Notes

Our preprint on single-molecule flow cytometry (smFC) is now live on bioRxiv. We integrated high-NA oblique plane microscopy with microfluidics to push flow cytometry sensitivity down to single molecules on live cells that enables detecting as few as ~2 labelled receptors per cell.

The key insight was combining optical sectioning from the OPM light sheet with super-bright, large Stokes-shift fluorophores to overcome cellular autofluorescence which is the main bottleneck in conventional flow cytometry at low expression levels. We validated the system on c-kit receptor distribution in triple-negative breast cancer cells and found a subpopulation invisible to standard FC.

Future work will be on extending smFC to multicolour detection and higher throughput. Feedback very welcome!

→ Read the preprint on bioRxiv

Working with solvatochromic dyes like Nile Red and Laurdan at sub-zero temperatures throws up some surprising challenges. How membrane fluidity changes if you cool mammalian cells toward 0°C? and the changes we see are from the dye or actual fluidity is changing? cause lowering temperature also affect how the dye partitions and reports.

The peak emission wavelength of solvatochromic dyes shifts with the polarity of their local environment. In disordered membranes, the fluorophore sits in a more polar environment and shifts one way; in ordered membranes, it shifts the other. The problem at very low temperatures is that most mammalian membrane lipids start to phase-separate or gel, and the spectral shift you'd normally interpret as "ordered membrane" now partially reflects a kinetic freezing-in of the dye rather than true equilibrium partitioning.

So if I prepare membrane models and image them at low temperature, it should be interpretable of how the dye is behaving on the membrane at cold and we can use this as the calibration of membrane dyes at cold. More notes to follow as this project develops.

A recent Nature Communications paper presents soTILT3D, a single-objective tilted light-sheet approach for whole-cell 3D SMLM. It is a clever engineering solution to an aged problem! But why not just have an high-NA OPM?

The key idea is to steer a light sheet at a tilt angle through the sample using only the primary high-NA objective, combined with nanoprinted microfluidics to hold the sample. This avoids the stitching artefacts and drift issues that plague dual-objective setups. The results show super-resolution imaging of whole mammalian cells with reduced background compared to TIRF or HILO (Boring!).

A recurring debate in imaging labs: should you build your own microscope or buy a commercial system? The honest answer depends on what question you are trying to answer, but I've come to think the framing itself is slightly wrong.

Commercial systems are optimised for robustness and reproducibility They are excellent for asking well-defined questions on well-behaved samples. The moment your sample or your question is at the edge of what the instrument was designed for, flowing cells, sub-zero temperatures, nanometre-precision focus over long timescales, you are fighting the machine rather than using it.

Building your own system is slow and often frustrating, but it forces you to understand every degree of freedom in the measurement. When something goes wrong (and it always does), you know where to look. More importantly, when you want to do something the machine was not designed for, you can. The PiFocus project started because I needed focus stabilisation compatible with different objective lenses and my custom-built OPM.

My working rule: buy commercial for routine measurements, build custom for novel modalities. And never underestimate how much you learn from building.