You can see a lot by looking – confocal microscopy imaging of laminins (etc)

In the last few weeks Lee and I have spent a lot of time on microscopes imaging laminins and laminin-binding proteins in a variety of cellular contexts. These images are part of our research efforts, they tell us a story about our cells and allow us to answer questions. However, they are also pretty in their own right. Most of these won’t make it into papers and so I’d like to share them with a little description of what you are looking at.

hTCEP 24h RG13-G LMa3bR-03.czi (RGB)
A little colony of corneal cells (6 magenta shapes outline where the cells are). Over a period of 24h these guys have moved around each other leaving a rose-like laminin (green) pattern beneath the cells.
NOK WT 24h GB3-03_ FIRE
No this is not a wormhole or some other celestial formation, rather its laminin again! This time the cells depositing the protein are oral mucosa keratinocytes and there is a lot more of them in frame here. Note that the camera captures images in black and white, the colour you see here (and elsewhere on this page) is added afterward. Here I have chosen a pseudocoloring scheme that helps to emphasise features; yellow indicates more intense, brighter staining compared with purple.
LaNt OE 24h Gb3-04.czi (RGB)
Laminin again! Of course it is. The magenta pattern here is more like paving stones. Same protein, same cells, same time frame so what’s different? The green you are seeing in the cells here is actually a marker we are using to visualise a protein that binds to laminins, in this experiment we have used genetic tools to increae expression of this protein, and our data (including this pic) indicates that it changes the way the laminin is organised
LaNtOE 24h FN G-04.czi (RGB)
Not a laminin. The white is fibronectin, another extracellular protein that cells bind to. Compared with the laminin stain, we can tell that these cells don’t make as much fibronectin, but the stuff they do make is assembled into little fibres mostly around the front of the cell. If you did this sort of staining in a different cell type you might get long rope-like fibres everywhere.
W31 24h 3E11 LMa3b-1
This one is entirely different; some sort of abstract charcoal drawing perhaps? No, you’re looking at the cell skeleton. I’ve just inverted the colour scheme so it is easier to see the fibre like pattern. Note the stuff in black is inside the cell. These fibres define the cell’s shape and influence things like cell movement.

A few more that I like from the set

Want to know more about the science behind these pics?

We have an intro page talking more about laminins including some more staining images. Although these pics are cells in a dish, laminins are really important to holding tissues together. In tissues, laminins are found in structures termed basement membranes, the link will take you to our very friendly introduction to these structures.

How do we prepare the samples?

The images on this page were generated using a procedure called indirect immunofluorescence microscopy (IIF, or sometimes immunocytochemistry). Basically we have grown our cells on thin pieces of glass (coverslips) then used methanol to hold everything in place (to fix the cells) and to allow our antibodies to permeate the cells. We then have used antibodies that specifically recognise our protein of interest, washed off unbound antibodies then used a second antibody that recognises the first antibody. The secondary antibody has a fluorescent tag attached to it which we then use the microscope to visualise (hence IIF comes from “indirect” = via the secondary antibody, immuno = using an antibody, fluorescence = the antibody has a fluorescent dye). For the imaging, we have shone laser light onto our slide and wherever the secondary antibody is we excite the fluorescent dye which  gives off light which we can capture on the microscope camera. Here we have used a laser scanning confocal microscope.

All that may have sounded complicated but it’s a really common, widely used technique and quite simple really! The hardest parts are making the primary antibodies, the ones that recognise the protein that you care about, and then working out the best way to use those antibodies (what dilution, what fix, what permeabilization chemistry etc). All antibodies are imperfect, so if you use too high a concentration you will get signal that isn’t real but if you use too low you may not get any signal that you can detect. So, although it only takes a few hours to process everything and then a couple of hours to image the slides, it might have taken a few weeks or months to get the conditions nailed down for how best to do it and to work out what is real and what is artefactual.

Fluorescence microscopy is still my favourite thing to do in the lab. I now have a team of scientists working with me and in addition to supervising them, I have teaching and administration commitments as well as grant and manuscript writing to do. The practical upshot of this is that I don’t get the chance to enter the lab as much as I would like. However, whenever there is a chance for some ‘scope time I am always happy. Plus, while there are caveats and limitations to these techniques, but you can often learn a lot by looking!

Like this?

You might enjoy our previous “You can see a lot by looking” post on live-TIRF microscopy

Our next post in this series will probably be about tissue staining…sign up for updates via the follow button

Uncategorized you can learn a lot by looking

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