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Access to the Titan Krios Cryo鈥慣EM

The Titan Krios Cryo鈥慡TEM (鈥淜rios鈥�) is a flagship, high鈥憈hroughput cryo鈥慹lectron microscope capable of autonomously collecting extremely large datasets at high resolution. Because it is both costly to operate and in exceptionally high demand among regional, national, and international research groups, access to the Krios is carefully managed to ensure fair use and maximize scientific output.

Operational Model

To make the most efficient use of this instrument, FEMR staff perform the majority of Krios operations on behalf of users. This includes:

• Grid clipping and cassette preparation

• Loading and unloading of autogrids

• Cryogenic transfers

• Microscope alignments and calibrations

• Low鈥慸ose imaging setup

• Automated data collection

• Troubleshooting and system monitoring

Following extensive training and demonstrated proficiency, some users may be authorized to perform selected tasks independently. Such authorization is granted at the discretion of FEMR staff.

1. Project Submission & Assessment

All projects begin with:

1. Submission of the FEMR Project Form

2. Attendance at an Assessment Interview with FEMR staff

Only projects deemed appropriate for high鈥憆esolution cryo鈥慐M will proceed to the Krios screening stage.

2. Cryo Grid Quality Control (Mandatory)

No sample may be loaded into the Krios without passing both negative鈥憇tain TEM screening and cryo鈥慐M screening.

Negative鈥慡tain TEM Screening

Performed using uranyl acetate or uranyl formate on:

• FEI Tecnai Spirit 120 keV TEM, or

• Glacios 2 Cryo-TEM

This step evaluates sample integrity, monodispersity, and suitability for vitrification.

Cryo鈥慐M Screening

Vitrification is performed using the TS Vitrobot Mk IV or Leica EM GP2, followed by imaging on:

• Glacios 2 Cryo-TEM, or

• Tecnai Spirit 120 kV TEM (with cryo鈥慴ox)

This step assesses ice thickness, particle distribution, and overall grid quality.

Requirements for Krios Imaging

To qualify for loading onto the Titan Krios, users must provide:

1. Low鈥憁agnification images showing high鈥憅uality ice of appropriate thickness

2. High鈥憁agnification cryo鈥慐M images demonstrating:

   • Clear particle visibility

   • Minimal aggregation

   • Absence of contamination

   • Consistent ice quality

3. A full cryo鈥慐M grid map (Atlas) generated using SerialEM

4. A completed Project Form 鈥� Cryo鈥慐M

Recommended Workflow for Successful Krios Sessions

To maximize the likelihood of successful data collection:

1. Optimize Vitrobot blotting and sample concentration using the Glacios 2 Cryo-TEM or Tecnai Spirit.

2. Once optimal conditions are identified, freeze ten new grids using the same parameters.

3. Screen one or two of these grids.

4. If screening confirms good ice and particle distribution, submit six grids for Krios loading.

5. If none of the four grids are suitable, users may submit another set of six grids for an additional fee of $500.

This process ensures that only high鈥憅uality grids are loaded, maximizing productive microscope time.