The Optical Imaging Core has a variety of options for imaging samples. Equipment in the OIC can accommodate a wide range of sample sizes, ranging from flowering plantlets, developing animals, tissue sections, and biofilms to cells, organelles and large molecules.
The microscopes in the OIC can accommodate a variety of culture dishes, slides and flow cells. An amazing breadth of options for excitation of fluorescent labels from 405nm up to 1060nm are available. There are objective lenses from 0.7X up to 100X, including some water and oil immersion options.
The Fluorescence Activated Cell Sorter (FACS) in the OIC is capable of counting objects as small as bacteria up to large mammalian and plant cells, high quality sorting into four tubes or multiwell plates, and side scatter, forward scatter and fluorescent characterization.
Two separate computers are available for image and flow cytometry data analysis These provide users the ability to make simple image adjustments and annotations as well as complex intensity measurements, movie creations and object counting with statistics, and direct logging to data management programs.
Olympus Fluoview 1000 Confocal/multiphoton microscope
A variety of objective lenses, including oil and water immersion, allow for viewing of slides, open well dishes and flow cells on the upright Olympus system. A motorized stage provides quick scanning of large areas. Spectral overlap is also easily resolved by control of the width of emission in the final filter. The efficient detectors provide fairly quick acquisition times.
Olympus Fluoview 1000 Confocal/Multiphoton microscope technical specifications:
- Microscope platform – upright (BX61). Accepts slide mounted material and open culture dishes or biofilm flow cells.
- Magnification options for slide samples - 10X, 20X, 60X.
- Magnification options for wet samples - 20X, 40X, 60X.
- Laser excitation options for confocal imaging - 405, 440, 488, 515, 559 and 635nm wavelengths.
- Laser excitation options for multiphoton imaging – tunable Coherent near-IR laser from 690-1040nm wavelengths.
- 3 fluorescent channels, 1 transmitted/DIC channel
- Acquisition software - FluoView ASW
Nikon/Andor Spinning Disk Confocal Microscope
The spinning disk system includes a stage-top environmental control chamber on an inverted microscope for long time-lapse imaging. Dynamic imaging can be automated in 3 dimensions (X, Y, Z) over time (t) at multiple locations on the slide or culture dish using multiple fluorescent labels and DIC. Two cameras and 6 laser excitation options provide choices in resolution, speed of acquisition and fluorescent probes.
Nikon/Andor Spinning Disk Confocal microscope technical specifications:
- Dry objective lens options @ 10X, 20X, 40X and 40X Extra Long Working Distance Plan Fluor
- Water Immersion objective lens options @ 40X (LWD) and 60X.
- Oil Immersion objective lens options @ 40X, 60X and 100X.
- Widefield LED excitation for DAPI, CFP, GFP, YFP, mCherry, Cy5
- Laser excitation options: 405, 440, 488, 515, 561 and 640nm.
- Andor Zyla sCMOS camera: up to 100 frames/second, 5.5 mpixel sensor, 6.5um pixel size, 16,000:1 dynamic range @ 30 fps.
- Andor DU888 EMCCD camera: high quantum efficiency, low read noise, 13um pixel size, 26 fps, 8500:1 dynamic range @ 30 fps.
- OKO Stage top incubator with temperature set point up to 50C, humidity and CO2 control.
- Photoactivation device @ 405nm for optical highlighters & optogenetics studies.
- NIS-Elements acquisition software for 6 dimensions, well plate scanning, ratio/fret module and image stitching.
- Perfect Focus System for maintaining focus during long imaging sessions.
Nikon Eclipse 1000 Fluorescent Microscope
The Nikon Eclipse has filter sets for standard DAPI, FITC and TRITC fluorescence, with additional sets for Violet excitation and choices for different GFP emission requirements. MetaMorph software (Molecular Devices) controls the Hamamatsu C4742-95 12-bit monochrome camera and filter set movements. Further processing and annotation of images, as well as counting and tracking of user-identified objects, can be done on the MetaMorph program. Journals can be created to automate many of these routine measurement steps and resulting data is logged directly to data management software.
Nikon Eclipse 1000 Fluorescent Microscope
- Microscope Platform – upright. Ideal for slide mounted samples.
- Magnification options - 4X, 10X, 20X, 40X, 60X, 100X.
- Fluorescence filter sets - DAPI, Violet, FITC, multiple GFPs, TRITC.
- Phase contrast imaging - 20X, 60X.
- Camera - High resolution Hamamatsu 12-bit C4742-95
- Acquisition software - MetaMorph (Molecular Devices)
Leica Fluorescent Stereoscope
The Leica MZ16F Stereo Fluorescence microscope with DAPI, GFP and DsRed filter sets provides imaging of surface structure of larger samples, such as nematodes, seeds and flowering plants. Fluorescence and standard reflected white light imaging is achieved using a Leica DFC420 color digital camera and Leica Application Suite software. Images can be easily organized, adjusted, annotated and made ready for publication in the Leica Application Suite software.
Leica Fluorescent Stereoscope Technical Specifications
- Magnification range – 7.1X to 115X
- Fluorescent filter sets – DAPI, GFP, DsRed
- Camera – Leica DFC420 color digital camera
- Acquisition Software – Leica Application Suite
BD Biosciences FACSAria Flow Cytometer
Cells or microorganisms in solution can be counted, characterized and even sorted with the FACSAria flow cytometer. If desired, samples can be sorted into separate tubes based on chosen features. Any particle 0.2-85 micrometers in size is suitable for analysis. The flow cytometer measures relative size, granularity or internal complexity of cells or particles and, if labeled with a fluorescent marker, fluorescence intensity. The fluidics, optics and electronics of the FACSAria system are very customizable. The system in the Optical Imaging Center contains 2 lasers. Lasers interact with the cells and determine their relative size, complexity and presence of blue and red excitation of fluorescent labels. The FACSAria has fixed alignment of the laser, an automated cleaning system and high resolution, allowing for efficient collection and analysis.
FACSAria Technical Specifications
- 488 nm, 20mW, solid state laser
- 633 nm, 18 mW, gas laser
- High numerical aperture (1.2) fluorescent objective lens
- Fluorescence filters for FITC, PE, PerCP Cy5.5, PE-Texas Red, PE-Cy7, APC, APC-Cy7
- 70 and 100 um nozzles for different sized cells/particles
- FACSDiva software for automation of acquisition, sorting, cleaning and analysis
- BD FACS Accudrop software for automation of the drop-delay determination
Image and Flow Cytometry Analysis
The IBEST Optical Imaging Core provides software for analysis of images and flow cytometry data. Each of the individual instruments has limited processing and analysis capacities within the acquisition software. Other data analysis packages are also available.
Image Analysis Station – Basic image adjustments (contrast, brightness, gamma) and annotations (scale bar, morphometric labels) are common to all of the image acquisition and analysis programs. NIS Elements AR and MetaMorph programs provide additional user defined morphometric measurements, such as shape, size or optical density. NIS Elements AR provides deconvolution and 6D analysis. Journals can be created for automation of repetitive measurements and data can be directly logged to a data management program, such as Excel. Imaris (Bitplane) software creates excellent 3- dimensional visualizations of confocal and multiphoton z-series.
Flow Cytometry Analysis Station - Detailed analysis of data from the flow cytometer is available using FlowJo software. FlowJo facilitates automation of repetitive analytical steps and produces statistics tables and graphical reports.