Cell and Tissue Imaging Core

Because new technologies allow high resolution imaging of living cells and tissues that may be infected with viable microorganisms this Cell and Tissue Imaging Core (CTIC) will be in BSL4 containment. Existing facilities for electron and more conventional microscopy of fixed, non viable specimens are part of the research infrastructure at BUMC that will be available to NEIDL investigators. The Cell and Tissue Imaging Core (CTIC) at the NEIDL will offer multiple imaging systems to analyze specimens using state-of-the-art technologies. The availability of different high resolution microscopy solutions will allow NEIDL investigators to integrate fine-scale topography of fixed tissues gathered from transmission or scanning electron microscopy with information gathered from multi-probe, live cell analyses using deconvolution or laser confocal microscopy.

Functions and Services

Various types of microscopes, such as laser confocal and deconvolution, are capable of acquiring and rendering 3-D images. The actual mechanism of image acquisition differs greatly, giving each microscope advantages in certain applications. Confocal microscopes use a system of pinholes placed before the collection aperture, which have the net effect of blocking out all light from the sample except for the light originating directly from the current plane of focus. This greatly increases the signal-to-noise ratio in a sample, especially when visualizing a single fluorophore, labeled molecule, or rare cellular particle in a sea of extraneous light-scattering organelles or other cells. Yet, the confocal has disadvantages, in that it is laser-based and pinholes must be correctly aligned with the plane of focus to achieve a meaningful image, and for each image in a Z-series, the focus has to be reset. When collecting a Z-series with the deconvolution microscope, the microscope does not discriminate between light emanating from inside or outside the plane of focus. An object can be focused near the center of the region of interest, and settings for upper and lower limits of Z-travel, number of images to take in a range, and distance between images (with a resolution capability of 0.1 micrometers in Z) are chosen. The microscope automatically takes a series of images, many of which will be completely out of focus. The deconvolution software is then used to reconcile out-of-focus information and integrate information from multiple sections into a composite 3-D image. The newest high resolution, image restoration microscopy system from Applied Precision is the DeltaVision Spectris, which provides live imaging capabilities to study dynamic processes within the cell or organism. This real-time imaging feature, combined with the advanced point-visiting technique of the system, enables researchers to study multiple cells simultaneously. The system accommodates combining spatial and temporal resolution to the cell structure and rate of activity. Fast exposures also reduce cell-damaging phototoxicity effects as well as minimizing photobleaching, both of which can limit observation time in live-cell imaging applications. The system integrates stage and filter positioning, fluorescence optics, and high-powered deconvolution software. It can enhance a broad range of research projects, and typical biological samples range from yeast, bacteria, plants and Drosophila, to cultured mammalian cells. Since the system is not laser based, a wide range of excitation and emission wavelengths can be employed, allowing for maximum flexibility in fluorescent probe selection - up to 5 probes in one data set. DeltaVision has the ability to use any wavelength form 340 nm to 700 nm, depending on the filter sets used. The DeltaVision system employs optimized illumination and CCD-based detection, yielding higher resolution images at substantially lower illumination levels than those of confocal laser microscopes. It has Expanded Intensity Quantification that is10 times the linear range of confocal technology and can differentiate both low and high intensity regions within the same image. The i ncrease in sensitivity permits this system to be used for time-lapse and 4D (3-D + time) experiments. It has the highest resolution available, and can resolve structures <175 nm, such as centrioles, microtubule decoration, or a single nuclear pore. Finally, an important consideration for a deconvolution microscope system is the amount of time required to digitally process stacks of images. DeltaVision deconvolution microscopy uses Fourier transform mathematics to remap the out-of-focus components of 3-D data sets. By using mathematical methods, the system achieves superior sensitivity versus confocal methods while producing higher resolution images. In addition to the DeltaVision instrument this containment core will have a scanning confocal microscope.