Breakout Groups Summary
Key Discussion Points
- Importance of getting the sample preparation right to avoid artefacts whilst conducive to the requirements for each specific technique
- Requirements of appropriate standards and reference materials that can be used not only with one technique but the possibility of across techniques for quantitative bioimaging and determining LODs
- Establishing the correct sequence of experiments & analyses
- Determining appropriate methods of correlation between techniques, which may influence sample preparation
- Integrating contextualising imaging in the form of tagging or other morphological imaging approaches
- Addressing current inherent limitations in sample size/throughput
Several major challenges were identified and discussed:
- Sample preparation for MMI approach
- Sample numbers and throughput required for statistically meaningful conclusions
- Biological context in elemental mapping (what are you actually looking at)
There is a need to work on defining and comparing different sample preparation strategies similar to the work of Matteo. If done properly this could potentially lead to a set of recommended approaches for other projects and users in the field and would make comparing scientific studies easier. Also a need to develop a set of standards that can be tested and perhaps even used across imaging modalities. There could be an opportunity to collaborate with LGC on this one (been speaking about it for years now).
XRF microprobe seems to be the most promising, yet still has limitations in throughput and LODs. How to address the throughput limitations? Always will be the need for a parallel bulk quantification approach until the techniques allow for a throughput that would be considered statistically significant with quantification approaches that are well characterised and robust.
There is a need to have parallel capacity to image the biological context directly or close to the locations where elemental mapping is being conducted. This could be as simple as confocal microscopy (although cryo confocal is ideal under many cases) or molecular imaging. Being able to label specific organelles, specific cell types and a general requirement to identify the context in which the elemental mapping is taking place. Often the best and only approach is sequential sections as sample prep lends itself to artefacts in the case of labelling approaches. In this context would be important to determine specific sample prep workflows that can be done, which do not influence metal distribution (and/or identify which specific metals are influenced in what ways).
Also discussed 3D XRF tomography at I18 for zebrafish and requirements and ideas on sample preparation (ie high pressure freezing difficult due to the size of the embryos)
An explanation of phenotype
- Mechanisms underlying biological observation
- Differential metal tolerance
- Dietary deficiency
- Differential sensitivity of life stages
- Differential species sensitivity
- Use of genetic modification and imaging to explore phenotype and function.
- Transport at Barriers and Interfaces
- Egg Chorion
- Dynamic movement of metals
- Influence on uptake of Ligands
- Biotransformation at barriers
- Intestinal epithelium
- Uptake of essential micronutrient (ie Colbolt for Vit B12)
- Interaction between metals
- Dynamic in vivo metal distribution
- Paramagnetic metals (Mn, Gd and Fe)
- Sample preparation
- 2D imaging: Chemical Vs cryo / morphological integrity vs speciation of metal integrity
- 3D Tomography
- Influence of cellular environment
- Co Measurement of redox and pH
- Sequence of experiments
Having different methods but one doesn’t know what workflow to use.
Different instruments need different type of samples.
- Major issue: Sample preparation
Different methods require different sample preparation. Eg.: EM needs coating, SIMS doesn’t.
Different instruments require different type of samples
- Reference materials
How do they behave in distinct environments? Same or different?
- Sample size
- Post analysis
- NEED STANDARDS -> would be very valuable in guiding scientists trying to use distinct methodologies
How do we do quantitative biology from imaging?
- Some methods don’t have good resolutions
How do you orientate yourself?
- Using markers that are specific to a particular organelle or location in the cell
- Could have a reference point and measure the distance from there to orientate yourself
After everyone introduced him/herself in the group, we started with discussing manganese localization in the Golgi. If one does not consider subcellular fractionation and wants to use in situ imaging, there are issues with localization and sensitivity and correlation with microscopy. We discussed metal-labelled antibody (AB) technology of permeabilized and non-permeabilized cells. AB tagged with lanthanides and gold nanoparticles were mentioned. We then spent quite a bit of time on discussing sample preparation for synchrotron radiation-based measurements. It was noted that additional instrumentation for multimodal imaging is rarely available at the synchrotron and therefore sample integrity during transport is an issue. We talked about spectroscopy with synchrotron radiation as mainly being a non-destructive technique and therefore to be used before labelling or other analytical techniques are employed. However, there is an issue with interference from other metals in some cases, damage to the sample, in particular to changing valence states of metal ions, and therefore the need to monitor sample integrity. In terms of sample preparation we discussed cryopreservation and some of its complications in transporting samples, also calling for need to prepare samples at the location. Chemical fixation is useful for some tightly protein-bound metals such as zinc and copper, but washout has been observed for alkali and earth alkali ions and is likely an issue if pools of metals ions that are bound less tightly are to be investigated, including the metal ions stored in cellular vesicles, lysosomes etc. We also discussed that metal contamination is always an issue to contend with.