CMtO Services
Instruments
Cytometry and Microscopy to Omics operates one Main Facility (ERML) and three Satellite Facilities (RAL, Burill Hall, Everitt Lab) all of which are Biosafety Level Two (BL2).
The Main Facility consists of an instrument lab, computer room, wet lab and cell culture area. Facility staff are available to assist with instrument operation, training and protocol development Mon-Fri 9am-5pm.
The three Satellite Facilities provide trained users 24/7 access to flow cytometry instrumentation.
Click on the instrument names below for specific details.
Have a look at our protocols page for more information on panel design and different protocols used in flow cytometry
Please fill out our New User Registration Form and a staff member will reach out to you for training on our available instruments
Sorters (FACS)
BD FACS Melody
- Four- way cell sorter
- Gel filled Quartz cuvette.
- 3 laser system: 640nm, 561nm, 488nm
- Temperature control for sample input by software: 4°C, 20°C, 37°C & 42°C
- Nozzle size: 100um, 20 psi
- Sort collection 4-way sorting: 1.5-, 2.0-, and 5.0-mL tubes
2-way sorting: 1.5-, 2.0- and 5.0-mL tubes
1-way sorting: 6-, 24-, 48-, 96- and 384-well plates, 96-well PCR tray, microscope slide
BD FACS Melody configuration:
Please Click for More Information:
- BDFACS Melody Technical Specifications.pdf
- BDFACS Melody User Brochure.pdf
- Fluorochrome reference guide.pdf
- Fluorochrome laser guide.pdf
- BD Biosciences spectrum viewer
- Designing a panel based on fluorochrome performance: Spillover vs Resolution
Manufacturer: BD Biosciences
Equipment Model: FACS Melody
Location: Main Facility at 231 Edward R. Madigan Laboratory
Contact: Kerishnee Naicker or Marcin Wozniak: cmto-core@mx.uillinois.edu
BigFoot Invitrogen Thermofisher Spectral Cell Sorter
Key System Specifications
- Six-way sorter.
- Sort rates exceeding 70,000 events per second, and analysis rates greater than 100,000 events per second.
- Multi-well sorting into 96-well, 384-well plates. Straight-down sorting into 1536-well plates.
- Excitation lasers at 349 nm, 405 nm, 488 nm, 561 nm, 640 nm.
- Detection parameters up to 55 fluorescence with 5 scatters.
- 70, 100, 120, and 150 µm ceramic nozzle tips with adjustable pressure.
- Capable of output holders with 1.5, 5-, 15-, and 50-mL tubes
CMtO Bigfoot Cell Sorter Configuration:
Please Click for More Information:
BigFoot QuickStart Guide.pdf
BigFoot Technical Specifications.pdf
BigFoot User Brochure.pdf
Fluorophore and reagent selection guide
Designing a panel based on fluorochrome performance: Spillover vs Resolution
Manufacturer: ThermoFisher Scientific
Equipment Model: Bigfoot
Location: Main Facility at 231 Edward R. Madigan Laboratory
Contact Person: Marcin Wozniak: cmto-core@mx.uillinois.edu
Flow/Cell Analyzers
Bio-Rad Cell Counter
Key System Specifications
- Compatible with a variety of cell types. Including cell lines, primary cells, and stem cells.
- Auto-focusing technology removes variation from manual focusing.
- Precise cell counts in 30 seconds.
- Stores up to 100 counts for easy data access.
- Utilizes multifocal plane analysis to analyze cells accurately.
Please Click for More Information
Manufacturer: BIO-RAD
Equipment Model: TC20 Automated Cell Counter
Location: Main Facility at 231 Edward R. Madigan Laboratory
Contact Person: Kate Janssen: cmto-core@mx.uillinois.edu
Amnis ImageStream
Key System Specifications
- Analyzes up to 5,000 cells per second.
- Intuitive user interface for easy plotting and gating.
- Assisted compensation wizard.
- Up to 6 lasers to analyze sample volumes between 20-200 µL
CMtO Amnis ImageStream Configuration:
Please Click for More Information:
- IDEAS Data Analysis Software Manual.pdf
- Compensation Quick Start.pdf
- User Operation Quick Start.pdf
- Sample Preparation Guide.pdf
- Cytek ImageStream Brochure
Manufacturer: Cytek
Equipment Model: ImageStream Mk II
Location: Main Facility at 231 Edward R. Madigan Laboratory
Contact Person: Marcin Wozniak: cmto-core@mx.uillinois.edu
FACSymphony A1 Configuration ERML
Key System Specifications
- 16 fluorescent detectors available.
- Four high-powered 100 mW lasers for excitation boost.
- Utilizes BD FACSDiva™ software.
- Small particle side scatter detector for particles up to 90 nm.
CMtO FACSymphony A1 Configuration:
Please Click for More Information:
- Symphony System Brochure.pdf
- Symphony FACSFlow User Guide.pdf
- Symphony User Guide.pdf
- Fluorochrome reference chart.pdf
- Fluorochrome laser guide.pdf
- BD Biosciences spectrum viewer
- Designing a panel based on fluorochrome performance: Spillover vs Resolution
Manufacturer: BD Biosciences
Equipment Model: FACSymphony A1
Location: Main Facility at 231 Edward R. Madigan Laboratory and 264 Roger Adams Lab
Contact Person: Marcin Wozniak: cmto-core@mx.uillinois.edu
FACSymphony A1 Configuration RAL
Key System Specifications
- 16 fluorescent detectors available.
- Four high-powered 100 mW lasers for excitation boost.
- Utilizes BD FACSDiva™ software.
- Small particle side scatter detector for particles up to 90 nm.
CMtO FACSymphony A1 Configuration:
Please Click for More Information:
- Symphony System Brochure.pdf
- Symphony FACSFlow User Guide.pdf
- Symphony User Guide.pdf
- Fluorochrome reference chart.pdf
- Fluorochrome laser guide.pdf
- BD Biosciences spectrum viewer
- Designing a panel based on fluorochrome performance: Spillover vs Resolution
Manufacturer: BD Biosciences
Equipment Model: FACSymphony A1
Location: Main Facility at 231 Edward R. Madigan Laboratory and 264 Roger Adams Lab
Contact Person: Marcin Wozniak: cmto-core@mx.uillinois.edu
Luminex 200 Plate Reader
Key System Specifications
- Simultaneous analysis of multiple analytes within a low volume sample.
- Up to 80 samples analyzed within one single 96-well micro-titer plate.
- Luminex xPONENT v3.1 software
- 96-well plate read time of only 45 minutes.
- Equipped with 2 lasers: 635 nm and 532 nm.
Please Click for More Information:
Manufacturer: BIO-RAD
Equipment Model: TC20 Automated Cell Counter
Location: Main Facility at 231 Edward R. Madigan Laboratory
Contact Person: Kate Janssen: cmto-core@mx.uillinois.edu
BD Symphony A1 with HTS
Key System Specifications
- 16 fluorescent detectors available.
- Four high-powered 100 mW lasers for excitation boost.
- Utilizes BD FACSDiva™ software.
- Small particle side scatter detector for particles up to 90 nm.
- Acquisition in less than 15 minutes for 96-well plates
- 2-10 µL sample volume (HTS mode)
CMtO FACSSymphony A1 Configuration:
Please Click for More Information:
- BD High Throughput Sampler User’s Guide_1.pdf
- HTS Quick Reference Guide_0.pdf
- BD FACSFlow Supply System User’s Guide.pdf
- Symphony System Brochure_0.pdf
- BD FACSymphony™ A1_ User’s Guide.pdf
- Fluorochrome reference chart.pdf
- Fluorochrome laser guide.pdf
- BD Biosciences spectrum viewer
- Designing a panel based on fluorochrome performance: Spillover vs Resolution
Manufacturer: BD Biosciences
Equipment Model: FACSymphony A1
Location: 2117A Everitt Lab
Contact Person: Marcin Wozniak: cmto-core@illinois.edu
Microscopy
LSM 980 Airyscan 2
Key System Specifications
- Full spectral flexibility, ideal for multi-color experiments
- LSM Plus assists during live cell experimentation
- 32 detector elements to provide super-resolution results.
- Zen microscopy provides AI Sample Finder to locate region of interest
Sample Data:
Please Click for More Information:
Manufacturer: Carl Zeiss
Equipment Model: LSM 980 with Airyscan 2
Location: Main Facility at 231 Edward R. Madigan Laboratory
Contact Person: Mayandi Sivaguru, (217)778-2423, cmto-core@mx.uillinois.edu
Zeiss Axiolab 5
Key System Specifications
- New white LED illumination
- Smart Microscopy technology to provide ease in image capturing
- Automatically applied scaling
- Enhanced ergonomics
Sample Data:
Please Click for More Information:
Manufacturer: Zeiss
Equipment Model: Axiolab 5
Location: Main Facility at 231 Edward R. Madigan Laboratory
Contact Person: Mayandi Sivaguru, (217)778-2423, cmto-core@mx.uillinois.edu
Confocal Raman Spectroscopy, Atomic Force Microscope and Scanning Nearfield Optical Microscope- from WITec Alpha 300 Series
The WITec Alpha 300 RA Raman-AFM-SNOM system provides a state-of-the-art confocal Raman imaging, together with AFM and SNOM capabilities in one system. The system based is on an upright microscope from 10-100x objectives with VIS and NIR spectrometers 450nm-1100nm. It has three excitation wavelengths 488, 532 and 785nm. One can use any other microscopy system in the Core Facilites including Superresolution systems and overlay Raman spectroscopy images for correlative purposes. Examples are provided below.
The software helps analyze the data efficiently to identify peaks and interpret.
Offline full-version of the image analysis software is available in the Yellowstone computer in the Core Facilities. Please fill out the training request form here.
Sample Data
Correlative Superresolution Microscopy and Raman Spectroscopy of Human Kidney Stones
Sample Data
Correlative High-Resolution Widefield Brightfield Microscopy and Raman Spectroscopy of Human Kidney Stones
The below image composite shows individual color coded Confocal Images of Raman components and corresponding spectra and the merged components overlaid on high resolution color brightfield images at different magnifications.
Correlative Raman and Atomic Force Microscopy
Contact and AC Modes
Correlative Raman and Scanning Near-Field Optical Microscopy
Superresolution microscopy down to 60 nm can be achieved with the WITec scanning near-field optical microscope (SNOM) alpha300 S. SNOM, as compared to other optical superresolution techniques such as STED or STORM which is not dependent on fluorescent tags or specialized laser excitation sources. The operation of the system is straightforward as various measurement procedures such as a high-speed automatic cantilever approach and adjustment can be controlled through the intuitive software.
TrueSurfaceTM Microscopy
With TrueSurface module Raman spectroscopy on rough, inclined, irregularly shaped and curvy samples are possible as it keeps focus on those contoured surfaces. It uses an advanced optical profilometer integrated with the system that provides simultaneous acquisition of topographic and chemical/molecular information.
Sample Data
Surface of a 5mm2 polished concrete without TrueSurface (left) where the sample is focused only at the center and TrueSurface image on the right where the entire sample in focus retrieving the curvature of the sample.
Brochure and Manuals (pdfs)
Raman Protocols and Procedures
Free WITec Offline Software Download link-Please fill out the form at the end of this link to receive a download instructions.
https://www.witec.de/products/accessories/software-witec-suite/
Manufacturer: WITec Scientific Instrument and Technology GmbH, Germany
Equipment model: WITEC Alpha 300 RS
Location: Room 124 D, Carl R. Woese Institute for Genomic Biology
Contact Person for Correlative and Confocal Raman: Austin Cyphersmith, 217-244-9654, ajcypher@illinois.edu
Contact Person for AFM and SNOM: Austin Cyphersmith, 217-244-9654, ajcypher@illinois.edu
Training Request: https://www.igb.illinois.edu/corefacilities/training-request
North Star Imaging X5000 2D Digital Radiography and 3D Computed Tomography
North Star Imaging X5000 2D Digital Radiography and 3D Computed Tomography
The X5000 system boasts a large scanning envelop and excellent ergonomics for loading sizable objects while still maintaining the sensitivity to inspect even the smallest of samples.
Key System Specifications and Advanced Modules
- Geometric Magnification – >3000x
- Overall Maximum System Resolution – 1 micron
X-Ray Source and Tube
- Voltage Range – 10 kV to 225 kV
- Minimum Focal Spot Size 500nm
- Microfocus Tube
X-Ray Detector
- Varex 2520DX
- 8″ x 10″ (203mm x 254mm) image area
- Cesium Scintillator
- 127 micron pixel pitch; 1536 x 1920 pixels (approximately 3 megapixels), 16-bit resolution
- 30 fps (2×2 pixel binning) image acquisition; 12.5 fps at full resolution
- Tungsten shielded to 225kV
Vortex Scan
This technique scans elongated objects that traditionally cannot fit into a single exposure. The scan does not require volume stitching and the object length is only limited by the system scanning area.
mosaiX and subpiX
mosaiX is used to stitch multiple images forming one seamless image with a much larger field of view. Also, with the mosaiX an subpiX modalities, the effective imaging field of view is no longer limited by the detector panel size. This results in images with improved resolution that is typically double of what the detector alone is capable of achieving (note: this reduces the focal distance by 4″ or 10.16cm).
4D Computed Tomography Module
4D CT is an application that combines time and motion with traditional 3D CT volumes. The resulting dynamic data sets allows for the study of form, structure and function. It is an ideal application for failure analysis, reverse engineering and quality assurance.
Manufacturer: North Star Imaging, MN
Equipment Model: X5000
Location: Room 5, Carl R. Woese Institute for Genomic Biology
Contact Person: Glenn Fried, (217) 333-4328, gfried@illinois.edu
Sample images:
Fossil earwig in amber
The image shows the 3D CT reconstruction of a high performance cylinder head cast from scrap aerospace aluminum alloy AA7075
Omics
10x Visium Spatial Transcriptomics
DV-200 Analysis
https://www.agilent.com/en/promotions/2100-bioanalyzer-dv200-determination
Invitrogen Qubit 4-RNA Quality Check (RIN)
Cryostat for Fresh Frozen 10x Visium workflow
The System
https://epredia.com/solutions/cryotomy-solutions/
Operating Manual/Brochure
https://epredia.com/pdf/cryotomy/cryostar-NX70-en/
10x Fresh Frozen Sample Prep Videos
https://pages.10xgenomics.com/sup-how-to-visium-tissue-prep.html
10x Support for Fresh Frozen Visium
https://www.10xgenomics.com/support/spatial-gene-expression-fresh-frozen
10x Demonstrated Step by step Protocols for Fresh Frozen Visium
https://www.10xgenomics.com/support/spatial-gene-expression-fresh-frozen/documentation/steps/tissue-prep
Microtome for 10x FFPE-CytAssist workflow
The System
https://epredia.com/solutions/microtomy-solutions/
Operating Manual/Brochure
https://epredia.com/pdf/microtome/HM355S-en/
Typical 10x workflow involving CBC core infrastructure at Illinois
Visium Spatial transcriptomics libraries (10x Genomics) will be constructed at the University of Illinois Roy J. Carver Biotechnology Center’s (CBC) Cytometry and Microscopy to Omics Facility. Fresh frozen OCT embedded tissues will be sectioned and placed onto a Tissue Optimization Slide with eight capture areas each having thousands of spots with poly-dT capture probes. Tissues will be permeabilized for 3-30 minutes (time course). mRNAs are annealed to oligos and converted to cDNA with a fluorophore and evaluated with a Zeiss LSM 980 Airyscan II super-resolution microscope. The permeabilization conditions producing the brightest image with the least diffusion will be chosen for processing of tissues. Tissue sections will be placed on a Visium Spatial Gene Expression slide which contains four capture area square (6.5×6.5mm), with ~5000 spots/square barcoded with poly-dT oligos. Sections will be fixed, stained with H&E and visualized with the same Zeiss LSM 980 Airyscan II super-resolution microscope equipped with a Zeiss Axiocam 712 color camera. After permeabilization, mRNAs will be converted into spatially-barcoded cDNA and transferred to the CBC DNA Services laboratory where they will be amplified, assessed for quantity and quality, and converted into sequencing -ready, dual-indexed libraries. The final libraries are quantitated on Qubit and the average size determined on the AATI Fragment Analyzer (Agilent, CA) and diluted to 5nM concentration and further quantitated by qPCR on a Bio-Rad CFX Connect Real-Time System (Bio-Rad Laboratories, Inc. CA). The libraries are then pooled by qPCR value and capture spot coverage and sequenced on an Illumina NovaSeq 6000 to a length of 28 nt (read 1, contains the spot barcode and unique molecular identifier used for removing PCR duplicates), 10 nt for each index (libraries contain unique dual indexes to prevent index switching) and 150 nt for read 2 (the cDNA read) to a minimum depth of at least 50,000 cDNA reads per spot. Fastq.gz will be demultiplexed with SpaceRanger 2.0.0 and sent to the HPCBio facility of the CBC for further analysis. Data are processed and visualized using Space Ranger Analysis Pipelines and Loupe browser using the appropriate reference transcriptome. Visium uses the cDNA barcodes to associate the transcripts to an X–Y coordinate on the slide, which will then be used to overlay the H&E-stained image with the transcript information from a spatial viewpoint. The R package Seurat will be used to read in both the count data and the images. Principal component (PC) analysis will be performed and at least the top 30 PCs were used in both nearest neighbor cluster calling and Uniform Manifold Approximation and Projection (UMAP) dimension reduction as the first steps in the statistical analyses. Further steps are tailored to the needs of each research project and could include optimization of the cluster calling based on tissue geography, differential gene expression testing between clusters and/or between samples, identification of spatially variable genes using tissue locations as input, and down-stream data mining of identified gene sets.
10X FFPE CytAssist Visium
- Sample workflow of 10x Visium involving CBC-CMtO, DNA Services, Proteomics and Metabolomics
- 10X FFPE CytAssist Visium (website)
- CytAssist Short Brochure (pdf)
- 10x CytAssist and how it works video?
10x Visium CytAssist FFPE Service
Key System Specifications
- Whole tissue section profiling, eliminating need for sections of interest
- 1-10 cell resolution, depending on type of tissue
- Accommodating for diverse organ samples among various species
- Human, mouse, rat, and others
- Two capture area sizes for compatibility with larger tissue samples
- 6.5 x 6.5 mm and 11 x 11 mm
- RNA-templated litigation (RTL) probe allows for high specificity and sensitivity for entire transcriptome of human and mouse
- Compatible with histological and immunofluorescent sections
High Resolution Histology section obtained image from LSM 980 and the corresponding CytAssist image acquired in the CytAssist system at the time of RNA extraction, to be overlaid for spatial transcriptomic analysis.
Section location and alignment requirements for CytAssist FFPE
Please Click for More Information:
CytAssist FFPE Workflow (pdf)
NOW OFFERING - 10X VISIUM HD
Visium HD empowers a new era of spatial discovery, enhancing proven whole transcriptome spatial analysis with single cell-scale resolution, enabling continuous tissue coverage, and delivering best-in-class data with innovation probe-based chemistry and a Visium CytAssist-enabled workflow.
What type of samples are compatible with the visium HD?
Currently, Visium HD is compatible with human and mouse formalin-fixed paraffin-embedded (FFPE) tissue sections containing mRNA. This includes archived FFPE blocks and pre-sectioned tissue on glass slides.
Click any of the links below for more information:
10x genomics Visium Cytassist HD
Visium HD in FFPE human breast cancer at single cell scale.pdf
Grant application resources for visium products.pdf
Visium_HD_Flyer_Launch_Letter_Digital.pdf
Can we do plant samples on Visium?
Manufacturer: 10X Genomics
Equipment Model: 10x Visium CytAssist
Location: Main Facility at 231 Edward R. Madigan Laboratory
Contact Person: Mayandi Sivaguru, sivaguru@illinois.edu
CurioBioscience Fresh Frozen
10x Xenium In situ System
Xenium is an end-to-end platform from 10x Genomics that provides highly sensitive, targeted gene and protein expression information at subcellular resolution. The Xenium Analyzer combines the power of single molecule RNA and protein detection with powerful optics, data acquisition, and decoding technology to rapidly detect analytes at high plexy and at subcellular resolution across entire tissue sections. The platform is designed to be compatible with both fresh frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) tissues
Xenium In Situ workflow overview
Xenium In Situ: High-performance in situ from the single cell leader
Xenium Sample information | |
Imageable Sample area | Total sample area inside the fiducials is 10.45mm x 22.45mm. |
Tissue compatibility | Fresh Frozen, FFPE, and Fixed frozen |
Sample type | Mouse and human brain, human lung, human breast, human skin, human colon, as well as multi-tissue panels for both human and mouse samples are available. Human Immuno-Oncology Profiling Panel is now available |
Restricted thickness | Fresh Frozen = 10 µM; FFPE = 5 µM |
Data analysis | Xenium explorer, Xenium ranger, third party tools |
Downstream processing | Enables tissue morphology (H/E) staining, or Visium spatial transcriptome analysis and immunofluorescence (IF), on the same tissue section. |
Xenium supported panels:
Post-Xenium In Situ Applications: Immunofluorescence, H&E, and Visium CytAssist Spatial Gene Expression
For more information about the Xenium platform:
Gene expression panels on xenium
Xenium software updates and important notes
FFPE Human breast tissue on Xenium
Manufacturer: 10X Genomics
Equipment Model: 10x Xenium Platform
Location: Main Facility at 231 Edward R. Madigan Laboratory
Contact Person: Mayandi Sivaguru, sivaguru@illinois.edu
Software
Andor Imaris with Cell
FlowJo
- FlowJo Introduction: https://docs.flowjo.com/flowjo/
- FlowJo University: https://www.flowjo.com/learn/flowjo-university/
FCS Express 6 & 7
- FCS Express Features: https://denovosoftware.com/full-access/features/
- Quick Start Resources: https://denovosoftware.com/full-access/quick-start/
- FCS Express Webinars: https://denovosoftware.com/videos/
IDEAS AI Analysis ImageStream Data
- ImageStream Software: https://www.luminexcorp.com/imagestreamx-mk-ii/#software
- ImageStream Resources: https://www.luminexcorp.com/imagestreamx-mk-ii/#videos
To guarantee availability, instrument time should be scheduled one week in advance. Please contact us to schedule an appointment or discuss a future project. (Cmto-core@mx.uillinois.edu)
