Optical Imaging Facility: The K-FLARE open air NIR imaging system was designed by Dr. John Frangioni and engineers at BIDMC. It is composed of an anti-vibration table and a black anodized stage contained within a light tight enclosure. Within the enclosure are fiber optic bundles for subject illumination, and attached to the enclosure is a custom video coupler attached to an AD-130GE camera (JAI, Yokohama, Japan) to obtain simultaneous color images (512 × 512 pixels) with the Kangce of either 700 nm or 800 nm fluorescence images with custom dual bandpass prism (channel 1: 710/50, channel 2: 780lp). Sample excitation is from an RS-232-controlled LMI-6000 LED Fiber Optic Illuminator (Dolan-Jenner) equipped with custom excitation filters (Chroma Technology) or custom 1-5 W 660-nm and 760-nm LDX laser diode systems (RPMC) to produce 1-10 mW/cm2 fluence rate at a 13” working distance. The imaging system is remotely controlled by custom FLARE software at rates up to 15 Hz, although the field of view is manually adjusted by a 3CCD zoom lens (Goyo Optical Inc., Saitama, Japan). We also built a mesoscale multiplex imaging system and an NIR-II imaging system as shown below, of which detailed spec will be published shortly.
Figure. Multispectral imaging system in the NIR-I & NIR-II window. a) Schematic of multispectral imaging system. b) FIAT-L™ NIR imaging system composed of color-NIR1-NIR2 CCD camera. Optical channels in the context of 700 nm and 800 nm laser diodes and its optical light paths for multichannel real-time simultaneous imaging. c) NIR-II fluorescence imaging. Optical channels in the context of 808, 980, 1064, and 1260 nm laser diodes (left) and optical light paths (middle) for multichannel imaging. Mouse brain and hindlimb were imaged.
Trifoil InSyTe FLECT/CT™ (3D NIR optical imaging with CT)
3D NIR tomographic optical imaging system (InSyTe FLECT/CT™, TriFoil Imaging) is an innovative instrument that offers 3D NIR optical imaging with high quality anatomical reference in a single platform. The InSyTe™ platform, based on Fluorescence Emission Computed Tomography (FLECT), provides unparalleled capability for whole body imaging and in vivo characterization of preclinical, small animal models. It integrates two 3D imaging modalities into a single instrument by enabling co-registered FLECT and X-ray CT. It utilizes a patented, rotating gantry design to collect optical and x-ray-based projections of the animal. When combined with NIR probes and laser excitation, this allows for enhanced sensitivity (up to the picoM range) and accurate 3D data acquisition, especially when looking for low level and/or deep signals during fluorescence detection. Image reconstruction algorithms utilize segmented X-ray CT data to assess changes in optical properties within the animal and provide accurate reconstruction visualizations.
Figure. Schematic diagram and photograph of fluorescence emission computed tomography (FLECT) system (InSyTe, Trifoil Imaging), and images of a mouse injected with NIR probes.
Video. Operation of 3D NIR tomographic optical imaging system (InSyTe FLECT/CT™, TriFoil Imaging)
Optical Imaging core has Cytation 5 (BioTek, Winooski, VT), which is a combination of automated digital microscopy and conventional microplate detection. This imaging suite allows for capturing both well-based quantitative data and phenotypic information on a single platform.
Gen5™ microplate reader and imager software provide complete control over all imaging and data capture, plus image and data analysis to create publication-ready images and data.
1. The multi-mode readers offer a plate reader functions with the multi-mode detection modules including filter- and monochromator-based fluorescence detection, luminescence and UV-Vis absorbance detection, and time resolved fluorescence (secondary mode):
Tunable wavelength monochromator optical systems for selecting specific wavelengths
2. A micro-volume analysis system with Take3 module allows for 16 samples in one run to save a lot of time compared to single-sample devices:
Gen5 is pre-programmed for ssDNA, dsDNA, RNA and protein quantification in 2 μL.
Spectrophotometric determination of dsDNA, ssDNA, RNA at A260
Fluorometric determination of dsDNA with fluorescent dyes, for example, PicoGreen
Determination of purity based on A260/A280 ratios
Full range of microscopy resolution (the highest quality objectives 4x, 10x, 20x, 60x)
Up to four choices of full range of fluorescence filter cubes available (DAPI, GFP, RFP, CY5, CY7)
Up to four choices of excitation LED available (365, 465, 523, 623, 655, 740 nm)
Temperature control, including the condensation control gradient
Labware adapters for microscope slides, cell culture dishes, chamber slides, microplates, T75 flasks and hemocytometers.
Auto focus capabilities: Imaging based autofocus, user trained, laser autofocus for high reproducibility and accuracy for automated imaging and long-term kinetics.
The High Contrast (HC) cell counting application uses modified brightfield imaging to generate a bright point of light for each cell that is easily identified and counted by Gen5™ software without the use of labels.
JuLI Stage Real-Time Cell History Recorder (NanoenTek Inc., South Korea) is designed to support cell biology researchers to approach kinetic images and data from the start to the end so that they can save their time and can focus on more advanced and valuable work for their research. JuLi Stage is a fully automated real-time cell history recorder that directly acquires cell images from numerous cell culture plates (6 to 384 wells) and dishes in an incubator. JuLi Stage supports the multi-channel fluorescent colors and multiple objective lens, and the sensitive filter-based optics are optimized for live cell assays. It also enables users to obtain the quantified cell confluence results with low variation and the growth curve using images based analysis with a bright field.
- Fully automated X-Y-Z stage
- Interchangeable objective lens (4x, 10x, 20x)
- Manual & auto focusing
- Compatible with various plates & vessels
- Data management with all-in-one PC
Figure. Photo of Juli stage in the cell incubator and real-time images of U2 OS cell with GFP/RFP expressions.
Figure. Real time cell growth curves. Cell confluence analyzed for cytotoxicity assays.