SCHOOL OF MEDICINE

Department of Medicine

Indiana Center for Biological Microscopy

Current Facility Equipment

Leica SP8 Resonant-scanning confocal/multiphoton microscope – This microscope is configured for both multiphoton and standard confocal imaging. The scanner is mounted on an upright fixed large-platform motorized stage equipped for confocal excitation at 405, 448, 488, 552 and 633 nm with multiphoton fluorescence excitation provided by a fully automated Spectra-Physics MaiTai DeepSee titanium-sapphire laser, providing tunable illuminations from 710 to 990nm. . The sytems 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.

 

Leica SP8 confocal/multiphoton microscope – Recently purchased with funding from an NIH Shared Instrumentation Grant, this is an additional Leica SP8 confocal/multiphoton microscope system. The confocal capabilities will be equivalent to those of the existing system, but the new system is mounted on an inverted microscope stand, a configuration that is better suited to intravital microscopy. The new system is also equipped with new features that will extend our capabilities for intravital microscopy. First, it is equipped with two lasers, a Spectraphysics MaiTai DeepSee laser and a Spectraphysics Insight laser system. The Insight laser extends the range of wavelengths that can be used for multiphoton excitation out to 1300 nm, significantly improving our capability for intravital microscopy of red probes, and particularly yellow and red fluorescent proteins. The two-laser system will also facilitate simultaneous excitation of multiple, spectrally separated probes as well as excitation ratio multiphoton microscopy. Second, this system is configured with 4 spectral detectors on the non-descanned pathway (2 PMTs and 2 HyD detectors), improving our ability to detect and distinguish multiple probes in living animals. Finally, this system is equipped with a two channel ISS Fast FLIM fluorescence lifetime system.

Olympus Fluoview 1000 confocal/multiphoton microscope system - Funding obtained from the NIH via a shared instrumentation grant in 2008 was used to purchase this system. Fluorescence emissions may be collected either with internal (descanned) detectors (one photomultiplier tube and two spectral detectors) or with an 4-channel external (non-descanned) detector module. Confocal fluorescence excitation is provided at 405, 488, 515, 559 and 635 nm and multiphoton fluorescence excitation is provided by a Spectraphysics DeepSee tunable titanium-sapphire laser, providing tunable illuminations from 710 to 990nm. The system is mounted on an IX-81 inverted microscope stand and is equipped with an automated stage, supporting automatic time course studies of multiple fields.

Nikon live cell imaging system – Assembled in 2015, this microscope is configured for high-speed live cell imaging of fluorescent proteins, FRET and immunostained samples in three dimensions. Built around a Nikon TiE inverted microscope stand, it is a combined confocal and widefield microscope system. Confocal microscopy is based upon a Yokogawa 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 microscopy can be conducted using a Xenon lamp source, 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. Heated high-resolution water and oil immersion objectives are available on this microscope including fluorite lenses for imaging below 400 nm.

Keyence/Akoya CODEX microscope system – Acquired in the Spring of 2019, the Keyence microscope is an automated high-content imaging system with advanced automated image collection and analysis capabilities. The Akoya CODEX system is a robotic system that 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.

OpenSPIM Selective Plane Illumination Microscope (SPIM) system – Assembled in the Spring of 2015, , based upon a design provided by the Open-SPIM project (www.openspim.org). Equipped with lasers providing excitation at 442, 488, 514, 568 and 647 nm, the system is capable of imaging up to 5 different proteins in the same sample. Unlike laser scanning approaches, SPIM is based upon deconvolution of images collected by wide-field microscopy, with focal sectioning provided by a thin light-sheet projected orthogonally to the imaging plane. As such, SPIM collects image volumes very rapidly, and is ideally suited to studies of 3D organoids and embryonic development.

Other microscope systems – The ICBM has 2 additional epifluorescence microscope systems, each equipped with a cooled CCD detector capable of high-sensitivity, high resolution epifluorescence and DIC microscopy.

Image analysis, data management and storage - Image analysis, data management and storage - The Center is equipped with two workstations loaded with software necessary for image analysis, including Metamorph and ImageJ for quantitative analysis, Imaris, Amira and Voxx for analysis of volumetric image data, and Adobe Photoshop and Premiere, for image editing and movie making, respectively. Web and remote file services are available via a failover linux cluster. All microscopy systems and computers are connected to a 1000Base-TX Ethernet switch. Microscope image data may be stored on microscope host computers for up to two days, and on a 4TB Raid Array server for up to one month. It is the responsibility of the investigator to copy data to their own computer systems or to CD, DVD or BluRay disc for extended storage. Data stored on the server is backed up to tape weekly, and is thus available, but resources for data retrieval by IUSM are limited, limiting the speed with which archived data may be restored.