Integrated Light Microscopy Core

Lightsheet Microscopy

Single Plane Illumination Microscopy
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Selective / Single Plane Illumination Microscopy (SPIM) aka Lightsheet Microscopy

What is SPIM? Selective / Single Plane Illumination Microscopy is particularly suited to long-term timelapse imaging of larger living specimens, such as zebrafish, Drosophilia embryos, and small plants. This technology has also been used to image living and fixed 3D cell cultures, including organoids, cysts and 3D cell migration assays.

 

 

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
Zeiss Lightsheet Z.1

The Zeiss Lightsheet Z.1 was provided by the Institute for Genomics & Systems Biology (IGSB).

​Overview: The Zeiss Lightsheet Z.1 is one of the first commercially available Single Plane Illumination microscopes. It is particularly suited for temperature controlled, long-term imaging of living samples such as zebrafish or Drosophila embryos. Samples can be kept in the chamber and imaged for hours or days. Sensitive cameras and single plane illumination allow for gentle sample illumination, reducing photodamage.

Location: KCBD 1250C

Training Contact: Christine Labno

Fluorophores this microscope can image:

  • Cyan (CFP)
  • Green (GFP, Alexa 488)
  • Near Red (DsRed, mCherry)

Excitation Light Sources:

  • solid state 445nm laser
  • solid state 488nm laser
  • solid state 561nm laser

Emission Detection and Filters:

  • Two chilled PCO-Edge scMOS 16-bit cameras with 1920 x 1920 pixels for fast, detailed, simultaneous two color imaging.
  • BP 460-500nm (CFP),
  • BP 505-545nm (GFP/YFP),
  • SP 550nm (GFP/YFP),
  • LP 585nm (mCherry)

Objectives:

  • 5x / NA 0.16 dry (uses 5x NA 0.1 dry illumination objectives)
  • 20x / 1.0 water dipper (uses 10x NA 0.2 dry illumination objectives)
  • 40x / 1.0 water dipper (uses 10x NA 0.2 dry illumination objectives)

Sample Chamber:

  • Sample is held in a buffer-filled chamber during imaging. PBS, seawater and nutrient buffers (e.g. E3) have all been used successfully.
  • Water cooled/heated Peltier block for chamber temperature settings from 10C-42C. Temperature feeds back to the software from both the block and the sample chamber.

Special Features:

  • Green/red or cyan/red simultaneous imaging is possible. Cyan/green or three color serial imaging is also possible.
  • 360 degree sample rotation plus programming for imaging from multiple angles. Incorporation of beads allows for multi-angle 3D reconstructions.
  • Off-line version of Zeiss Zen software available on facility workstation.
LaVision Ultramicroscope II Large Format Upright Lightsheet

Overview: The LaVision Ultramicroscope is an upright lightsheet microscope ideal for large, fixed and cleared fluorescent samples. For comparisons of clearing techniques, Visikol has a nice chart detailing the basics of many commmon methods.

Location: KCBD 1250B

Training Contact: Christine Labno

Fluorophores this microscope can image:

  • Cyan (CFP)
  • Green (GFP, Alexa 488)
  • Near Red (Alexa 543, Alexa 594)
  • Far Red (Alexa 647)
  • Near IR (Alexa 700)

Excitation Light Sources:

  • solid state 440nm laser
  • solid state 488nm laser
  • solid state 561nm laser
  • solid state 640nm laser
  • solid state 785nm laser

Emission Detection and Filters:

  • cyan emission filter at 480/40
  • green emission filter at 525/50
  • red emission filter at 620/60
  • far red emission filter at 680/30
  • near IR emission filter at 845/55
  • Andor Zyla 4.2 megapixel sCMOS camera with 6.5um pixels

Objectives:

  • 1.3x NA 0.08, 9mm working distance, dry
  • 4x NA 0.3, 6mm WD, dipper with refractive index matching collar from 1.30 (water) to 1.55 (DBE)
  • 12x NA 0.53, 10mm WD, dipper with two interchangeable RI-matching collars from 1.414-1.574 and 1.487-1.647

Sample Chamber:

  • Upright format with dipping objectives
  • Unique large, cuvette-based imaging chamber to allow for imaging of cleared specimens in clearing solution or other medium
  • Inner dimensions of cuvette are 72 x 74 x 35mm
  • Interchangeable sample holding platforms can accommodate samples up to 1cm^3

Special Features:

  • Maximum lightsheet width = 17mm
  • Total xy travel distance = 10mm
  • Maximum scan area (xy) = 27mm^2 in a single field of view
  • XYZ tiling imaging with Python-based stitching (Terastitcher) for mosaic-based imaging of large specimens
3i Lattice Lightsheet Microscope with Bessel Beam Illumination

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. All samples MUST be on 5mm round coverslips. Please be sure to arrange all sessions in advance with Core Staff as use of this microscope requires careful calibration of the system before each use.

Location: KCBD 1250B

Training Contact: Vytas Bindokas Note: use of this microscope requires training in calibration of the system as well as use for sample imaging.  Core staff is available to help with system calibration, as it must be done before each session.

Fluorophores this microscope can image:

  • Cyan (CFP)
  • Green (GFP, Alexa 488)
  • Near Red (mCherry, Alexa 594)
  • Far Red (Cy5, Alexa 647)

Excitation Light Sources:

  • solid state 405nm laser
  • solid state 488nm laser
  • solid state 561nm laser
  • solid state 642nm laser
  • LED for brightfield illumination

Emission Detection:

  • Hamamatsu Flash4v2+ sCMOS high-sensitivity, low noise camera (100nm pixel size)

Objective:

  • Nikon 25x NA 1.1 water immersion objective

Sample Chamber:

  • Due to sample holder configuration all samples MUST be on 5mm round #1.5 glass coverslips
  • Heated bath chamber for live cell work. Perfusion is possible but we have no CO2 at this time

Features:

  • High speed, low light 3D timelapse imaging.
  • 100nm pixel size (xy)