High-Content Imaging
Microscopic images for scientific research.

Mouse stem cells from Phinney lab at UF Scripps.
Mouse stem cells from Phinney lab at UF Scripps are tagged with fluorescent dye to reveal specific cell structures.

Our aim is to provide cutting edge instrumentation for High-Content Imaging (HCI), which allows the acquisition of microscopic images in high-throughput. This enables investigators to monitor cellular or subcellular morphology in response to libraries of small molecules or gene-perturbing molecules.  The core also offers resources for primary cell culture and automated liquid handling.


Equipment

Imaging Flow Cytometry: Image Stream mkII

The Amnis ImageStream X MkII System from Luminex combines Flow Cytometry with Microscopy enabling the individual cells signals detected with the high-throughput of the flow stream to be resolved at the sub cellular  level under magnification. As a result, the microscopic phenotypes observed are supported by the robust statistical analysis conferred by the large sample sizes. The technology has applications in Cell Signaling, internalization/Phagocytosis, Co-localization, Cell shape changes, Chemotaxis, Cell-cell interaction, Cell death, Autophagy, Cell Cycle, Stem Cell Biology, Microbiology, Parasitology. Other emerging applications are: Micro-nuclei assay, Netosis, Exosome Internalization, Protein aggregation and more.

Instrument web page

Researchers can be trained to become certified independent users of the instrument. Trainings are free. Please contact us to schedule. Independent users must comply with posted standard perating procedures at all times when using the instrument.

Rates

UF Scripps Community: $60/hour (Operator-assisted); $45/hour (User-operated)
External Academic: $60 per hour plus indirect costs.

Data Analysis

Raw image files (.rif) are loaded into the IDEAS software for analysis. Guided wizards are available to help with common analysis pipelines such as co-localization, translocation, shape change, and internalization. Users may also build their own templates to suit their needs by combining image masks, features, and populations. Image masks are used to identify regions of interest within the image such as cells, features quantify aspects of these masks such as fluorescent pixel intensities or shape, and populations are used to group images of similar features. Each of these categories can be combined and operated on in many ways to allow highly flexible analyses. The resulting population feature values can then be organized and exported for further analysis and hypothesis testing. The IDEAS software is available for free download through the Flow Core.

Objectives

  • Magnification 20X: Numerical aperture 0.5, field of view 120 um, width covered per pixel 1 um
  • Magnification 40X: Numerical aperture .75, field of view 60 um, width covered per pixel .5 um
  • Magnification 60X: Numerical aperture .9, field of view 40 um, width covered per pixel .33 um

Cameras

The instrument has two CCD cameras with time delay integration. Light transferring between neighbor pixels is in exact synchrony with the velocity of the cell stream to prevent blur and maintain a 0.5 um pixel resolution.

Five Lasers Per Brightfield

Violet 405 nm, Blue 488 nm, Yellow-green 561 nm, Red 642 nm, and side scatter 785 nm lasers.

Compatible Cell Vessels

Eppendorf Tubes

12 Channels

Nine-colors fluorescence plus two brightfield and side scatter.


High Content Imaging: IN Cell 6000

The IN Cell 6000 analyzer is an automated high content confocal imager. Image acquisition relies on the instrument’s laser-based, auto-focus function to determine the exact position of the target, followed by hands-free canning according to user-designed protocols. In focus fields are scanned in 2D or 3D and the image files produced are promptly uploaded as .xdce (image stacks) to a server. An environmental control unit renders the platform suitable for multi-hour image acquisitions of live cells. Downstream analysis pipelines, uniquely built by the researcher drive software-based segmentation of signals of interest, which are objectively quantitated in high-throughput. By generating multi-parametric, spatially and morphologically resolved data, the IN Cell 6000 makes up a powerful research tool in applications from RNAi, CRISPR and drug libraries screens to phenotypical assays such as live/dead and cell cycle, apoptosis, translocation, DNA damage, angiogenesis, neurite outgrowth, cell migration, mitochondrial morphology and function and many yet to be imagined applications aimed at exploring the inner workings of the cellular environment.

Objectives

  • Magnification 4X: Numerical aperture 0.2, No spherical aberration collar, working distance 20 um
  • Magnification 10X: Numerical aperture 0.45, No spherical aberration collar, working distance 4 um
  • Magnification 20X: Numerical aperture 0.75, Yes spherical aberration collar, working distance 1 um
  • Magnification 60X: Numerical aperature 0.95, Yes spherical aberration collar, working distance 0.15 um

Cameras

Scientific Grade sCMOS Camera, 2560 x 2160 pixels (5.5 Megapixel)

Large Field of View (Will capture about 80% of well area of a 96-well plate at 4X on a single field).

Lasers

  • 405 nm (UV)
  • 488 nm (blue)
  • 561 nm (green)
  • 640 nm (red)

Emission Filters

DAPI Emission

  • Laser line: 405 nm (UV)
  • Channel: DAPI
  • Center wavelength: 455 nm
  • Bandwidth 50 nm

FITC Emission

  • Laser line: 488 nm (blue)
  • Channel: FITC
  • Center wavelength: 525 nm
  • Bandwidth 20 nm

TRITC Emission

  • Laser line: 561 nm (green)
  • Channel: TRITC
  • Center wavelength: 605 nm
  • Bandwidth 52 nm

Cy5

  • Laser line: 640 nm
  • Channel: Cy5
  • Center wavelength: 706.5
  • Bandwidth: 72

Environmental Control

Temperature and CO2 modules maintain live cells at physiological culturing conditions during multi-hour scans.

Compatible Vessel Formats

The IN Cell 6000 can hold slides and most commercially available microplate formats. It is highly recommended that imaging grade plates be used. Please, see reference to book chapter and links to the main vendors of imaging-grade microplates below. (Resources.)

Resources

Springer Link’s book: “High Content Screening. A Powerful Approach to Systems Cell Biology and Phenotypic Drug Discovery”, edited by Paul A. Johnston and Oscar J. Trask. is an excellent resource for those new to High  ontent Imaging, while also containing chapters on more advanced applications. High Content Screening is freely available for downloading.

Of interest, chapter 6 “Guidelines for Microplate Selection in High Content Imaging” has useful information on Imaging-grade microplates. For high content imaging microplate suppliers, see links:

Contacts

Melissa Kazantzis

SCIENTIST
Phone: (561) 228-2898
Mailing Address:
120 SCRIPPS WAY
JUPITER FL 33458

Mayra Caramazana

Lab Admin Coordinator II
Phone: (561) 228-3221
Mailing Address:
130 SCRIPPS WAY # 1B2
JUPITER FL 33458

Alta S Johnson

Flow Cytometry Core Manager
Phone: (561) 228-2764
Mailing Address:
130 SCRIPPS WAY # 2C1
JUPITER FL 33458
Physical Address:
130 SCRIPPS WAY RM C276
JUPITER FL 33458

operator

Emily Fox, Ph.D.


(561) 228-3221
Location: C276