Major Microscope Systems and Equipment

Leica TCS SP8 Resonant-scanning confocal/multiphoton microscope is configured for both multiphoton and confocal imaging on an upright stand. Confocal fluorescence excitation at 405, 448, 488, 552 and 633 nm with multiphoton fluorescence excitation provided by a Spectra-Physics MaiTai DeepSee titanium-sapphire laser, providing tunable illuminations from 690 to 1040nm. The system is configured with 4 internal (descanned) PMTs and 4 external (non-descanned) detectors – 2 PMTs and 2 HyD hybrid detectors. The SP8 MP includes both standard galvanometric scanning for imaging at up to 1800 Hz and resonant scanning for imaging up to 8000 Hz, supporting full frame imaging at up to 30 fps. The automated stage supports automatic time lapse studies of multiple fields and tile acquisitions. 

Leica TCS SP8 Dive confocal/multiphoton microscope has confocal capabilities equivalent to those of the system described above, but is mounted on an inverted stand. This system is also equipped with several features that extend our capabilities for intravital microscopy. First, it is equipped with two IR lasers, a Spectraphysics MaiTai DeepSee laser and a Spectraphysics Insight laser system that extends multiphoton excitation out to 1300 nm, improving intravital microscopy of red fluorescent proteins. The two-laser system also facilitates simultaneous excitation of multiple, spectrally separated probes. Second, this system is configured with 4 spectral detectors on the non-descanned pathway (2 PMTs and 2 HyD detectors), improving detection and distinction of multiple probes. Finally, this system is equipped with a two channel 2-photon ISS Fast FLIM fluorescence lifetime system and a motorized stage supporting tile acquisitions and time-lapse multiple filed studies.

Nikon live cell imaging system is configured for high-speed imaging of living samples. Built around an inverted Nikon Eclipse Ti stand, it supports both confocal and widefield imaging. Confocal microscopy is based upon a Yokogawa CSU-X1 spinning disk, with laser illumination provided at 442, 488, 514 and 561 nm via an Agilent launch with images collected using a Photometrics Prime 95B Scientific CMOS. Widefield epifluorescence microscopy can be conducted using a Xenon lamp source, with optics suitable for excitation from 340 nm to 700 nm, with images collected by a Hamamatsu Orca Flash 4.0LT Scientific CMOS. It is equipped with a Perfect Focus system, a motorized stage that supports automatic time course studies of multiple fields and a stage-top incubator for maintaining a heated and CO₂ dependent environment to ensure stable long term imaging of cultured cells.

Keyence BZ-X810 microscope system is an automated high-content imaging system with advanced automated image collection and analysis capabilities, supporting a variety of cellular and biochemical assays of living or fixed specimens mounted in dishes, flasks, multi-well plates, and microscope slides or chambers. System supports imaging in fluorescence, brightfield, and oblique illumination with a highly sensitive, cooled CCD camera and a built-in darkroom, generating publication-quality images. It is equipped with DAPI, GFP, RFP, Cy5, and Cy7 epi-filter cubes, 4x/0.1, 10x/0.3, and 20x/0.75 objectives, automated stage, and a stage-top environmental incubation system supporting time-lapse imaging of live cells. Automated quantification can be performed with the Keyence analysis application augmented with three additional module licenses (Hybrid Cell Count, Time-lapse, Advanced Observation).

Akoya Phenocycler-Fusion multiplex fluorescence imaging system is an integrated high-speed imaging coupled with automated fluidics control covering a full workflow from staining to analysis. It supports multiplexed fluorescence imaging of 40+ probes in the same sample, using a proprietary process of repeated sequential imaging and stripping of fluorescently-labeled DNA-barcode probes. The Fusion microscope is also capable of high throughput slide scanning with filters for Brightfield and the proprietary Opal dyes between 440 nm and 780 nm.

The ICBM offers two phenocycler systems.

Alpenglow Aurora 3Di Light-sheet microscope — Purchased in August 2024 with funding from an NIH Shared Instrumentation Grant, the Aurora 3Di is a high-throughput, open-top light-sheet microscope specifically designed for imaging optically cleared large samples (up to 12 cm x 7.5 cm x 1 cm) or multiple smaller specimens, thanks to its open-top geometry. The Aurora 3Di features two acquisition light paths: low-resolution SCOUT mode (3x magnification) and high-resolution ZOOM mode (40x magnification), both utilizing a dedicated Hamamatsu ORCA-Fusion BT CMOS camera (16-bit, 2048x2048 pixels).

Equipped with four laser lines (405 nm, 488 nm, 561 nm, and 638 nm) and a set of filters (Zoom: 447/60-25, 525/50-25, 618/50-25, 647R-25; SCOUT: QUAD 432/515/595/730-25 bandpass), the system supports imaging of commonly used fluorophores. Its “scout and zoom” capability allows users to quickly scan an entire specimen at low resolution and then re-scan regions of interest at high resolution without manually changing or calibrating objectives. The system includes 10 custom-designed sample holders (RI 1.56), supporting multiple or single specimen imaging. Currently calibrated for a refractive index (RI) of 1.56, the system can be adapted for other RI clearing solutions (upon request only).

The Aurora 3Di workflow is supported by two high-specification workstations and a direct data transfer system. When integrated through this system with the microscope acquisition computer, these workstations form a three-node image collection, processing, and analysis (Imaris v.10.2) system dedicated to the Aurora microscope.

Image Analysis, Data Management and Storage — Three computer workstations, each equipped with multicore Xeon processors, up to 24 GB of GPU memory, 256 GB of RAM, and up to 4 TB of solid-state disk storage, run a variety of image processing software, including Metamorph v7.7, Imaris Core Facility Package v.10.2, Halo, NIS Elements, and Leica LASX. Specialized image analysis software is also available on the image acquisition computers (VistaVision, Keyence BZ-X800 Analyzer, and the CODEX software suite). All microscopy systems and computers are connected to a 1000Base-TX Ethernet switch. Additionally, the ICBM is supported by the supercomputing infrastructure provided by Indiana University.