Confocal and Specialized Microscopy

The Confocal and Specialized Microscopy Shared Resource (CSMSR) provides advanced microscope systems for multidimensional optical imaging of living and fixed cells and tissues. The CSMSR offers a wide range of state-of-the-art microscopy technologies, from super-resolution microscopy to laser capture and microirradiation.

Our full-time staff provide consultation, assistance, training, and technical support. Trained in cell biology, biophysics, microbiology and image analysis, with in-depth expertise in light microscopy, we can help with experimental design, image acquisition, and data interpretation.

Technologies Offered

  • Confocal microscopy (scanning and spinning-disk)
  • Two-photon microscopy
  • Wide-field fluorescence microscopy
  • Digital deconvolution
  • Transmitted-light imaging (phase, DIC, histology)
  • Super-resolution imaging (STORM and SIM)
  • Cell surface imaging with <100 nM z-resolution (TIRF)
  • Laser-capture microdissection
  • Microfluidic cell culture while imaging
  • Software for image analysis, processing, and presentation

Application Examples

  • Live-cell imaging
  • Fluorescence Recovery after Photobleaching (FRAP)
  • Förster Resonance Energy Transfer (FRET)
  • Colocalization analysis
  • Cell and particle tracking studies

Locations and Contacts

Lasker Biomedical Research Building
3960 Broadway, 3rd Floor, Room 320 (Entrance on 166th St.)
Tel: (212) 342-3604

Liza A. Pon, PhD, Director
lap5@cumc.columbia.edu

Tel: (212) 305-1947

For consultation and training:

Theresa Swayne, PhD, Manager
tcs6@cumc.columbia.edu
Tel: (212) 342-3604

Laura Munteanu, PhD, Assistant Manager
elm2157@cumc.columbia.edu
Tel: (212) 342-3604


Online Reservations

The CSMSR uses the iLab Core Management System for service requests and billing. Before you can request services, you must log into iLab and be approved as a lab member.


User Fees and Policies

New Users

  • Before making a reservation, you must consult with the staff to discuss your experimental needs. This free consultation will help us identify the best instrument needs for your study.
  • To set up a consultation, please contact us and provide experimental details:
    • What is the goal of the imaging? What question do you want to answer, or what phenomenon do you want to illustrate with images?
    • Do you know which microscope you would like to use?
    • What is the sample?
    • How is the sample mounted?
      • Slide
      • Chambered slide
      • Chambered coverslip
      • 35 mm glass-bottom dish
      • 35 mm plastic dish
      • Multiwell plate
      • Some other format
    • For fluorescence imaging, what labels are you using?
    • Do you need transmitted-light (bright-field) imaging?
    • Is there anything else we should know about your sample or project?

Assisted Use and Training

You may choose to be assisted at the microscope by a staff member, or request training for independent microscope use. Assisted use is available from 9:30 a.m. to 5:00 p.m., Monday through Friday. Please request appointments for assisted use or training at least 2 days in advance.

Certified independent users may use the microscopes at any time.

Hourly Rates

Initial consultation

  • No charge

Assisted microscope use or training, peak hours

  • Cancer Center Members*: $99.30
  • Non-Cancer Center Members: $132.40
  • External users: $211.84

Unassisted use, peak hours

  • Cancer Center Members*: $54.45
  • Non-Cancer Center Members: $72.60
  • External users: $116.16

Independent Use, off-peak hours

  • Cancer Center Members*: $45.38
  • Non-Cancer Center Members: $60.50
  • External users: $96.80

Image processing/analysis service, training, or programming

  • Cancer Center Members*: $45.00
  • Non-Cancer Center Members: $60.00
  • External users: $122.40

Image analysis workstation, unassisted

  • Cancer Center Members*: $12.38
  • Non-Cancer Center Members: $16.50
  • External users: $26.40

Technical support

  • Included in user fee

Note: Peak hours are 9:00 a.m. to 5:00 p.m., Monday through Friday. Reduced rates apply for overnight experiments.

*Members of the Pathology Department, which contributed funds for the super-resolution systems, pay the Member rate for use of those systems (SIM and STORM).

Time Limits

You may reserve time up to 7 days in advance. Shared Instrumentation Grant (SIG) lab affiliates can reserve the relevant instrument up to 10 days in advance. For the Nikon A1RMP (Orange), SIG labs are Clynes, Costantini, Doetsch, Mason, Pon, Vallee, and Wang. For the N-SIM, SIG labs are Gundersen, Mao, Pon, Vallee, and Worman.

You may sign up for a maximum of 4 peak hours per day or 10 peak hours per week. Peak hours are Monday through Friday, 9:00 a.m. to 5:00p.m.

If you have a special need to reserve an instrument further in advance or for a longer period, please contact us.

Cancellation Policy

If you’re more than 30 minutes late and you haven’t notified us, anyone may take over your time slot.

If you need to cancel, do so at least 1 day in advance. If you cancel on the day of your appointment, you will be charged for any time that is not used by someone else.

Acknowledgement and Co-authorship Policy

We request that the Confocal and Specialized Microscopy Shared Resource and its funding (NIH #P30 CA013696) be acknowledged in all publications that use our instruments, software, and/or expertise. This helps ensure our continued funding and ability to acquire new instruments.

In addition, we welcome opportunities to provide significant intellectual and practical contributions that will lead to shared authorship.


Microscopes and Supporting Equipment

Methods

  • Scanning confocal
  • Spinning-disk confocal
  • Wide-field fluorescence
  • 2-photon or second harmonic generation
  • Histology (color brightfield imaging)
  • Laser capture microdissection
  • Spectral confocal
  • Super-resolution localization microscopy (STORM/PALM)
  • Super-resolution structured illumination microscopy (SIM)
  • Total internal reflection fluorescence (TIRF)
  • UV microirradiation, targeted DNA damage

Instruments and Accessories

Nikon A1 Confocal

  • Microscope stand: Nikon Ti Eclipse inverted
  • Features:
    • Motorized XY Stage, allowing mosaic images and multi-point time-lapse imaging,
    • Autofocus using the Perfect Focus System
    • Targeted photobleaching, FRAP, FRET and FLIP capability
  • Samples: Slides, 35 mm  round culture dishes, chamber slides, chamber coverslips, multiwell plates
  • Fluorescence excitation: 405, 488, 561, 638 nm
  • Compatible fluorophores: Similar to DAPI, DyLight 405, FITC, TRITC, Cy5
  • Objectives: Nikon Objectives
  • Software: NIS-Elements

Nikon A1RMP Multiphoton

  • Microscope stand: Nikon Ti Eclipse inverted
  • Features:
    • Motorized XY Stage, allowing mosaic images and multi-point time-lapse imaging
    • Fast live imaging with resonant scanner and piezoelectric focus
    • Spectral unmixing of overlapping fluorophores or autofluorescence
    • Autofocus using the Perfect Focus System
    • Tokai Hit stage-top incubator and objective heater
    • Targeted photobleaching, FRAP, FRET and FLIP capability
    • 2-photon microscopy with non-descanned detector: Best for deep (up to 1mm) or long-term imaging
    • Spectral fingerprinting, multiphoton excitation fingerprinting, unmixing of multiple fluorophores/autofluorescence sources
  • Samples: Slides, 35 mm round culture dishes, chamber slides, chamber coverslips, multiwell plates
  • Fluorescence excitation:
    • 1-photon: 458, 488, 514, 561, 639 nm
    • 2-photon: Software-tunable Chameleon II laser (680-1040 nm)
  • Compatible fluorophores:
    • 1-photon: Similar to DAPI, DyLight 405, CFP, FITC, YFP, mOrange, TRITC, Cy5
    • 2-photon: Compatible with most red, green and blue fluorophores
  • Objectives:
    • 1-photon: Nikon Objectives
    • 2-photon: 25x/1.10 coverslip-corrected water-immersion IR lens
  • Software: NIS-Elements

Nikon N-SIM

  • Microscope standNikon Ti Eclipse inverted
  • Features:
    • SIM provides double the resolution of the conventional confocal microscope (lateral ~58-110nm; axial ~200-250nm)
    • Autofocus using the Perfect Focus System
    • 3D Axial Range: up to 20μm
    • Imaging speed: 0.6 sec/frame (2D SIM); 1.0 sec/frame (3D SIM)
    • Motorized Stage and Piezo Z
  • Samples: Slides, 35 mm round coverslip-bottom culture dishes, chamber slides, chamber coverslips
  • Fluorescence excitation: 405, 488, 561, 647 nm
  • Compatible fluorophores: DAPI, FITC, TRITC, Cy5
  • Objectives: Nikon Objectives
  • Software: NIS-Elements

Nikon STORM/Spinning Disk/TIRF

  • Microscope stand: Nikon Ti Eclipse inverted
  • Features:
    • Tokai Hit stage-top incubator and objective heater
    • Motorized Stage and Piezo Z
    • EMCCD Camera (512x512) (Andor DU-897 and Evolve)
  • Features-STORM: 
    • Allows imaging fixed samples with a lateral resolution of ~20-30nm and an Axial resolution of ~50-60nm
    • 2D and 3D STORM capabilities
  • Features-Spinning-disk confocal imaging: 
    • Yokogawa CSU-X1
    • Allows very fast imaging of bright samples (>50 frames per second)
    • Lower photo damage than scanning confocal imaging
  • Features-Total Internal Reflection Fluorescence:
    • Helpful for imaging events at the cell surface (~100nm) with very good signal/noise.
  • Samples: Slides, 35 mm round culture dishes, chamber slides, chamber coverslips, multiwell plates.
  • Fluorescence excitation: 405, 488, 561, 647 nm
  • Compatible fluorophores: DAPI, FITC, TRITC, Cy5
  • Objectives:
    • For TIRF and STORM, 60x/1.49 and 100x/1.49
    • For spinning-disk: Nikon Objectives
  • Software: NIS-Elements

Zeiss MicroBeam IV Laser Capture/Histology/Fluorescence

  • Microscope standInverted Zeiss AxioObserver.Z1
  • Features:
    • Automated stage for multi-location time-lapse or mosaic imaging
    • Autofocus using hardware (Definite Focus) or software
    • Pecon stage-top incubation
  • Features-Wide-field fluorescence:
    • Bioptechs objective heater
    • Fast switching LEDs for multicolor live imaging
    • Orca ER cooled CCD camera
  • Features-Microirradiation and Microdissection:
    • Non-contact laser catapulting for capturing live cells or material from frozen or fixed tissue
    • Captured material can be used for isolation of DNA, RNA, or protein, or for cell/tissue culture applications
    • 355-nm solid-state laser can also be used for:
      •    creating localized double-strand breaks in DNA
      •    ablating cells or subcellular structures
      •    photobleaching or photoactivation
  • Features-Color histology imaging:
    • Axiocam ICc for color imaging and histological staining
  • Samples: Slides, 35 mm round culture dishes, chamber slides, chamber coverslips, multiswell plates
  • Fluorescence excitation: Mercury halide lamp with fluorescence filters; LEDs at 390, 455, 470, 505, and 590 nm
  • Compatible fluorophores: DAPI, FITC, TRITC, CFP, GFP, YFP, RFP (DsRed)
  • Objectives: Zeiss Objectives
  • Software:
    • Zen Blue
    • PALM Robo

Accessories

A lab bench with dissecting microscope, minifuge, vortexer, water bath, and hotplate are available for basic sample preparation. For live-cell imaging experiments, a cell culture incubator with CO2 is available in the main microscope room. We also have a laminar flow hood for sterile tissue culture procedures. Stagetop incubators provide temperature, humidity, and CO2 control during experiments. A syringe pump and peristaltic pump are available for microfluidics and perfusion experiments. The CellAsic ONIX microfluidic system offers the opportunity for stable cell culture with simultaneous high-quality, long-term imaging of mammalian cells, yeast, or bacteria.

Nikon Objectives

Magnification

Numerical Aperture

Immersion

Notes

10x 0.4 air

OK for plates

20x 0.75 air

high-res, low-mag

25x 1.1 water

2-photon

40x 0.6 air OK for plates
40x 0.75 air

highest resolution without oil

40x 1.3 oil  
60x 1.49 oil

TIRF, super-resolution

100x 1.45 oil  
100x 1.49 oil TIRF
100x 1.45 oil

TIRF, super-resolution

Zeiss Objectives

Magnification Numerical Aperture Immersion Notes
2.5x 0.075 air  
5x 0.16 air  
10x 0.3 air

OK for plates

10x 0.5 air

high-res, low-mag

10x 0.4 air

OK for plates

20x 0.4 air laser capture, OK for plates
20x 0.6 air laser capture, OK for plates
40x 0.75 air

highest resolution without oil

40x 1.3 oil  
100x 1.3 oil  

Image Analysis and Presentation

We offer a Windows workstation for image analysis and processing, and a color laser printer for documentation. We provide consultation and training on the use of software for image processing, analysis, and presentation. Facility software includes:

  • Volocity Restoration (deconvolution), Visualization and Quantitation
  • NIS Elements
  • ImageJ and Fiji
  • Apple Quicktime Pro

Director

Liza Pon, PhD

Director