Integrated Light Microscopy Core

Superresolution Microscopy

Sub-diffraction fluorescence imaging
Check Microscope StatusNew User Training

Hands-on Microscopes

To access a microscope, contact the Technical Director listed in the description and schedule a hands-on training session. Trained users have 24/7 access to the facility and are given permission to schedule their own microscope sessions through our online scheduling software

Stimulated Emission Depletion (STED)

STED micrograph courtesy of V. Cloud, Doug Bishop lab

What is STED-CW? Stimulated Emission Depletion (STED) is an advanced form of laser scanning confocal microscopy that allows for a 3-4 fold improvement in resolution over traditional laser scanning confocal; from a smallest resolvable feature size of 150-200nm FWHM to approximately 50nm FWHM. This allows us to see objects that were previously below the limit of optical resolution for light microscopy (Abbe diffraction limit). CW (continuous wave) technology means that the depletion laser is continuous, not pulsed as in previous STED microscopes. This allows for superresolution at higher speeds than previously possible. See the Leica website for more information and example images.

For best results in STED-CW imaging: we recommend placing samples on a #1.5 coverslip (0.170mm thickness) mounted with ProLong Gold (Invitrogen) antifade mounting medium or Thiodiethanol (TDE). Ideally, samples should contain high quantum yield, photostable fluorophores such as: AlexaFluor 450/488/514, Dylight 488, Atto 425/488, eGFP, mCitrine, mVenus, or mCerulean. For information on STED imaging, or to set up a training session, contact Christine Labno or Vytas Bindokas.

Leica SP8 Laser Scanning Confocal with White Light Laser, 3D STED and FLIM

Overview: The Leica SP8 is a laser scanning confocal capable of fluorescence lifetime (FLIM), 3-color 3D STED and tau-STED imaging.

It features a white light laser (WLL) capable of excitation in any wavelength from 470nm to 670nm, and an acousto-optical beam splitter (AOBS) to select/introduce up to eight laser lines (at 8nm intervals) at a time. In addition to the WLL, there are Argon and UV lasers for imaging, photobleacing and photoactivation, so exciation spans the spectrum from UV to deep red. There are also three depletion lines for STimulated Emission Depeletion (STED) superresoluion: 775nm (depletes red and far red dyes), 660nm (depletes orange and yellow dyes) and 592nm (depletes green dyes). The acousto-optical tunable filters (AOTF) make it possible to detect a wide range of emission wavelengths with unlimited range on each of three HyDs (hybrid GaAsP detector) two PMTs (photon multiplier tube). Spectral scanning / unmixing in exciation and/or emission is available. Time gated fluorescence detection is possible on the three HyD detectors.

Location: KCBD 1250G

Training Contact: Christine Labno

Fluorophores this microscope can image:

  • Violet/near UV (ex: DAPI, Alexa 405)
  • Cyan (ex: CFP)
  • Green (ex: GFP, Alexa 488)
  • Yellow (ex: mCitrine, YFP)
  • Orange (ex: Cy3, Alexa 543)
  • Near Red (ex: Texas Red, Alexa 594)
  • Far Red (ex: Cy5, Alexa 647)
  • Near IR (ex: Alexa 700)

Excitation Light Source(s):

  • White light laser -- ANY wavelength from 470nm - 670nm. Up to 8 lines at once with 8nm spacing
  • Near UV laser at 405nm
  • 5 line Argon laser at 458, 476, 488, 496 and 514nm

Lasers for STED depletion:

  • pulsed 775nm (depletes red and far red dyes)
  • continuous wave 660nm (depletes orange and yellow dyes)
  • continuous wave 592nm (depletes green dyes)

Emission Detection:

  • Custom emission detection of any wavelength range between 410nm and 800nm
  • 2 chilled photon multiplier tubes (PMTs)
  • 2 chilled, single-molecule sensitive hybrid GaAsP/PMT detectors (SMD-HyDs) with optional time gating
  • 1 non-SMD hybrid GaAsP/PMT detector (HyD) with optional time gating
  • Transmitted light detector
  • 8-, 12-, or 16-bit grayscale output

Objectives (base magnification, additional optical zoom is possible):

  • 10x / NA 0.4 dry
  • 20x / 0.7 multi-immersion (water, oil or glycerol)
  • 40x / 1.25 oil
  • 63x / 1.4 UV oil
  • 100x / 1.45 oil (STED-rated)
  • 93x / NA 1.3 glycerol (must be installed prior to use, STED-rated, motorized correction collar)
  • 86x / NA 1.20 water (must be installed prior to use, STED-rated, motorized correction collar)

Sample Chamber:

  • Inverted platform for imaging of slides or dishes
  • Automated XY stage for tiling / multipoint scanning
  • "SuperZ" galvo focusing stage, 1.5 mm range. Demonstrated capability of three cell volumes per second (12 x 1-mm optical slices each)
  • Navigator software for non-rectangular tiling. Increases speed for large tiling projects. Includes a fast "stage overview" mode
  • Chamber slide users see our chamber slide use warning before starting your cultures!

Special Features:

  • Capable of Fluorescence Lifetime Imaging (FLIM) and lifetime enhanced STED (tau-STED) imaging
  • Capable of 2D or 3D superresolution imaging in XY, XZ or XYZ.
  • AOBS (acousto-optical beam splitter) plus sequential scanning capability allows for rapid sequential scanning of fluorophores with minimal bleed-through or cross-talk
  • Hybrid GaAsP detectors (HyDs) and chilled SMD HyD detectors feature digital time gating (0.1 nanosecond increments, 3.5 nanosec. (min) to 12 nanosec (max) range) and photon counting mode.
  • Transmitted light detector features DIC polarizer/analyzer with prisms for 20x, 40x and 63x.
  • Notch filters for 488, 561, 594 and 633 from the WLL
  • Spectral scanning across excitation, emission, or both, allowing for separation of fluorophores with similar ranges (e.g. GFP and FITC) through spectral unmixing.
  • Time-gating on HyDs allows for differentiation of fluorophores with similar spectra or enhancement of STED resolution from the pulsed 775nm laser.
  • Tandem scanner with dual scanning galvanometer mirrors allows for either high speed scanning (max 16,000 Hz scan rate) 25 images/sec at 512x512; strip scans to 333 fps (5 channels) OR high pixel density scanning (imaging to 4k x 4k [16 megapixels] per channel in galvo mode).
  • Standard galvo scanner includes beam park for FRAP, bleaching and photoactivaiton
  • Optical zoom for sampling to 5nm pixel size
  • Wizards for FRAP, FRET and Live Data Mode
  • 3D/4D reconstruction software built in to LAS_X image collection software
  • LAS_X Leica confocal software on Windows 10
  • Off-line version of LAS_X software available on facility workstation
Leica SP5 STED Laser Scanning Confocal

​Overview: The SP5 II is an advanced, high speed laser scanning confocal platform. It includes an acousto-optical beam splitter (AOBS) to select/introduce most excitation laser lines. There are eight excitation lines available, spanning the spectrum from green to far red. The acousto-optical tunable filters (AOTF) make it possible to detect a wide range of emission wavelengths with unlimited range on each of three PMTs (photo multiplier tubes), two HyDs (hybrid GaAsP detector) or two APDs (avalanche photodiodes).

Location: KCBD 1250F

Training Contact: Christine Labno

Fluorophores this microscope can image:

  • Cyan (ex: CFP)
  • Green (ex: GFP, Alexa 488)
  • Yellow (ex: mCitrine, YFP)
  • Orange (ex: Cy3, Alexa 543)
  • Near Red (ex: Texas Red, Alexa 594)
  • Far Red (ex: Cy5, Alexa 647)
  • Near IR (possible but not ideal, ex: Alexa 700)

Excitation Light Source(s):

  • 5 line Argon laser at 458, 476, 488, 496, and 514nm
  • DPSS laser at 561nm
  • orange HeNe laser at 594nm
  • red HeNe laser at 633nm

Emission Detection:

  • Custom emission detection of any wavelength range between 410nm and 800nm
  • 3 chilled photon multiplier tubes (PMTs)
  • 2 hybrid GaAsP/PMT detectors (HyDs) with optional time gating
  • 2 internal avalanche photodiode detectors (APDs) for high sensitivity (green/red and CFP/YFP filters available)
  • Transmitted light detector with DIC polarizer/analyzer plus prisms for most objectives available
  • 8-, 12-, or 16-bit grayscale output

Objectives (base magnification, additional optical zoom is possible):

  • 10x / NA 0.4 dry 2.74mm WD
  • 20x / 0.7 multi-immersion (water, oil or glycerol) 0.26-0.17mm WD
  • 40x / 1.25-0.75 oil 0.22mm WD
  • 63x / 1.4-0.6 UV oil 0.14mm WD
  • 100x / 1.40 oil (STED-rated) 0.13mm WD
  • 50x / 0.9 dry WD=0.28mm (must be installed prior to use)
  • 63x / 1.3 glycerol CORR WD=0.30mm (must be installed prior to use)

Sample Chamber:

  • Full wrap incubator box with warm air heating for live samples
  • Inverted platform for imaging of slides or dishes
  • Automated XY stage for tiling / multipoint scanning
  • "SuperZ" galvo focusing stage, 1.5 mm range. Demonstrated capability of three cell volumes per second (12 x 1-mm optical slices each)
  • Chamber slide users see our chamber slide use warning before starting your cultures!

Special Features:

  • Continuous wave depletion laser at 592nm allows for single or dual color super resolution (STED method) with either the resonant (high speed) or galvo (high pixel density) scanners.
  • STED mode allows for resolution of particles down to 50nm FWHM (cyan, green, yellow fluorophores only)
  • Tandem scanner with dual scanning galvanometer mirrors allows for either high speed scanning (max 16,000 Hz scan rate) 25 images/sec at 512x512; strip scans to 333 fps (5 channels) OR high pixel density scanning (imaging to 8k x 8k [64 megapixels] per channel).
  • Standard scanner includes beam park for FRAP, bleaching and photoactivaiton
  • Sequential scanning capability allows for rapid sequential scanning of fluorophores with minimal bleed-through or cross-talk
  • Wizards for FRAP and FRET
  • AOTF (acousto-optical tunable filters) for spectral scanning, allowing separation of fluorophores with similar ranges (e.g. GFP and FITC) through spectral unmixing
  • LAS_AF Leica confocal software on Windows
  • Off-line version of LAS_AF software available on facility workstation

Ground State Depletion (GSD)

Depth-coded GSD image courtesy of Jiping Yue, Xiaoyang Wu lab

What is GSD? Ground State Depletion is one of the “pointilist” techniques for superresolution microscopy. This group also includes Stochastic Optical Reconstruction Microscopy (STORM) and Photoactivated Localization Microscopy (PALM).”Pointilist” superresolution microscopy techniques use various methods to manipulate a fluorescently labeled sample into returning only a handful of diffraction limited (200nm) fluorophore signals at one time. These signals are then mathematically processed to localize them to sub-diffraction (20nm) spots. Thousands of frames worth of these localized spots are collected over a potentially minutes-long imaging time and integrated on the fly to form a complete image. Up to three fluorophores (green, red, deep red) can be imaged serially to create multi-color images. TIRF illuminated, 2D epifluorescent and 3D superresolution imaging are all possible.

Leica GSD/TIRFM Ground State Depletion Superresolution Microscope

The Leica GSD was provided by the Institute for Genomics & Systems Biology (IGSB) and the Institute for Molecular Engineering (IME).

Overview:The Leica GSD is a four-color Total Internal Reflection fluorescence (TIRF) and a three-color Ground State Depletion (GSD) microscope. It is not a confocal microscope. There are four excitation lines available, spanning the spectrum from violet to far red. All can be used for TIRF; green to far red can be used for GSD.

Location: KCBD 1250B

Training Contact: Christine Labno or Vytas Bindokas

Fluoropohores this microscope can image:

  • Blue (ex: DAPI, Alexa 405) - TIRFM ONLY, NO GSD
  • Green (ex: GFP, Alexa 488)
  • Orange (ex: Cy3, Alexa 543)
  • Near Red (ex: Texas Red, Alexa 594)
  • Far Red (ex: Cy5, Alexa 647)

Excitation Light Source(s):

  • solid state violet laser at 405nm (used to "backpump" which increases fluorophore blinking, can also be used for TIRF excitation)
  • solid state blue laser at 488nm
  • green fiber laser at 532nm
  • red fiber laser at 642nm

Excitation and Emission Filters:

  • Green: simultaneous excitation at 400-410 and 483-493nm with dichroic at 496nm and emission at 425-475nm and 505-605nm
  • OPTIONAL manual green band pass filter for emission at 503-547nm
  • Red: simultaneous excitation at 400-410nm and 527-537nm with  dichroic at 541nm and emission at 425-475nm and 550-650nm
  • OPTIONAL manual red band pass filter for emission at 582-636nm
  • Far Red: simultaneous excitation at 400-410nm and 637-647nm with dichroic at 649nm and emission at 425-475nm and 660-760nm
  • OPTIONAL manual long pass filter for far red emission at 664nm (664 LP)
  • Quad: ex 400-410nm; 483-493nm; 27-537nm; 637-647nm dichroics: 417, 496, 544, 655 em: 421-477nm; 497-519nm; 547-621nm; 666-732nm

Emission Detection:

  • iXon Ultra EMCCD camera for high frame rate, low light imaging with low read noise
  • Option to use a Photometrics Prime95B camera and Hamamatsu w-Gemini image splitter operated by a second imaging computer. This allows simultaneous two-color GSD acquisition and/or development of biplane 3D localization

Objectives:

  • 160x / 1.43 oil 0.07mm WD - this is a state-of-the-art, adhesive-free objective developed by Leica exclusively for GSD imaging
  • 10x, 20x, 40x and 60x DRY objectives are available for ocular viewing and widefield images ONLY

Sample Chamber:

  • Inverted platform
  • Small laser safety chamber for imaging on #1.5 coverslips or 35mm glass-bottom dishes (sorry, NO heated stage or CO2 at this time)
  • Manual XY stage movement
  • PiFoc precision focusing / Z step control

Special Features:

  • Supressed Motion (SuMo) stage which locks the 160x objective to the stage to minimize sample drift
  • Camera-based, software-driven TIRF laser focusing and alignment.
  • Software-based TIRF angle setting allows for your choice of multiple laser angles, creating varying evanescent wave penetration depths. TIRF imaging direction (north, south, east or west) can also be selected
  • Two off-line workstations for data processing

Proper Sample Preparation for Ground State Depletion: Samples should be mounted or grown on #1.5 thickness coverslips (high tolerance Schott glass if possible) and stained with high quantum yield fluorophores such as: AlexaFluor 647, 555, or 488, Atto 647 or 488, Rhodamine 6G, Cy5, Cy3b or bodipy (this is not an exhaustive list). DO NOT use DAPI! Also, DO NOT mount the coverslips to slides. Bring unmounted coverslips in the holding medium of your choice (PBS works well) for imaging. We will provide MEA mounting buffer and apply it just before imaging. For more information on GSD sample prep contact Christine Labno or Vytas Bindokas.

Structured Illumination Microscopy (SIM)

Lattice Lightsheet Bessel Beam Illumination Microscope

Overview:This is a commerically-produced clone of the Betzig system described in Science October 2014 and currently running at the HHMI Janelia Campus’s Advanced Imaging Center. It is designed for low-light 3D time lapse imaging. This is NOT like the Zeiss Lightsheet.Z1 meant for large preps! Imaging is by means of water-immersion objectives and is limited to less than 100 micrometers depth of penetration.

Sample Preparation: All samples MUST be on 5mm round coverslips. Use of this microscope requires a Staff person to operate the system. Please be sure to arrange your session in advance with Core Staff. Contact Vytas Bindokas or Christine Labno (information at left).

Special Features:

  • High speed, low light 3D timelapse imaging.
  • Heated bath chamber for live cell work. Perfusion is possible but we have no CO2 at this time
  • Hamamatsu Flash4v2+ sCMOS high-sensitivity, low noise camera
  • 100nm pixel size (xy)
  • Nikon 25x NA 1.1 water immersion objective
  • 405nm (DAPI), 488nm (GFP), 561nm (mCherry) and 642nm (Cy5) lasers
  • LED for brightfield illumination