DRAFT_Specifications_for_Microscope.pdf

Spinning Disk Confocal/Total Internal Reflection Fluorescence (TIRF) Microscope with

Photo-manipulation

Overview of the Microscopy System:

The system described below defines the cutting edge of quantitative light microscopy that gives our department the edge in working out the molecular mechanisms underlying rapid and long-term cellular or tissue level processes in living cells or tissues. In order to achieve our goals of high-resolution and/or super-resolution (per individual user’s and experimental needs), sustained quantitative live cell imaging, we are requesting a combined spinning disk and TIRF confocal microscope equipped with a photo-manipulation laser launch that can perform quantitative techniques including FRET, FRAP, FLIP, optogenetics and photoactivation. This system should be easily upgraded to add on STORM for single molecule tracking in live cells.

The idea is to have a single, modular, versatile unit that allows one to perform long-term, sustained quantitative high- and super-resolution microscopy on the same sample of live cells, and allows for photomanipulation during image acquisition.

Software:

For sustained imaging of cells in culture or in tissues, the software should allow for complex decision making during timelapse. The programming interface should allow users to create unique experimental conditions using image analysis based “If/Then” type statements. This allows one to track individual cells even as they move beyond the field of view and to rapidly build montages of large XYZ fields. Other features in the software should allow for photo-manipulation or photostimulation on the fly while evaluating the fluorescence intensity of a region of interest for quantitative light microscopy, or complex high content image acquisition in multi-well dishes for screens.

The system should also provide a passive license software key for off-line analysis (USUHS to supply a dedicated workstation/monitor for this second key).

Combined Applications and Hardware Tools:

Combined spinning disk and TIRF: This combined system should integrate the spinning disk confocal with the motorized TIRF hardware through the program software in order to perform high resolution and super-resolution on the same sample, individually or simultaneously. This allows one to acquire different levels and types of information from the sample with minimal perturbation of this sample. Each mode (spinning disk and TIRF) must be equipped with an individual EMCCD camera (min of 512x512, 16um2 pixel size, peak QE 97%, full frame rate

56fps) and a high-speed emission filter wheel (<150ms adjacent positions with a minimum of 8 positions) for uncompromised flexibility and sensitivity in every experiment. Additionally, the unit should be a fully motorized inverted microscope, equipped with two separate laser launches, one for imaging and one for photostimulation.

Photostimulation: In order to perform cutting edge quantitative light microscopy techniques including FRAP, FLIP, optogenetics and photoactivation, the dedicated photostimulation tool must be an ultra-fast beam steering unit for the most effective and precise delivery of high energy laser light into multiple user defined regions of interest. Additionally, this system must provide true SIMULTANEOUS stimulation wherein the image collection process is not interrupted while the stimulation or bleaching is occurring. This must apply to imaging in both spinning disk confocal and TIRF imaging modes.

Lasers: We require two separate laser launches: (1) a no alignment and no maintenance laser source with a minimum of 6 illumination wavelengths (405/20mW, 445/20mW, 488/70mW, 514/40mW, 561/70mW and 647/125mW) for both the spinning disk and TIRF modules. This unit must be ready for STORM super-resolution in order to do single particle tracking, (2) a no alignment and no maintenance laser source that includes three (405/488/561) illumination wavelengths dedicated for the photo-manipulation/photostimulation tool.

Focus Drift Correction: A key aspect of long-term sustained high and super-resolution imaging of live cells is a real-time Focus Drift Correction to counteract focus drift due to subtle changes in the environment including temperature shifts and the addition of pharmaceutical agents in the media. The Focus Drift Correction system must perform corrections continuously in real time during the image collection process without requiring a pause for evaluation and correction of focus. The speed should be on the order of 30Hz to avoid loss of time-sensitive data due to delay during adjustment. To achieve this, the Focus Drift Correction system should be autonomous as part of the microscope’s optical hardware yet must be able to operate independently of the software while being able to be integrated into complex experiments through the software.

Microscope Hardware:

· Fully automated inverted microscope platform for live cell imaging studies

· Triggerable Transmitted light LED light source for the merging of transmitted light images/TIRF with filter to prevent autofluorescence

· Linear encoded XY Stage with joystick and software control for large image tiling and precise multipoint experiments

· Fast Piezo-Z Stage with a minimum of 100um travel for the acquisition of fast z-stacks with holders for well plates, slides and Petri dishes 35-65mm.

· Stage top environmental incubator chamber with mixer for control of CO2, Humidity and

Temperature with holders for 35mm dishes, Slides, Chamber Slides and Chambered

Coverglass samples.

· Motorized TIRF with automated control of critical angle for up to 4 laser lines.

· Widefield, software-controlled white light LED with individual filter cubes for

DAPI/GFP/mCherry in a motorized filter turret.

· Minimum objective specification:

o 10x Plan Apo 0.45 NA/ 4mm WD o 20x Plan Apo 0.75 NA / 1mm WD o 40x Plan Fluor 1.30 NA oil / 0.20mm WD o 60x Plan Apo 1.40 NA oil / 0.13mm WD o 100x Apo TIRF 1.49 NA oil / 0.12mm WD with Correction Collar

· Advanced Imaging Workstation capable of controlling the acquisition and processing of large data sets. Min specs: 8-core Xeon processor, 64 GB DDR4 RAM, 256GB SSD OS Drive, 6GB Upgraded GPU Video RAM (the upgraded GPU is important)

· 30” Monitor

· 36x48’ Vibration Isolation Table, 4” Top

Triggered Device Control: The components of this system must be built for speed in order to capture the rapidly evolving dynamics of life. Triggering capability to reduce or eliminate software overhead and allow the system to capture at the fastest frame rates achievable by the hardware offers unique value for our work.

Upgradeability: The microscope should start with a modular optical base that can accommodate any number of additional upgrades. In order to maximize the value of this system and keep it at the cutting edge over the next decade with minimal expenditures, the combined CSU/TIRF/photostimulation system should be built upon a platform that allows in-the-field upgrades to any of the following:

· Super-resolution o STORM super-resolution o SIM super-resolution o Hybrid Simultaneous High-resolution Point Scanner/High-speed Resonant

Scanner o spectral or multi-photon confocal and dedicated High Content Analysis Tools.

· Point Scanning Confocal o spectral confocal o Multi-photon confocal

· HCA

o True Integrated High Content Collection and Analysis assays including robotic plate loading

· Customizable microscope illumination tools:

o Additional wavelength specific TIRF inputs o Digital Mirror Device photostimulation tools o STORM super-resolution o Fast channel switching LED illumination

Support:

Given the complexity and high usage of this system, there should be a local technical representative team at USUHS to provide rapid service. To maximize the value of this system, we would favor a vendor that provides on-site unlimited technical support and training for the life-time of this microscope.