Integrated Cellular Imaging
Emory University
Contact: Neil Anthony, PhD
The Integrated Cellular Imaging (ICI) core provides state-of-the-art light microscopy and image analysis platforms. ICI offers confocal, spinning disk confocal and live cell imaging, multi-photon animal and tissue imaging, widefield with deconvolution, super resolution, light sheet and image analysis. To effectively implement these technologies, ICI provides consultations, expert training, and support for all our systems. We assist our users from bench to publication, starting with designing experiments tailored to the specific microscope, through training and data acquisition, to data analysis and interpretation.
Confocal Imaging
Leica SP8 (Winship): The Leica SP8 confocal laser scanning unit, attached to the latest Leica inverted DMi8 body, comprises an infinitely flexible prism based spectral detector system and multiple excitation laser lines (405, 488, 514, 561 and 633nm) for a vast variety of experimental and labeling options (coupled with LASX’s unmixing software for experiments with potentially 10 or more colors). Four channel detection, via two PMT’s and two extremely sensitive Leica HyD detectors (GaAsP based), together with resonant scanning provides high speed imaging. A transmitted light detector allows for simultaneous laser scanned DIC or phase imaging. A stage and objective warmer facilitates numerous live cell imaging approaches such as FRET, FRAP, and photoconversion. High speed automated tiling/stitching and multipoint location acquisitions are also possible in combination with the above.
Olympus FV1000 (HSRB): This FV1000 confocal laser scanning microscope is mounted on an inverted Olympus IX81 microscope with a motorized x-y-z stage. It has a three channel PMT detection system with grating based spectral detectors on channels 1 and 2 (allowing for spectral unmixing). In addition, a transmitted light detector provides laser scanned DIC for label free imaging. These coupled with 6 excitation laser lines (405, 458, 488, 515, 559, and 635nm) facilitate highly flexible imaging combinations.
Olympus FV1000 (Whitehead): This FV1000 laser scanning microscope is identical to the HSRB FV above, except it’s filter based detection on all channels and a different combination of excitation laser lines (405, 488, 515, 543, 635). Having near identical systems in two locations provides easy secondary options without additional trainings during maintenance and repairs.
Olympus FV1000 Upright (Whitehead): This FV1000 laser scanning microscope is mounted on an upright Olympus BX61 microscope, with the same 3 channel filter based setup as above. Slightly different laser lines (405, 488, 514, 543, and 635nm) provide different experimental options, again without additional training. A large area stage and water dipping objective lenses allow for larger and aqueous based sample mounting options.
Live cell confocal imaging
Nikon A1R HD25 (Whitehead): This live cell confocal laser scanning microscope is optimized for imaging cellular dynamics at high speed and a large view of view (25mm). A hybrid dual scanning confocal system with both galvanometric and resonant scanners, together with a piezo z controller, can acquire 1024x1024 images at 15 fps, 512x512 images at 30 fps, or with a reduced size up to 720 fps. A stage top environmental chamber at 37° C and 5% CO2 required for live cell imaging, coupled with a Perfect Focus system, provides extremely stable long-term imaging without drift. Flexible laser scanning control for photokinetics experiments can be conducted in multiple user-defined regions of interest. Six laser lines (405, 440, 488, 514, 561, & 640nm) and four channel PMT (2x GaAsP, 2x Multi-Alkali) detectors, in addition to a transmitted light channel for laser scanned DIC provide flexible live cell experimental options. A DUVB-2 GaAsP spectral detector unit provides arbitrary 10 – 320 nm wide spectral windows between 400 and 720 nm for complete spectral flexibility. NIS Elements packaged with analysis options coupled to JOBS tailorable data-based control system.
Nikon Ti2 – Crest X-Light Spinning Disc Confocal (Winship): A Nikon controlled Ti2 with a Crest Optics X-Light large FOV ultrafast spinning disk confocal provides a 18mm FOV utilizing a sensitive high speed sCMOS camera (~100fps or more for cropped ROI). A stage top environmental chamber at 37° C and 5% CO2 required for live cell imaging, coupled with a Perfect Focus system, provides extremely stable long-term imaging without drift. A fast z-piezo and motorized xy stage provides high speed automated multi-location and tiling/stitching acquisitions. Seven laser lines (408, 445, 473, 518, 545, 635, & 750nm), DIC, and phase acquisitions are available. NIS Elements packaged with analysis options coupled to JOBS tailorable data-based control system.
Super-resolution imaging
Nikon N-SIM (Whitehead): The Nikon Structured Illumination Microscope (SIM) doubles the resolution of widefield microscopy in all three dimensions (down to 120 nm x-y and 300 nm z resolution), and can image samples prepared with conventional techniques (same as confocal or widefield). A sensitive iXon EMCCD camera and three laser line system (488, 514, and 561nm) provides high speed multi-color acquisitions. A SIM-TIRF mode is also available. The system also includes a 37°C 5% CO2 and humidity-controlled stage insert, together with heated objectives for the option of live cell super resolution acquisitions.
Deltavision OMX BLAZE (HSRB): The DeltaVision OMX imaging platform is an advanced multi-mode, super-resolution microscope system representing the next generation of microscopy. DeltaVision OMX provides 4 color super-resolution imaging using 3D structured illumination (3D-SIM) as well as ultra-fast widefield-deconvolution acquisition (3x sCMOS cameras for 300 fps imaging). The OMX Blaze also has Ring-TIRF capabilities to image biological processes at the cell surface-coverslip interface. The laser scanning optics of the TIRF beam path allow for fast switching of photoactivation and photokinetics in combination with live cell widefield-deconvolution ,3D-SIM imaging or TIRF. The system also includes a 37°C 5% CO2 and humidity-controlled stage insert, together with heated objectives for the option of live cell super resolution acquisitions.
Lattice Light Sheet (Winship): The Intelligent Imaging Innovations (3i) lattice light sheet is a transformative new technology for live cell imaging with unprecedented spatiotemporal resolution. It combines custom water dipping objectives and structured illumination to provide a resolution 3-4x better than traditional confocal microscopy in SIM mode, or fast volumetric imaging with ~100 times less phototoxicity/photobleaching than confocal in differed mode. The system has four-laser options (405, 488, 561, and 642nm) operating in sequential mode, but without spectral separation for speed optimization. Samples are loaded onto 5mm coverslips and immersed in an 8ml bath below the inclined objective lenses.
Multiphoton
Zeiss 710 NLO (Clinic B): This multiphoton allows for intravital (live animal) imaging with high magnification and resolution. This microscope is equipped with a Chameleon Vision S tunable multiphoton laser (680 nm to 1050 nm) that can penetrate farther into tissue than a traditional confocal laser. This system is mounted on an upright Zeiss Axioexaminer microscope, together with a flexible large area stage for imaging live mouse single cells, thick tissue sections, and cell lines. The confocal optics of the 710 house spectral detectors coupled with six lasers lines (405, 458, 488, 514, 543, and 633nm) for flexible acquisitions. Two non-descanned detectors (NDDs) provide ultrasensitive detection of multiphoton signals. Oil, air and water immersion objective lenses are available.
Leica SP8 (Whitehead): The Leica SP8 is an upright confocal and multiphoton microscope with a Coherent Chameleon Vision II laser tunable from 680 to 1080nm. Two highly sensitive non-descanned GaAsP based HyD detectors provide imaging of multiphoton processes. The confocal unit houses an infinitely flexible prism based spectral detector system and multiple excitation laser lines (458, 476, 488, 496, 514, 561, 594nm) for a vast variety of experimental and labeling options (coupled with LASX unmixing software for experiments with potentially 10 or more colors). Four channel detection, via two PMT’s and two extremely sensitive Leica HyD detectors (GaAsP based), together with resonant scanning provides high speed imaging. A CCD camera allows for brightfield and Dodt Gradient Contrast (DGC) for label free imaging and sample navigation. A stage and objective warmer facilitates numerous live cell imaging approaches such as FRET, FRAP, and photoconversion. A resonant imaging mode provides high speed scanning at up to 428 fps and there is automated tiling and image stitching. Oil, air and water immersion objective lenses are available.
Widefield fluorescence
Zeiss Axioplan 2 (Winship): The motorized Zeiss Axioplan 2 is equipped for routine widefield epifluorescence and transmitted light applications (DIC, phase, and brightfield) using a range of high-end Zeiss Plan-Apo objectives. A Zeiss Axiocam color camera allows for histology imaging or fluorescence imaging via multiple filter sets.
Olympus IX71 (HSRB): The inverted IX71 can perform epifluorescence and transmitted light microscopy. It contains long working distance objectives that allow for visualization through plastic culture dishes or glass. A sensitive color QImaging CCD camera and multiple filter cube sets provides fluorescence imaging in monochrome mode, and histology images in color mode.
Olympus IX51 (Winship): The inverted IX51 can perform epifluorescence and transmitted light microscopy. It contains long working distance objectives that allow for visualization through plastic culture dishes or glass. An Infinity CCD monochrome camera allows for sensitive acquisitions from multiple filter set options.
BioTek Lionheart FX (Whitehead): The compact Lionheart FX widefield microscope provides flexible automated acquisitions in fluorescence, brightfield, and color imaging modes, together with multiple lenses and 37°C and 5% CO2 control allowing live cell multiple plate reading, to IHC, and widefield-deconvolution.
Image Analysis
Five image analysis workstations are available, ranging from Dell workstations to custom built towers designed to handle 500GB sized data sets. Users can analyze their data using a full suite of image analysis options that have recently been updated. These include Bitplane Imaris, Volocity 6.2 and 3-D analyses. 2D analysis can be performed with Nikon Elements, Fiji Is Just ImageJ (FIJI; open source), CellProfiler (open source), and Zeiss Axiovision. Deconvolution is available via Huygens Essentials. Custom Fiji macros and java plugins can be design and tailored to individual needs, and are available to download from our resources page, together with YouTube analysis tutorials. Matlab code can be design for stand alone usage, or for direct interface within Imaris.
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