Confocals

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

Pic
AX NSPARC front view

Lasers:

Launch: LUD-S4 

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

DIC imaging possible (laser scanning confocal only)

Emission Filters & molecules imaged: (All are Chroma)

Confocal scanning:

  • 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.

NSPARC scanning:

  • 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

NSPARC super-resolution lens:

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

Additional standard objectives:

  • 2x PLAN APO  λ 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  NA 0.80 WD 0.80 (mm), DIC
  • 40XC PLAN APO λ S  SILICONE 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
  • DUX-VB, 4 PMT-GA GaAsP
  • Nikon Spatial Array Confocal (NSPARC) pinhole array sensor

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 trael
  • Adjustable GFP and RFP emission bandwidth (confocal only)
  • TOKAI-HIT Cell Incubator 1 x 35 (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)
  • Nikon Perfect Focus System
  • NIS Elements AR 6.02.03 with JOBS, general analysis

Email, James.Orth@Colorado.edu

Reserve the NSPARC

For training email, James.Orth@Colorado.edu

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

Pic
Thor

 

Lasers:

Launch: LUNV6, high-output

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

Emission Filters & molecules imaged: (All 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)

Light source, filters & viewing by eye:

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

N-SIM super-resolution lens:

  • 100x 1.49NA OIL SR Apo TIRF

Additional standard objectives:

  • 4x 0.2NA Plan Apo λ 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 request: 

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

Laser Scanning Confocal:

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

N-SIM:

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

 

 

Sample Holders

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

Capabilities

  • 3D structured illumination, 'fast' 2D SIM
  • 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)

  • Nikon Perfect Focus System

    SIM imaging with 647nm laser:

    Thor, SIM, far red

Email James.Orth@Colorado.edu

Reserve THOR - Nikon TiE A1+/NSIM LSC

For training email, James.Orth@Colorado.edu

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

Pic

 

BlackWidow panarama

Full system

 

Lasers & Filters

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

Emission Filters & molecules imaged: (All are Chroma)

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

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 request: 

  • 4x 0.13NA Plan Fluor WD 17.2 (mm)
  • 60x 1.27NA water SR Plan Apo WD 0.17 (mm)
  • Inquire with James.Orth@Colorado.edu
  • Andor iXon Ultra 897 EM-CCD, 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 nm Coherent OBIS laser (50mW)
  • SOLA Light engine
  • Motorized X, Y, Z
  • Nikon Perfect Focus System
  • TOKAI-HIT Cell Incubator; 1 X 35 (mm) dish or chamber slide (coverslip bottom).

Reserve BLACK WIDOW - Nikon TiE SDC

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)

Pic
Full system

Lasers & Emission Filters

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

Emission filters are from Olympus:

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

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, Cy5 with triple-band emitter, brightfield, DIC

Disks

• Yokogawa CSU-W1 Dual SoRa and 50 micron pinholes disks

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)
  • 30x 1.05NA UPLSAPO 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,  WD 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)
  • 60x NA1.10; LUMFLN60XW LWD water immersion, with correction collar, WD 1.5 (mm)

2 ORCA-Fusion BT Gen-III sCMOS CAMERAS

MORE here:

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

NIH 1S10OD038323. IN PROGRESS. Equipped with 2 ultra-low noise, high efficiency, Hamamatsu ORCA-Fusion BT Gen-III sCMOS cameras camera, a Yokogawa W1 SoRa dual disk with 50 micron pinholes, Zero Drift Compensation (Olympus) autofocusing and software focusing. This microscope has minimal phototoxicity and photobleaching, and is 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. Full DIC optics for 20, 30, 40, 60 and 100, excellent brightfield imaging through the disk. 

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 cellSens 4.4.1

Viewer

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

Pic
CV1000

Lasers & Emission Filters

  • 405, 488, 561, Brightfield is provided by a deep red LED

Emission filters:

  • Emission filter: 417-477 (nm): DAPI, Hoechst, BFP, etc. (Semrock, custom)
  • CFP has been imaged using this microscope
  • Emission filter: 500-550 (nm): Alexa488, FITC, EGFP, etc. (Semrock)
  • Venus and YFP have been imaged using this microscope
  • Emission filter: 580.5-653.5 (nm): Alexa568, 594, TxRed, TRITC, mRFP, mCherry, mKO, TdTomato. etc. (Semrock)

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)
  • 60x NA1.10; LUMFLN60XW LWD water immersion, with correction collar, WD 1.5 (mm)
  • Inquire with James.Orth@Colorado.edu

Hamamatsu IMAagEM X2 1k back-thinned EMCCD

MORE here:

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

Chronos is equipped with an ultra-sensitive, high-resolution mode Hamamatsu EMCCD camera and a special Yokogawa X1 spinning disk with the option of dual microlens-enhanced Nipkow scanning disks with pinholes of 50 μm or 25 μm; disks can also be removed for widefield imaging. Zero Drift (Olympus) autofocusing and software focusing. This microscope has minimal phototoxicity and photobleaching, and is 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

Reserve CHRONOS Yokogawa SDC

For training email, James.Orth@Colorado.edu