CRYO-EM Quality Control And Workflow Optimisation

Identify samples suitable for Cryo-EM in just 4 minutes
I'm Interested
cryo-em

How does FIDA increase Cryo-EM scan success rates?

01

Reduce your time to publication

  • Key sample properties identified in just 4 minutes
  • Upfront rejection of samples of suboptimal quality samples reduces costs and saves time spent on samples that are not suited for Cryo-EM of droplet formation
  • Informed troubleshooting
02

Key sample properties determined

  • Ensure homogeneity with polydispersity index (PDI)
  • Quantify aggregation
  • Measure absolute viscosity
  • Detect and quantify stickiness – prior to cryo-EM scan
  • Check structural integrity - thanks to absolute size (Rh) measurement
03

Directly in Cryo-EM relevant concentrations

  • FIDA users do not need to dilute their samples to get key pre-cryo-EM parameters
04

Native conditions

  • No buffer constraints
  • No detergents, salt, pH or concentration constraints, provide increased flexibility.
  • Triple temperature control
  • UV-detector allows to work with unlabelled proteins.
05

Lower sample consumption

  • Only hundred nanolitres per condition in the screening stage
  • Down to 4 μL analyte, 40 nL indicator for characterisation
06

Boosted process efficiency

  • FIDA users reduce overall cryo-EM cost by discarding unsuitable samples upfront
  • FIDA users avoid wasting time and financial losses incurred by scanning samples of poor quality
  • Low energy consumption of the Fida instrument
  • Low sample usage (down to 4 μL analyte, 40 nL indicator)
  • Full automation saves time
  • Speed of QC process minimises time-related sample degradation
07

Improved structural resolution and data reproducibility

  • Pre-cryo-EM in-depth sample characterization
  • Flexible dilution options, native conditions
  • Optimize sample deposition through enhanced knowledge of your sample
  • Flexible buffer conditions allow FIDA users to work in a wide range of pH and salt concentrations
08

Automated and temperature controlled

  • Autosampler fits 2 x 96 well plates or 2 x 50 vials. Save your time for what really matters.
  • Keep samples stable at 5°C during the duration of an assay
09

Work with membrane proteins and nanodiscs

  • Assess membrane proteins and lipid nanodiscs thanks to FIDA’s detergent compatibility.

Success of CRYO-EM assay depends on the sample quality.

FIDA users select the adequate sample upfront thanks to fast and efficient pre cryo-EM analysis.

Quantitative data about viscosity, aggregation, homogeneity and oligomeric state,
measured in one assay, allows FIDA users to select only the best samples,
and avoid the time and cost loss incurred due to scanning samples of poor quality.

On top of that, having an in-depth knowledge of the sample makes it easier to perform grid preparation.

Absolute size (Rh)

  • Hydrodynamic radius measurement from 0.5 to 500 nm
  • Makes it possible to ensure the basic feasibility of a sample/that the sample is within practical size limits of cryo-EM
  • Globular size change (Rh) down to 5%

Aggregation

  • Quantified FIDA readout is delivered as a number of aggregates in the sample
  • Quantification of aggregates performed ubiquitously – all throughout the measurement 
  • Aggregates are presented visually as signal spikes on raw FIDA data
  • No constraints on size of aggregates

Stickiness – fully visible

  • Reveals sample hydrophobicity – more informed workflow helps to achieve optimal layer thickness
  • Awareness of sample stickiness helps to troubleshoot issues with ice thickness and ultimately deposit a suitable monolayer of macromolecules
  • FIDA is the only technology on the market that delivers stickiness readout

Sample polydispersity index

  • Ensure homogeneity & spot the optimal purification stage to avoid over-purification.
  • Measured in high concentrations – FIDA users have no need to dilute the sample to assess homogeneity
  • Insight into potential protein denaturation and/or deformation pre-vitrification

Viscosity as an absolute measurement

  • Quantified viscosity measurement – allows FIDA users to assess suitability of the sample to cryo-scanning and customise sample deposition settings.

Absolute size (Rh)

  • Hydrodynamic radius measurement from 0.5 to 500 nm
  • Makes it possible to ensure the basic feasibility of a sample/that the sample is within practical size limits of cryo-EM
  • Globular size change (Rh) down to 5%

Aggregation

  • Quantified FIDA readout is delivered as a number of aggregates in the sample
  • Quantification of aggregates performed ubiquitously – all throughout the measurement
  • Aggregates are presented visually as signal spikes on raw FIDA data
  • No constraints on size of aggregates

Stickiness – fully visible

  • Reveals sample hydrophobicity – more informed workflow helps to achieve optimal layer thickness
  • Awareness of sample stickiness helps to troubleshoot issues with ice thickness and ultimately deposit a suitable monolayer of macromolecules
  • FIDA is the only technology on the market that delivers stickiness readout

Sample polydispersity index

  • Ensure homogeneity & spot the optimal purification stage to avoid over-purification.
  • Measured in high concentrations – FIDA users have no need to dilute the sample to assess homogeneity
  • Insight into potential protein denaturation and/or deformation pre-vitrification

Viscosity as an absolute measurement

  • Quantified viscosity measurement – allows FIDA users to assess suitability of the sample to cryo-scanning and customise sample deposition settings.

01

FIDA may also be a useful tool in prevention of denaturation issues

Since denaturation changes the globular size of a molecule, FIDA’s absolute size measurement can indicate those changes in samples pre-vitrification.

This ensures that samples that denature pre-vitrification are excluded.

02

FIDA may also be a useful tool in prevention of denaturation issues

Secondly, FIDA can be used for buffer screening. FIDA’s buffer flexibility makes it possible to test the behaviour of the molecule in buffers with diverse pH or salt concentration.

Since buffers impact the stability of the protein (due to the buffer-related surface charge), altering buffer composition changes the molecule's behaviour upon freezing. Using FIDA to choose the right buffer can help to prevent denaturation.

03

FIDA may also be a useful tool in prevention of denaturation issues

On top of that, since FIDA does not require sample delusion, a sample is QCed at the same concentration as the sample used for vitrification, increasing the applicability of QC results obtained.

04

FIDA may also be a useful tool in prevention of denaturation issues

Moreover, FIDA’s triple temperature control makes it possible to maintain stable temperature during the quality check.

Benefits

Sample QC at the concentration used during vitrification
Identification of samples with a high likelihood of CRYO-EM success
Sample QC at the concentration used during vitrification
Direct impact on success rate and speed-to-publication
A QC assay in 4 minutes
Stability control
Informed troubleshooting

Features

Undiluted measurement
No buffer restrictions
Quantified aggregation  
Polydispersity index
PDB structure correlator
Just 120 nL of sample consumed for triplicate measurement
Samples can be analysed and stored at desired temperatures
Recoverable sample – can be used directly for vitrification

Measures

Accurate determination of dilute phase concentration
Relative droplet size distributions
Kinetics of droplet formation and maturation into amyloid fibrils
Determination of binding affinity between the polypeptide undergoing LLPS and LLPS-modulating compounds

This methodology is very sensitive and useful for assessing binding in a quantitative manner using small amounts of auto-fluorescent proteins. Using this method, we can observe clear changes in apparent hydrodynamic radius (Rh) and in fluorescence signal of EYA1 as a function of increasing concentrations of peptides or of small molecules

Dana Farber Institute
CANCER INSTITUTE

''Fida  Instrument provides a ''ruler'' to measure the length of fibrils''

Stefan Rüdiger Ph.D.
Professor of Protein Chemistry of Disease; Head of Department of Chemistry

''The FIDA is very effective for screening small molecules for solubility and interactions with protein targets. A major advantage has been its ability to clearly show drug binding to difficult-to-work-with targets such as intrinsically disordered proteins and amyloids''

Dr. Alexander Taylor
Research Fellow in Biochemistry/Biophysics

I would say that this instrument is quite useful for core facilities at KI or for Swedish research community in general:

  1. it was possible to measure unlabeled membrane proteins and soluble proteins at low concentrations in small volumes very fast (~6 minutes per measurement).
  2. I also like the temperature control and the autosampler which allowed us to run a lot of samples overnight.
  3. I would say that there is a lot of potential to perform other assays as well, such as the Kd determinations etc. (For this, I use ITC which is quite time-consuming and requires a lot of protein).
  4. In addition, the software and user interface for sample runs and data analysis is quite straightforward and intuitive to use.
Postdoctoral Researcher

"What we value about FIDA is the amount of information we can get from a tiny amount of protein in a single QC run. FIDA technology enables us to understand our proteins better - particularly when they behave in unexpected ways. We chose FIDA because it gives us that vital first result quickly - we don't waste our time optimising an assay that isn't going to work."

Duncan Smith
Lead Scientist

"I am convinced. I have never before been able to detect the ternary construct formation of my construct directly in cell lysate – it took less than two hours."

Thomas Smith
Associate Director, Chemical Biology & Therapeutics

"The Fida 1’s unique capability for measuring binding Kd from weak mM level to strong pM level really enhances our bioanalytical capabilities."

Sherry Zhu
Senior Scientist

"FIDA is an in-solution technique, and thus an ideal binding assay for structurally diverse bsAbs because the analysis does not rely on potentially obstructive surface immobilization and hence it is able to perform characterizations that are unbiased by bsAb format and spatial orientation of the binding domains."

Andreas V. Madsen
PhD Student

"After thorough comparisons, it was clear that the Fida 1, amongst others, presents unique opportunities in analysis of membrane proteins, which is essential to us."

Svend Kjæer
STP Deputy

''The Fida 1 system offers a new approach to detergent screening that has significant advantages allowing essential data to be obtained faster using less material (…) this work presents a new approach to characterize membrane proteins that allows users to reducing costs and time as well as to analyze protein expressed at low levels in a short time thus overcoming any stability issues.''

Isabel Moraes
Principal Research Scientist

"I like the ease of use and the straightforward data analysis and, that experiments can be performed in any type of buffers. We get data, where no other biophysical methods worked before."

Cathrine Birck
Head of Integrated Structural Biology Platform

"The method is easy to setup in any standard laboratory and can be adopted for a high throughput (HTTP) screening, which enabled mechanistic studies of RNA nuclease interactions, as well as efficient guide RNA lead discovery (…) Fida 1 offers straightforward assay development, walk away automation, absolute measurement and ultra low sample consumption."

Denny Truong
Principal Research Associate

"FIDA provides in-depth assessment of activity combined with local and global protein structural changes by measuring the overall hydrodynamic radius of the protein with minimal sample consumption. In addition, it is possible to measure the affinity constant for the analyzed interaction."

Dr. Melanie Hug
Research Associate

"The beauty of Fida 1 is that it allows us to conduct a lot of biophysical measurements in one system using a small amount of reagent and a rapid read-out time which is important for our projects and to our customers. (…) Providing a high-quality, protein characterisation QC package is certainly extending and future proofing our capabilities in biophysical characterisation of the proteins we produce."

Mark Abbott
Managing Director

"The Fida 1 has allowed us to assess the performance of MIPs with a much higher throughput than was previously possible on other platforms. MIPs can be developed in as little as 8 weeks, which is a significant improvement on antibodies, and now they can be fully characterized at an accelerated pace too."

Alan Thomson
CEO

"Fida 1 can be used for full characterization of ternary complex formation for targeted protein degradation. The Fida 1 platform only consumes 39 nL of sample for one measurement and thus allows elaborate condition screening using very small amounts of sample material."

Roman Agafonov
Associate Director of Biochemistry, Biophysics & Structural Biology

"The Fida1 is a very user-friendly system. With the Fida software we can quickly design new methods, set up experiments, and analyze data. By far this is a great instrument and technology that has allowed us to run binding assays for LNP in solution and with flexibility to run in any complex media which we were unable to do using traditional binding methods that required ligand immobilization. The FidaBio customer service is amazing, they are very knowledgeable and responsive whenever I run into issues and need assistance. I highly recommend this instrument for LNP or any other nanoparticle characterization. The system and technology are versatile with applications beyond nanoparticles and is a must in the biophysical tool kit."

Biophysical Analytical Department
Senior Scientist, Analytical Development

"Eventually, with FIDA, we managed to characterise our LNPs. And it was done in less than 2 days. We had for a long time been struggling with SPR."

Chemical Biology and Therapeutics
Associate Director

"We had been looking for ways of quantifying our exosomes, and tested many platform, without success. In less than 2 days FIDA had developed an assay and shown trustworthy quantifications directly in fermentation broth"

Rane Harrison
Director, Analytical Development

Become a user

Your laboratory instruments should serve you, not the other way around. We’re happy to help you.