Software
Image Aquisition
Commercial imaging systems of OMC are controlled by acquisition software specifically designed for the appropriate microscope, such as ZEN (Zeiss confocal microscopes), Nikon Elements (Nikon), Imspector (Abberior). Custom-built HILO instruments are controlled by Micro-Manager Open-Source Microscopy Software.
Image Processing
The next step, the most important for the scientist, includes image processing and/or quantification. Standalone workstations equipped with Zen, Nikon Elements, Imaris and Huygens are available in OMC for image processing such as deconvolution, filtering, surface rendering, segmentation, and for quantification of the protein content by fluorescence intensity, and for the super-resolution by SIM, DNA-PAINT and PALM, and nano-resolution by MINFLUX). Custom Matlab-based software is available for the estimates of parameters of protein dynamics by FRAP and Single Molecule Tracking, and macromolecular interaction by FRET.
Image Conversion
Finally, images may be prepared for publications; they are scaled from 16-bit to 8-bit notation, and/or converted to movies (Quick Time and AVI) on workstations of the Core. Final preparation of the figures for publication with Photoshop or PowerPoint should be done by the user in their own laboratory.
Conventional Techniques
OMC provides support in conventional microscopy techniques, from the multi-channel fluorescence imaging of fixed cells and tissue sections to the time lapse imaging of cells in 2D or 3D, and computational removal of out-of-focus light (deconvolution).
Muticolor 3D Imaging
Fluorescence imaging in fixed specimens is used for protein localization and colocalization in 3D. 3D imaging is an acquisition of a series of images from different focal planes through the specimen in multiple color channels. Multi-color imaging is necessary to observe colocalization of several proteins in the same cell. Many fluorescent proteins and very stable dyes are now available for multi-color labeling, and imaging of four to five different fluorophores is possible on most of our microscopes. These images contain out-of-focus light which reduces contrast and introduces blur, but the out-of-focus light can be reduced by using either confocal microscopy or deconvolution microscopy.
Time Lapse 4D Imaging
Live imaging of cells and tissues tagged with fluorescent markers provides a much deeper understanding of cellular processes than imaging of a fixed specimen, since live cell imaging permits analysis of the dynamics of biological processes. Time-lapse experiments typically investigate changes in fluorescently tagged protein localization over time, such as movement of a protein from the cytoplasm to the nucleus. We have environmental chambers that regulate temperature and CO2 on the microscope stage for the WF Nikon Ti2000 and LSM880-Airyscan and LSM780.
Deconvolution
Deconvolution is a computational approach to remove out of focus light from 3D images. It is an alternative to confocal microscopy which removes out of focus light optically. In many cases, particularly with dimmer or light-sensitive specimens, deconvolved images are superior to confocal images, at least in part because more sensitive cameras can be used on deconvolution microscopes. In our core we have commercially available deconvolution software Nikon Elements and Huygens.
Molecular Dynamics and Interactions in Live Cells
Single Molecule Tracking (SMT) provides parameters for the protein binding and dynamics of confinement in micro-compartments. We offer MINFLUX technology for molecular tracking with unprecedented spatio-temporal resolution, supported by Single Molecule Tracking (SMT), based on HILO technology, FRAP (Fluorescence Recovery After Photobleaching), and FCS (Fluorescence Correlation Spectroscopy. Interactions of macromolecules can be assayed in the cell by FRET.
FRAP (Fluorescence Recovery After Photobleaching) and FCS (Fluorescence Correlation Spectroscopy)
FRAP and FCS allow one to monitor and quantify the movement of proteins of interest fused with fluorescent proteins. In FRAP the fluorescent component of the protein fusion molecules contained in a specific region is “turned off” by means of photobleaching. If molecules are free to move, the exchange between bleached and non-bleached molecules results in the recovery of fluorescence within the region. The recovery data can be analyzed to provide information about the diffusion and binding rates of the tagged protein. Fast-scanning confocal microscopes can be used to apply FRAP and FCS to monitor fast protein kinetics. These methods are less precise than Single Molecule Tracking (SMT), yet they are indispensable for a preliminary characterization of the effects of mutations or environment on a macromolecule of interest.
Single Molecule Imaging by HILO, LLS and MINFLUX Technology
Single Molecule Tracking (SMT) is the method of choice for quantification of the dynamics of molecules moving through the cytoplasm or nucleus, or associated with intracellular targets such as DNA, intracellular bodies, or structural elements. Other microscopy methods, such as FRAP and FCS are based on population analysis and mathematical modeling, which adds additional difficulties in data analysis. The powerful technique of SMT derives data from direct observation of individual molecules. OMC adapts and develops SMT techniques for live cells. This includes the development of imaging and analysis protocols and Matlab-based routines for tracking and estimation of biophysical parameters, such as diffusion rate or the binding rate of molecules. Three types of imaging for SMT, complementing each other, are currently available at OMC. (1) Two custom-built microscopes are based on HILO illumination producing Highly Inclined Laminated Optical sheet. They are equipped with EMCCD cameras allowing acquisition of the movies of fluorescently labeled molecules with time resolution as low as 10 ms. (2) Lattice Light Sheet (LLS) microscope is available for single molecule tracking in 3D. This type of illumination reduces photobleaching and allows prolonged tracking of single molecules. This technique is currently under development. (3) MINFLUX (MINimising Fluorescence FUXes) nanoscope, based on a state of the art technique of precise localization by a minimum of fluorescence, allows acquisition of 2D and 3D molecular tracks with nano-precision and high temporal resolution as high as 0.1 ms. Imaging technique and analysis of MINFLUX data are under development.
FRET (Fluorescence Resonance Energy Transfer)
FRET is a technique to detect protein interaction and the formation of complexes between different proteins tagged with fluorescence markers. It also can be used to detect enzymatic activity by special reporters (FRET biosensors). In our core we have applied and further developed several different approaches for detection of FRET. We have performed FRET by either acceptor photobleaching or sensitized emission on a conventional confocal microscope. We used a conventional wide-field fluorescence microscope to perform FRET dynamics in studies of enzymatic activity with biosensors.
Super Resolution and Molecular Mapping
Nano-resolution provides information on micro-compartmentalization of macromolecules (molecular maps). We offer MINFLUX technology with unprecedented resolution up to 3-5 nm, and we support it with conventional Super-Resolution technologies, such as Single Molecule Localization Methods (SMLM, STORM/DNA-PAINT) and SIM (Structural Illumination Microscopy) technology.
Structured illumination (SIM)
Structured illumination (SIM) is a Super Resolution (SR) technique based on imaging of a set of images from the same focal plane with a shifting grid pattern. Information is extracted from the raw data to produce a reconstructed image having a lateral resolution of 100 nm (approximately twice that of conventional confocal and wide-field instruments) and an axial resolution ranging between 150 and 300 nanometers. Among the advantages of SIM are the abundance of dyes and fluorescent proteins for labeling specimens and the ease of conducting multicolor imaging. Acquisition is moderately slow but still acceptable for live imaging. Images are post-processed in batches to compensate for the length of processing time (~ 5 min for a full frame).
Single Molecule Localization Microscopy (SMLM) – STORM, PALM
SMLM is a Super Resolution (SR) technique based on the sequential photoactivation of the fluorescent molecules fused to the protein of interest. Most of those molecules are in inactive state, and only a few molecules at a time are active. The precise position of the center of the signal emitted by each single molecule is calculated. The image is a composite of all the single molecule coordinates. Expected spatial resolution is 20-50 nm in XY and 50 nm in Z. 3D version of SMLM achieved in Zeiss Elyra instrument by double-helix ramp allows 3d imaging of a 1.4 um optical slice. This process is recommended for the fixed specimens. Application of SMLM to live imaging is limited by necessity of obtaining thousands of sequential time-lapse frames to obtain one image. In OMC, STORM by “blinking” molecules and PALM by photoactivatable molecules may be achieved on Elyra system (Carl Zeiss).
MINFLUX Nano-resolution by blinking fluorophores and DNA-PAINT
New technology MINFLUX (MINimising Fluorescence FUXes) is based on excitation of the fluorescent molecules with doughnut-shaped beam, and photon-counting. Exact position of the molecule is detected by a minimum of fluorescence; thus, it requires low photon count and imaging is fast and non-photodamaging. Resolution is not dependent on wavelength and molecular tilt. This system may reach a level of resolution up to 3 nm, bridging the gap between the light and electron microscopy. MINFLUX may be used for molecular mapping of the intracellular structures such as organelles and high molecular complexes (centromeres, centrosomes, Cajal bodies, etc.) MINFLUX of OMC is one of the first in USA, this is a brand-new technique. OMC works on expanding the color palette for multicolor imaging, and adapting the techniques to a wide range of samples. Alternative to STORM and PALM, DNA-PAINT technique based on a periodic binding of a fluorescent imager to a docker oligonucleotide attached to antibody, is under development at OMC for MINFLUX.