Fluorescence Microscopy & Cell Imaging
Microscopy is at the heart of understanding tissue architecture, cell structure and dynamics, as well as molecular function. Fluorescence microscopy is routinely used to determine spatial and topological information about cells and tissues. Sophisticated laser scanning microscopic instrumentation, ultra sensitive digital cameras and specialized fluorescence probes make it possible to visualize cellular events in real time down to the molecular level.
The Fluorescence Microscopy and Cell Imaging Shared Resource advances cancer research excellence by making available state-of-the-art instrumentation and software for quantitative, multi-scale, and multi-modal fluorescence imaging.
Advancing Cancer Research Excellence
The Fluorescence Microscopy and Cell Imaging Shared Resource aids basic and physician researchers to image samples and publish high profile articles that:
- Elucidate cell and molecular mechanisms of cancer, immunologic, infectious, metabolic, neurologic and vascular diseases
- Evaluate therapeutic efficacy in cells and patient samples
- Test new nanotechnologies in cell based assays
- Quantitatively measure changes in tissue morphology and pathology
The Fluorescence Microscopy and Cell Imaging Shared Resource provides 猎奇重口researchers access to state-of-the art instrumentation for multiple fluorescence and transmitted light microscopy techniques:
- Laser scanning single and multi-photon microscopes and hyperspectral imaging systems enable simultaneous visualization and quantification of histochemical stains and fluorescent labels
- Total Internal Reflection Fluorescence (TIRF), Single Particle Tracking (SPT) and Single Molecule Localization Microscopy (SMLM) applications to quantify dynamic live-cell events and achieve resolution below the diffraction limit of light
- Ratiometric imaging allows quantification of real time changes in calcium and other ion concentrations in response to cell signaling
The Fluorescence Microscopy and Cell Imaging Shared Resource has resource staff who offer investigators:
- Expert consultation on experiment design and specimen preparation
- Training
- Imaging for a fee
- Ongoing assistance
Diane Lidke, PhD; Faculty Director
Department of Pathology
2325 Camino de Salud, CRF203A
猎奇重口Comprehensive Cancer Center
Albuquerque, New Mexico 87131
Phone: (505) 272-8375
Keith Lidke, PhD; Special Advisor
210 Yale Blvd NE, 2218
Physics and Astronomy Interdisciplinary Science Building
Albuquerque, New Mexico 87106
Phone: (505) 277-2616
To acknowledge use of this shared resource, please include the following in your publications:
This research was partially supported by 猎奇重口Comprehensive Cancer Center Support Grant NCI P30CA118100 and made use of the Fluorescence Microscopy and Cell Imaging shared resource.
Following publication, please send us one of your reprints or cover images for our records.
Instrumentation
Location: CRF212
Microscope Details
- Custom Olympus IX83 inverted microscope with Martzhauzer ultra-fast motorized XY stage with piezo Z insert.
- Cool LED pE-300Ultra LED multi-spectral LED (wide field) and Lumencor PEKA LED (bright field)
- 10X/0.4NA, 30X/1.05NA silicone oil, 40X/1.25NA silicone oil, 60X/1.3NA silicone oil 100x/1.35NA silicone oil
- DIC capable for 30X thru 100x optics.
- Olympus Real Time Controller for temporally accurate camera and excitation synchronization
- Yokogawa CSU-W1 dual camera spinning disk head with SD-SR 3X mag changer
- Hamamatsu ORCA-Fusion back-thinned Gen III sCMOS cameras
- 4 individual OBIS laser lines: 405nm (50mW); 488nm (100mW); 561nm (100mW) and 640nm (100mW)
- 4-color imaging (2 channel simultaneous) Emission filter FWHMs as follows: Blue (447/60); GREEN (525/50); RED 607/36 and Far RED (685/40)
- Non-photo destructive IR ZDC2 autofocus for extremely fast one shot or continuous autofocus capabilities
- Tokai Hit WSKMX live-cell imaging stage top incubator
- Olympus cellSens Dimension image acquisition software
- GPU enabled post-processing provided by Huygens Essentials for image restoration (deconvolution)
Possible Applications
- High speed, multi-dimensional live-cell imaging where phototoxicity is of concern
- 3D acquisition, time lapse and tiling
- Volumetric samples such as spheroids/organoids, zebrafish and drosophila embryo
Location: CRF219
System Details
- Custom Olympus IX83 inverted microscope with Asylum 3D XYZ piezo scanning stage
- Excelitas Turbo multi-spectral LED (wide field) and halogen lamp (bright field)
- 10X/0.3NA, 20X/0.7NA and 100X/1.4NA oil immersion objective
- Olympus XM10 monochrome CCD camera
- Asylum MFP Origin AFM head with extended travel range for biological preparations
- Asylum Research software for AFM controls
- Olympus cellSens software for light microscopy controls
- BioHeater for work with live cells/tissue
- Training probes provided while quantities last!
- AFM compatible dishes: WillCo cover bottom chambers (Ted Pella cat # 14025-20) and BD Falcon (#351006) polystyrene culture dishes
- Atomically flat surfaces (mica or HOPG) for high resolution scanning can be found at Ted Pella.com - please inquire for assistance
Possible Applications
- Alternating Contact (AC) Mode, aka Tapping Mode, for nanometer scale imaging in dry or wet preps
- Contact Mode for mechanical force measurements (Elastic Modulus, Adhesion, Indentation and Force Mapping) in dry or wet preps
Location: CRF216
Microscope Details
- Leica DMi8 inverted microscope with X-Y motorized stage
- Metal halide fluorescence lamp & halogen transmission lamp
- 10X, 20X Oil, 25X Water, 63X Water DIC & 63X Oil DIC objectives
- Laser Excitation for PA-GFP, DAPI, CFP, YFP, FITC, Rhodamine, & Cy5-like fluorophores
- Tunable & Pulsed White Light Laser: 470 - 670 nm
- Two additional lasers: 405 nm laser diode and a multi-argon (458, 477, 488, 514 nm lines)
- Six detectors: 2 Hybrid Spectral Detectors* (HyD); 2 Spectral SMD** HyDs, 1 standard Spectral PMT & 1 transmitted PMT (external)
- Galvo Z-stage
- Resonance scanner
- Pico Harp 300 Fluorescence Lifetime Microscopy (FLIM) Module
- LASX acquisition software
- SymphoTime64 (PicoQuant) FLIM Software
- Huygens Deconvolution software
- Tokai Hit Stage Top Incubator for Live Cell Imaging
* HyDs are specialized detectors utilizing GAsP photocathodes & avalanche photo-diodes
**Single Molecule Detectors (SMD)
Possible Applications
- Fixed specimens mounted on standard slide with #1.5 cover slips
- Live cell specimens (LabTekII #1.5 multi-well or 35 mm (#1.5) cover bottom)
- FLIM FRET (fixed or live-cell)
- FRET Acceptor Bleaching (fixed)
- FRET Sensitized Emission (live-cell)
- FRAP (live-cell)
- 3D acquisition (Z-Stacks) and Time Lapse imaging
- Multi-Spectral Unmixing
- Large area, multi-dimensional tile scans
- Super resolution via image restoration processing (~130nm)
Location: CRF226
Microscope Details
- Zeiss AxioObserver inverted microscope with X-Y motorized stage
- Metal halide fluorescent lamp & halogen transmission lamp
- 10x, 20x, 40x oil, 63x oil & 63x water immersion objectives
- DIC available with 10x, 20x, 40x & 63x objectives
- Laser Excitation for PA-GFP, DAPI, CFP, YFP, FITC, Rhodamine, & Cy5-like fluorophores
- Four solid state lasers: 405 nm, 488 nm, 561 nm, 640 nm
- Channel and Spectral detection – GaAsP PMT detectors
- Simultaneous detection in 3 channels available in Channel mode
- Airyscan array detector for 1.7X increase in XY resolution
- Transmitted light detector
- Live-cell incubation system: temperature, humidity and CO2 control
- Zen Blue Image Acquisition software
Possible Applications
- Fixed specimens mounted on standard slide with #1.5 cover slips
- Live cell specimens (LabTekII #1.5 multi-well or 35 mm (#1.5) cover bottom)
- FRET Acceptor Bleaching (fixed)
- FRET Sensitized Emission (live-cell)
- FRAP (live-cell)
- 3D acquisition (Z-Stacks) and Time Lapse imaging
- Multi-Spectral Unmixing
- Large area, multi-dimensional tile scans
- Super resolution via image restoration processing (~130nm)
Location: CRF226
Microscope Details
- Custom Olympus IX83 inverted microscope with ASI motorized XY stage with piezo Z insert.
- Excelitas Turbo multi-spectral LED (wide field) and Lumencor white LED (bright field)
- 20x/0.8NA oil, 60x/1.5NA oil TIRF, 60x/1.2NA water, and 100x/1.4NA oil objectives
- DIC capable for 60x and 100x optics.
- Olympus Real Time Controller for temporally accurate camera and excitation synchronization
- 4 laser line MITICO TIRF illuminator for independent pentration depths
- 4 individual laser lines for TIRF: 405nm (100mW); 488nm (100mW); 561nm (100mW) and 640nm (140mW)
- Direct Stochastic Optical Reconstruction Microscopy (dSTORM), Photoactivated Localization Microscopy (PALM) & Highly Inclined Laminar Optical (HILO) sheets imaging possible
- Hamamatsu Fusion sCMOS camera and Geminini W-View image splitter for 2 color simultaneous live cell acquisitons.
- Non-photodestructive IR ZDC2 autofocus for extremely fast one shot or continuous autofocus capabilites
- BiopTechs objective heater for short-term live cell applications
- Olympus cellSens Dimension image acquisition software
Possible Applications
- Fixed specimens with #1.5H cover slips
- Live cell specimens (LabTekII #1.5H multi-well or 35 mm (#1.5H) cover bottom)
- 3D acquisition (Z-Stacks) and Time Lapse imaging
- Specific applications include live-cell TIRF single molecule detection, HILO, 2D dSTORM or PALM
- GPU enabled post-processing provided by Huygens Localizer
Camera Location: CRF218
Microscope Details
- Inverted Olympus IX-71 inverted microscope
- Mercury fluorescence lamp and halogen transmission lamp
- Monochromator – 350nm-670nm excitation range
- Lasers: 405nm, 488nm,520nm and 642nm
- 20x, 60x H2O, 100x Oil, 150x Oil TIRF objectives
- Fluorescence filters for Quantum Dots, DAPI, FITC, & Rhod-like fluorophores, Fura-2, Indo-1
- Andor IQ Image Acquisition software
- Objective heater and Pathology Devices live cell incubation system w/ CO2, heat, humidity
- XYZ motorized stage
- Andor iXon EM-CCD camera
- Opti-split (dual) Image splitter
Possible Applications
- Fixed specimen mounted on slides
- Live cell imaging with objective warmer
- Single Particle Tracking
- Total Internal Reflection Fluorescence (TIRF)
- Ratio-metric Imaging (dual excitation/single emission or single excitation dual emission)
- Live cells in culture
- Widefield Fluorescence – many fluorophores possible with monochromator
- Z-stacks & time series
Camera Location: CRF212
Microscope Details
- Zeiss AxioObserver inverted fluorescence microscope
- Hamamatsu ORCA Flash4.0v2 sCMOS monochrome camera (100fps at full resolution 2048x2048)
- Metal halide fluorescent lamp & halogen transmission lamp
- 10x, 20x, 63xDIC Oil, & 100x Oil objectives
- Phase Contrast for 10x and 20x objectives
- Fluorescence filter sets include: DAPI, FITC, Rhodamine/TRITC/Cy3, Texas Red & Cy5
- DIC available with 63x objectives
- Objective heater (63x water objective) o Motorized nosepiece for Z-stacking
- Motorized X-Y stage for multi-position image acquisition (fixed or live-cell time lapse imaging)
- SlideBook6 image acquisition software
Possible Applications
- Fixed specimen mounted on slides
- Live cells in coverslip dish/heating with objective heater
- DIC (non-confocal) with 63x objectives, merged with fluorescence or alone
- Z-stacks, montage, and time series imaging
Located in CRF224 & CRF226
Software
- Huygens Deconvolution Scientific Volume Imaging (1)
- SlideBook (1)
- StereoInvestigator (1)
- Neurolucida (1)
- LASX (Leica confocal) (1)
- IQTL (Typhoon scanner analysis) (1)
- Zen (2)
- LSM510 (2)
- Nuance spectral imaging software (2)
Analysis Options
- Co-localization
- Deconvolution
- Spectral unmixing
- Intensity analysis
- Morphological analysis
- 3D rendering
- Object counting
- Particle Tracking
- Preparation for publication
Located in CRF214
Typhoon FLA 7000 is a fast and versatile laser scanner for biomolecular imaging applications including:
- sensitive and quantitative measurements of radioisotopic labels
- chemifluorescent Western blots
- single fluorescence (visible excitation)
- documentation of colorimetric stains (e.g., Coomassie blue and silver-stained gels)
Located in CRF214
Li-Cor Odessey Fc is a near infra-red scanner (680nm & 740nm) for biomolecular imaging applications including:
- sensitive and quantitative measurements of chemiluminescent Western blots
- documentation of colorimetric stains (e.g., Coomassie blue and silver-stained gels)
Microscopy Services Include
- Expert consultation on experiment design and specimen preparation
- User training
- Operator assisted imaging and acquisition of preliminary data for grant applications
- Annual open house to inform users of updates on new instrumentation, technologies and applications
- Microscopy course for graduate students and fellows on optics and instrument functionalities
- Annual workshops that give new users hands-on training in specimen preparation
Total Internal Reflection Fluorescence (TIRF) microscopy of basal FasL-GFP activity expression in RBL cells
Microscopy Services and Rates
Zeiss AxioObserver – Hamamatsu ORCA Flash4.0v2 sCMOS Monochrome Camera: $7/hour
Yokogawa Spinning Disk: $30/hour
Leica TCS-SP8/FLIM: $50/hour
Zeiss LSM800: $45/hour
Olympus IX71 Single Particle Tracking/Ratio-Metric Microscope (Rm. 218): $25/hour
Olympus IX83 TIRF/dSTORM Microscope (Rm. 226): $30/hour
All instrument Training, Imaging with Operator & Analysis Training: $125/hour
- Confocal (4 hours)
- Advanced Wide Field (2-3 hours)
- Atomic Force Microscopy (4-8 hours)
- Basic Wide Field (0.5-1 hour)
- Imaging with Operator and Analysis Training (Project Dependent)
Off-line Workstation, unassisted: $5/hour
Facility Funding
The University of New Mexico & Cancer Center Fluorescence Microscopy Shared Resource acknowledges the following funding for instrumentation purchase or facility support since its opening in 1999.
Current Funding
NCI 2P30 CA118100 (PI Willman, C.) “猎奇重口Cancer Center Support Grant” 9/25/05-8/31/20
NIGMS 5P50 GM085273 (PI Wilson, B.S.) “Center for the Spatiotemporal Modeling of Cell Signaling Networks (STMC)” 08/01/08-07/31/19
STMC P50GM085273: funding support for Leica TCS-SP8 and Olympus iX71
Past Instrumentation Funding
NCRR 1 S10 RR14668 (PI Wandinger-Ness, A.) “A Zeiss LSM510 Confocal Microscope for Health Sciences Research” 04/01/00-03/31/01
NSF MCB9982161 (PI Wandinger-Ness, A.) “Rab7 and Accessory Protein Function in Late Endocytosis” (Purchase of Hamamatsu digital camera for live cell imaging) 04/01/00-03/31/06
NCRR S10 RR016918 (PI Wandinger-Ness, A.) “Zeiss Axiovert 200 for Live Cell Imaging Project” 08/01/02-04/01/04
NCRR S10 RR19287 (PI Wandinger-Ness, A.) “META Detector and 405 nm Laser” 04/01/04-03/31/05
NCRR 1S10RR025540 (PI Wandinger-Ness, A., co-I Lee, R.) “A Confocal Stereology Microscope for UNM's Fluorescence Microscopy Core” ARRA 05/01/09-03/31/10
NSF DGE-0549500 (PI Osinski, M. co-I, Oliver Co-PI) “Nanotechnology for Cell Biology and Neuroscience” (Purchase of accessories for Olympus IX71) 6/15/06-5/31/11
Matthews Family Fund (PI Lee, R.) “Nuance Spectral Imaging System, Cri” 01/01/09-12/31/-09
NIDDK 3R301 DK050141 (PI Wandinger-Ness, A.) “Pathologic Mechanisms of Polycystic Kidney Disease” Purchase of Zeiss Axio Observer for 510 META) ARRA Administrative Supplement 09/30/09-09/29/11
NCRR P20 RR11830 (PI Oliver, J., co-PI Eaton, P.) “Enhancing Multidisciplinary Research in New Mexico” 9/30/96 ‑ 9/29/99
NCI P20 CA88807 (PI Willman, C.) “University of New Mexico Cancer Center Planning Grant” 07/01/00-06/30/05
Intramural funding 1999-Present: $437,266
University of New Mexico Health Sciences Center
University of New Mexico Cancer Center
University of New Mexico School of Medicine Department of Pathology