Working with the Gene Frame replacements

by Iren Grigoryan and Christian Rudolph

In my last post on the Gene Frame replacement, I left a number of questions deliberately open. I had sourced the Grace Bio-Labs SecureSeal frames, worked out how they handled, and reasoned my way through why their volume and drying behaviour would differ from the discontinued Gene Frames – but at that point nobody had actually put cells on one. Everything I said about imaging was inference.

We have now done real work with the frames, with the bench work carried out by the co-author of this piece, and several of the open questions can be closed.

They work

The headline first: for snapshot microscopy, the frames are excellent.

We imaged across six slides, and every one behaved. But the detail that matters most is this: the entire frame was in focus, edge to edge, with no fall-off at the margins. This is precisely the failure the frameless puddle method suffers from – the curvature gradient that leaves the centre sharp and the edges soft – and it did not appear here at all. A pad formed within a frame sits flat, and the flatness held right across the field, as illustrated by the images shown from three different locations within a frame. All three are fully in focus. That was true for all six slides we examined, and it is testament to the excellent evenness of the frames as well as the clean technique with which the pads were generated. A frame can only be as flat as the person generating it allows.

A second opinion on handling

I found the frames easy to work with in the last post, but I was the only person who had tried them, and I have twenty years of muscle memory that a new user does not. So it is worth reporting that my co-author liked them too, and rated the handling at least as good as the Gene Frames – probably better.

Her complaints were specific, minor, and worth passing on.

The first is the colour. The Grace frames are matt white rather than the old blue, and white is genuinely harder to see against a glass slide. When you are trying to place a frame precisely, or check that it has sealed, the lack of contrast is a small but real irritation.

The second is more practical, and I had not noticed it myself. The frame height matches the height of a microscopy slide quite closely. This sounds harmless, but it means alignment matters: if the frame is not placed squarely, a sticky edge stands proud of the slide, and you end up with an adhesive ridge catching on everything it touches. You have to line them up properly, which is precisely what is a little tricky given the colour.

Neither of these outweighs the improved handling, and the fact that her only complaints were a colour and an alignment tolerance is, if anything, a strong endorsement.

Volume, resolved

In the last post I suspected 70 µl would be about right, and that turns out to be better than 65 µl, leaving only a little space within the frame. We also tested 68 µl, which I suspect is the sweet spot, but in terms of imaging we did not observe any difference.

But a caveat I should make plainly, because it inverts the intuition: for snapshot work, the exact volume does not matter. A little more or less agarose changes nothing you can see in the image. Where the volume matters is time-lapse, where you actively want a small air gap – a few bubbles – within the sealed frame, to give the cells a little extra oxygen over a long acquisition. For a snapshot, 68 or 70 or 72 µl are all fine. For time-lapse, the margin is narrower, and that is a test still to come.

Drying, provisionally resolved

I had guessed 10 to 12 minutes at room temperature, down from the Gene Frame's 14, on the reasoning that a smaller volume in a shallower well dries faster.

We went with 10 minutes, and it worked very well. But after the recent heatwave the building is still unusually warm, and drying time depends on ambient conditions. In cooler weather 12 minutes may prove better. So call it 10 minutes for now, with the expectation that this number will need revisiting once the building returns to a normal temperature. It is a parameter, not a constant.

The thickness puzzle

One loose end from last time. I had the impression that the Grace frames were slightly thinner than the Gene Frames, despite both being specified at 0.25 mm.

The microscope has now settled the matter: the focusing settings required for the two were very similar. I suspect that the Grace frames are about 100 µm thinner than the Gene Frames, but I guess this still can be classed as minor. My impression that the frames felt thinner was almost certainly exacerbated by the thinner and more flexible covering plastic foil I noted in the last post. This thinner foil, together with a truly minor reduced thickness of the actual frame, makes the whole assembly feel less chunky in the hand, and that feeling transferred to the frame itself. The frame is basically as advertised.

What is still open

One thing remains genuinely untested: time-lapse microscopy.

Everything above concerns snapshot imaging, including long fluorescence exposures, and there the frames are a clear success. But time-lapse is the demanding case – it is where the volume, the air gap, the drying time, and the quality of the seal all have to hold together over hours rather than a single exposure. That test simply has not yet been done.

So the verdict, for now, is a confident and specific one. For snapshot work, the Grace SecureSeal frames are a genuine replacement for the Gene Frame – easy to handle, flat across the whole field, and validated by someone other than me. They cost far too much, as established last time. But they work well.

The time-lapse test, when it happens, will get a post of its own.

Similar and related posts:

• Gene Frame replacements
• Thermo Fisher discontinuing the Gene Frame
• An environmental chamber for time-lapse microscopy
• An excellent LED illumination system for microscopy