (nm); Alexa488, FITC, EGFP, Opal520, etc.
  • Emission filter: 570-616 (nm); Alexa555, 568, 594, TxRed, mRFP, mCherry, TdTomato, Opal570, etc.
  • Emission filter: 666-732 (nm); Alexa647, Cy5, Opal620, etc.

Light source, filters & viewing by eye

  • Nikon D-LEDI; 385, 475, 550, 621 (nm)
  • DAPI, GFP, RFP, Cy5 with quad-band emitter (Chroma)
  • Brightfield
  • DIC (10, 20, 40, 60x)

NSPARC super-resolution lens

  • 60X PLAN APO TIRF oil NA 1.49 WD 0.12 (mm), DIC

Additional standard objectives

  • 2x PLAN APO Ìýλ NA 0.10, WD 8500 (mm)
  • 10X PLAN APO λ D NA 0.45 WD 4.0 (mm), DIC
  • 20X CFI60 PLAN APO λ D ÌýNA 0.80 WD 0.80 (mm), DIC
  • 40XC PLAN APO λ S ÌýSILICONE NA 1.25 WD 0.30 (mm), DIC, correction collar (23-37 degrees C)
  • 60X PLAN APO IR SR water 1.27NA WD 0.18 (mm), DIC

Available upon request

  • 60x 1.40NA Plan Apo OIL VC λ WD 0.13 (mm)
  • 40x NA 0.95 WD 0.18 (mm)
  • Inquire with James.Orth@Colorado.edu

Laser scanning

  • DUX-VB, 4 PMT-GA GaAsP (photo-multiplier tubes)

Super-resolution

  • Nikon Spatial Array Confocal (NSPARC) pinhole array sensor; 5x5 array of 0.2 AU pinholes

Sample Holders

  • Slide, 35 (mm) dish, 50/60 (mm) dish
  • Multi-well plate

Capabilities

  • High frame-rate imaging using resonance scanner
  • Super resolution to 100nm XY using NSPARC detector
  • Queensgate (Prior QGNPS083-NI) Piezo Z-stage with 600 micron travel
  • Adjustable GFP and RFP emission bandwidth (confocal only)
  • TOKAI-HIT Cell Incubator 1 x 35 , 50 or 60(mm) dish, chamber side (coverslip bottom), multiwell plate
  • Motorized X, Y
  • Multi-point, time-lapse, optical sectioning, large-area montage with stitching
  • Advanced analysis tools via separate HP Z4 workstation, including spectral deconvolution, and full analysis suite (inquire with Director)
  • NIS Elements AR 6.02.03 with JOBS, general analysis
AX NSPARC front view

Email, James.Orth@Colorado.edu

For training email, James.Orth@Colorado.edu

THOR - Nikon TiE A1 laser scanning with structured illumination and TIRF (Porter B047A)

Lasers & Filters

Lasers

Launch: LUNV6, high-output

  • Excitation at: 405, 445, 488, 514, 561, 647 (nm)

Emission filters & molecules imaged (All filters are Chroma)

  • Emission filter: 425-475 (nm); DAPI, Hoechst, DyLight-405, Alexa405, etc.
  • Emission filter: 467.5-502.5 (nm); CFP, cerulean, etc.
  • Emission filter: 500-550 (nm); Alexa488, FITC, EGFP, Opal520, etc.
  • Emission filter: 521.5-554.5 (nm); YFP, Venus, etc.
  • Emission filter: 575-625 (nm); Alexa555, 568, 594, TxRed, mRFP, mCherry, TdTomato, Opal570, etc.
  • Emission filter: 650-720 (nm); Alexa647, Cy5, Opal620, etc.
  • DIC imaging possible (laser scanning confocal only, 20, 40, 60, 100x)

Light source, filters & viewing by eye

  • Lumencor SOLA
  • DAPI, GFP, RFP (Chroma)
  • Brightfield
  • DIC (20, 40, 60, 100x)

N-SIM super-resolution lens

  • 100x 1.49NA OIL SR Apo TIRF

Additional standard objectives

  • 4x 0.2NA Plan ApoÌýλ WD 20 (mm)
  • 10x 0.45NA Plan Apo λ WD 4 (mm)
  • 20x 0.75NA Plan Apo VC λ WD 1 (mm)
  • 40x 0.95NA Plan Apo λ WD 0.25-0.17 (mm)
  • 60X 1.27NA Water SR Plan Apo WD 0.17 (mm)

Available upon requestÌý

  • 60x 1.40NA Plan Apo OIL VC λ WD 0.13 (mm)
  • Inquire with James.Orth@Colorado.edu

Laser scanning

  • 2 x GaAsP high-sensitivity PMTs (CFP, GFP, YFP, RFP), photo-multiplier tubes
  • 2 x multi-alkali PMTs (DAPI, far-red), photo-multiplier tubes

N-SIM & TIRF

  • Andor iXON 897 Ultra EM-CCD (512x512)

Ìý

Ìý

Sample Holders

  • Slide, 35 (mm) dish, 50/60 (mm) dish
  • Multi-well plate

Capabilities

  • 3D structured illumination, 'fast' 2D SIM
  • TIRF
  • TOKAI-HIT Cell Incubator 1 x 35 (mm) dish, chamber side (coverslip bottom)
  • Motorized X, Y, Z and separate MCL Piezo Z with 200 micron range
  • Multi-point, time-lapse, optical sectioning, large-area montage with stitching
  • Advanced analysis tools via separate workstations, including spectral deconvolution, and full analysis suite (inquire with Director)

  • SIM imaging with 647nm laser:

    Thor, SIM, far red
  • ÌýAR 5.04; 64 bit
Thor

Ìý

Email James.Orth@Colorado.edu

For training email, James.Orth@Colorado.edu

BLACK WIDOW - Nikon TiE spinning disk with FRAP/PA (Porter B049)

Lasers, Filters & Disk

Lasers

Launch: LUNV4

  • 405, 488, 561, 640Ìý(nm)
  • Quad used for excitation transmission in photobleaching or photoactivation experimentsÌýis Semrock Brightline Di01-T405/488/568/647

Emission filters & molecules imaged (All filters are Chroma)

  • Emission filter: 430-480Ìý(nm); DAPI, Hoechst, DyLight-405, Alexa405, BFP, etc.
  • Emission filter: 500-540 (nm); Alexa488, FITC, EGFP, Opal520, etc.
  • Emission filter: 602-662Ìý(nm); Alexa555, 568, 594, TxRed, mRFP, mCherry, TdTomato, Opal570, etc.
  • Emission filter: 669-741Ìý(nm); Alexa647, Cy5, Opal620, etc.
  • Brightfield

Light source, filters & viewing by eye

  • Lumencor SOLA
  • DAPI, GFP, RFP, Cy5 (Chroma)
  • Brightfield

Disk

• Yokogawa CSU-10A, 50 micron pinholes

BlackWidow pinhole array

white light shining through the pinhole array (disk off)

Standard objectives

  • 10x 0.45NA Plan Apo λ WD 4 (mm)
  • 20x 0.75NA Plan Apo VC λ WD 1 (mm)
  • 40x 0.95NA Plan Apo λ WD 0.25-0.17 (mm)
  • 60x 1.40NA Plan Apo VC λ OIL WD 0.13 (mm)
  • 100x 1.45NA Plan Apo λ OIL WD 0.13 (mm)

Available upon requestÌý

  • 4xÌý0.13NA Plan Fluor WD 17.2 (mm)
  • 60xÌý1.27NA water SR Plan Apo WD 0.17 (mm)
  • Inquire with James.Orth@Colorado.edu
  • , 16bit, 512x512 16x16 micron pixels, binning and gain
  • Hamamatsu ImagEM EM-CCD, 16bit, 512x512 16x16 micron pixels (widefield), binning and gain

Sample holders

  • Slide, 35 (mm) dish, 50/60 (mm) dish
  • Multiwell plate

Capabilities

  • Fast Acquisition
  • Multipoint, montaging, stitching
  • Fast Z-sectioning via MCL Piezo Z-stage with 200 micron range
  • Single point FRAP/PA with 405 nmÌýlaser (50mW)
  • Motorized X, Y, Z
  • TOKAI-HIT Cell Incubator; 1 X 35 (mm) dish or chamber slide (coverslip bottom).

Ìý

BlackWidow panarama

Full system

Ìý

For training email, James.Orth@Colorado.edu

MAXIMUS - Evident Scientific IXPlore IX85 SpinSR spinning disk with super-resolution, 2 cameras and FRAP/PA, NIHS10OD038323 (Porter B059)

Lasers, Filters & Disks

Lasers

  • 405 (50mW), 445 (75mw), 488 (100mW), 514 (40mw), 561 (100mW) & 640 (100mW) (nm) from OBIS

Emission filters & molecules imaged (All filters are Semrock)

  • Emission filter: 417-477 (nm); DAPI, Hoechst, BFP, etc..
  • Emission filter: 464.5-499.5 (nm); CFP, etc.
  • Emission filter: 500-550Ìý(nm); Alexa488, FITC, EGFP, etc.
  • Emission filter: 525.5-630.5Ìý(nm); Venus, YFP, etc.
  • Emission filter: 580.5-653.5Ìý(nm); Alexa568, 594, TxRed, TRITC, mRFP, mCherry, mKO, TdTomato. etc.
  • Emission filter: 665-705 (nm); Alexa647, Cy5. etc.
  • Brightfield
  • DIC (20, 30, 40, 60, 100x)

Dichroic providing simultaneous 2 camera imaging

  • Yokogawa CSUW1P-DD03 Optional Dichroic Mirror 445/514/640

Light source and filters and viewing by eye

  • CoolLED; 385, 475, 550, 621 (nm) for DAPI, GFP, RFP with triple-band emitter (Semrock)
  • Brightfield
  • DIC (20, 30, 40, 60 and 100x)

Disks

• Yokogawa CSU-W1 Dual SoRa and 50 micron pinholes disks; W1 for spinning disk imaging, W1 and SoRa for super-resolution imaging

Standard objectives (All are Olympus)

  • 4x 0.16NA UPLXAPO WD 13 (mm)
  • 10x 0.40NA UPLXAPO WD 3.1 (mm)
  • 20x 0.80NA UPLXAPO WD 0.6 (mm)
  • 30xÌý1.05NAÌýUPLSAPO SIL with correction collar, WD 0.8 (mm)
  • 40x 0.95NA UPLXAPO with correction collar, WD 0.18 (mm)
  • 60x 1.50NA UPLAPO OHR OIL with correction collar, WD 0.11 (mm)
  • 100x 1.50NA UPLAPO OHR OIL with correction collar, ÌýWD 0.12 (mm)

Objectives available upon request

  • 20x 0.45NA LUCPLFLN, with correction collar, LWD 6.4-7.6 (mm)
  • 40x 0.60NA LUCPLFLN, with correction collar, LWD 2.7-4.0 (mm)
  • 40x 1.30 UPLSAPO OIL, WD 0.2 (mm)
  • 60x 1.20NA UPLSAPO water immersion, with correction collar, WD 0.28 (mm)
  • 60x 1.35NA UPLSAPO OIL WD 0.15 (mm)
  • 60xÌýNA1.10;ÌýLUMFLN60XW LWD water immersion, with correction collar, WD 1.5 (mm)

S

  • These are 2304 x 2304, 16 bit detector with 6.5 x 6.5Ìýmicron pixels
  • Back-illuminated sCMOS with >90% QE from 500-650 (nm)
  • Ultra-low noise
  • High-speed imaging

NIH 1S10OD038323. Equipped with 2 ultra-low noise, high efficiency, Hamamatsu ORCA-Fusion BT Gen-III sCMOS cameras allows for simultaneous 2 wavelength imaging. Uses a Yokogawa W1 with 50 micron pinholes and SoRa disk for optical super-resolution. Continuous focus using Zero Drift Compensation (Olympus) and software focusing.ÌýThis microscope has minimalÌýphototoxicity and photobleaching, andÌýis therefore great for live cell imaging at high temporal resolution, multi-point, and multi-day time-lapse. Multi-point montaging of large tissue samples in X-Y-Z.Encoded Marzhauser stage and Piezo Z stage with 500 micron range. Full DIC optics for 20, 30, 40, 60 and 100x, excellent brightfield imaging through the disk. 1X, 1.6 and 2X system magnifiers. Stage navigation and mapping. Advanced multi-point FRAP/PA using OXIS system with 100mW OBIS 405nm laser. Sample incubation and humidified atmosphere. More.

Standard sample holders

  • Standard slides, multi-well slides
  • 35 and 50/60 (mm) dish
  • 3 slides (coming soon, custom-made)
  • Multi-well plates; e.g. 6, 12, 24, 48, 96 and 384 wells

Environmentally controlled chamber

TOKAI-HIT STX with high capacity, warmed external bottle

  • Single 35 (mm) dish
  • Single 50/60 (mm) dish
  • Chamber slide
  • Multi-well plates

Acquisition

  • Evident Scientific

Viewer

  • Evident Viewer available here:
  • FIJI
Full system

For training email, James.Orth@colorado.edu

CHRONOS - Yokogawa CellVoyager 1000 spinning disk confocal scanner (Porter B047A)

Lasers, Filters, & Disks

LasersÌý

  • 405, 488, 561

Emission filters & molecules imaged (All filters are Semrock)

  • Emission filter: 417-477 (nm) (custom); DAPI, Hoechst, BFP, etc.
  • CFP has been imaged using this microscope
  • Emission filter: 500-550Ìý(nm); Alexa488, FITC, EGFP, etc.
  • Venus and YFP have been imaged using this microscope
  • Emission filter: 580.5-653.5Ìý(nm); Alexa568, 594, TxRed, TRITC, mRFP, mCherry, mKO, TdTomato. etc.
  • Brightfield is provided by a deep-red LED

Dichroic Mirror

  • Triple, 405/388/561 (Semrock, custom)

Disks

• Yokogawa CSU-X1 Dual Nipkow disks, 25 and 50 micron pinholes - special from Yokogawa for the CV1000

Standard objectives (All are Olympus)

  • 10x 0.40NA UPLAPO WD 3.1 (mm)
  • 20x 0.75NA UPLSAPO WD 0.65 (mm)
  • 20x 0.45NA LUCPLFLN, with correction collar, LWD 6.4-7.6 (mm)
  • 40x 0.60NA LUCPLFLN, with correction collar, LWD 2.7-4.0 (mm)
  • 40x 1.30NA UPLFLN OIL WD 0.2 (mm)
  • 100x 1.40 UPLSAPO OIL WD 0.13 (mm)

Objectives available upon request

  • 60x 1.20NA UPLSAPO water immersion, with correction collar, WD 0.28 (mm)
  • 60x 1.35NA UPLSAPO OIL WD 0.15 (mm)
  • 60xÌýNA1.10;ÌýLUMFLN60XW LWD water immersion, with correction collar, WD 1.5 (mm)
  • Inquire with James.Orth@Colorado.edu

Hamamatsu IMAagEM X2 1k back-thinned EMCCD high-resolution mode camera

  • 1024 x 1024, 16 bit detector with 13 x 13Ìýmicron pixels
  • High ÌýQE from 500-650 (nm)
  • Binnig and gain to boot signal
  • High-speed imaging

ChronosÌýis equipped with an ultra-sensitive, high-resolutionÌýmode Hamamatsu EMCCD cameraÌýand a special Yokogawa X1 spinning disk with the option ofÌýdual microlens-enhanced Nipkow scanning disks with pinholes ofÌý50 μm or 25 μm; disks can also be removed for widefield imaging. Zero Drift (Olympus) autofocusing and software focusing.ÌýThis microscope has minimalÌýphototoxicity and photobleaching, andÌýis therefore great for live cell imaging at high temporal resolution, multi-point, and multi-day time-lapse. Sample mapping, multi-point montaging of large tissue samples in X-Y-Z and low-to-medium throughput screening. Excellent brightfield imaging through the disk. Can reload previous sample map and imaging positions to re-image the same locations at different magnifications or over time.

Standard sample holders

  • Standard slides, multi-well slides
  • 1 x 35 (mm) dish, incubation chamber
  • 3 x 35 (mm) dish, incubation chamber
  • Multi-well plates; e.g. 6, 24, 48, 96 and 384 wells
  • Multi-well plates, incubation chamber; e.g. 6, 24, 48, 96 and 384 wells - must be low profile plates, contact Director for help

Environmentally controlled chambers

  • Selectable temperature
  • Selectable carbon dioxide concentration
  • Humidified

Acquisition

  • Yokogawa CV1000 1.6

Image Viewing

  • Yokogawa CV1000 1.6 Viewer (PC only), contact Director
  • FIJI
  • Other tif viewing applications, e.g. Imaris, Nikon NIS Elements, Evident Scientific cellSens
CV1000

For training email, James.Orth@Colorado.edu