SCHOOL OF MEDICINE

Department of Medicine

Indiana Center for Biological Microscopy

Current Facility Equipment

Custom multiphoton/confocal microscope modified from Olympus FV1000 – The ICBM modified a FV1000 confocal microscope system to support multiphoton fluorescence microscopy, using illumination provided by a Spectraphysics Mai-Tai HP Titanium-Sapphire laser and controlled via a Conoptics Pockels electro-optical modulator. Fluorescence or second harmonic emissions may be collected either with internal (descanned) detectors (one photomultiplier tube and two spectral detectors) or with a custom designed and fabricated 3-channel external (non-descanned) detector equipped with multi-alkali and/or gallium arsenide-phosphide high sensitivity detectors. Confocal fluorescence excitation is provided at 488, 514, 568 and 633 nm and IR illumination provided from 710 to 990mm. The system is also equipped with a two-channel ISS FastFLIM fluorescence lifetime imaging system, supporting collection of fluorescence lifetime data. 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.

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, which is similar to the Fluoview system described above, except that it is equipped with a Spectraphysics DeepSee tunable titanium-sapphire laser and an Olympus-designed 4-channel external detector module. 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. Confocal fluorescence excitation is provided at. 405, 488, 515, 559, and 635nm.

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.

Leica SP8 Resonant-scanning confocal/multiphoton microscope – Acquired in 2014, this microscope is configured for both multiphoton and standard confocal imaging. 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 laser. 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. The system is also equipped with a LSMTech objective inverter system, supporting studies that require an inverted microscope configuration. The system is ideally suited to intravital microscopy and can be equipped with an objective heater, heating pads and a thermal blanket.

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 - 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. 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.