I previously tweeted about fabricating chambers for Fluorescent Correlation Spectroscopy (FCS). Given the response, I thought I would give a little more detail in a post.
FCS is a great technique for studying the diffusive and physical characteristics of fluorescent (or fluorescently-labelled) molecules. I’m not going to go into it here especially as the Wikipeda page has lots of details and pretty pictures.
One of the problems of FCS is that measurements are highly sensitive to solution viscosity, which means that changes in concentration and temperature will affect the precision of your measurements.
What we set out to do was fabricate a simple and cheap chamber in which to do FCS measurements using a 40x objective on an inverted confocal microscope.
Version 1: Keep it simple
The first incarnation (as with future iterations) relied on using parafilm to make a chamber between a slide and coverglass (see below). These can be loaded by capilliary action (from either side) very easily. The chamber height is dictated by the thickness of the parafilm.
Unfortunately, when mounted on the microscope, the evaporation was quickly evident, based on the creep of the fluid edges inward. Clearly a design with less surface available for evaporation is needed.
Version 2: Bring out the hole punch
Version two worked on the idea that a closed system might be the best. In fact this was my initial design but Jennifer beat me to it with version 1. To make this chamber, a square coverglass was overlaid with a piece of parafilm, that had had a hole punched out of the middle (using a regular office holepunch).
The problem with version 2 is that while the chamber is closed (ideal from the standpoint of eliminating evaporation), capilliary action draws the liquid out between the parafilm and glass.
Version 3A: Fancy scissor-work
Back to having an ‘open’ chamber, we tried a U-shaped chamber which we called a lateral-U because of the orientation:
The other subtle difference in version 3 is that the chamber was placed onto a hot plate (set at around 100ºC) for about 10 seconds. This had the effect of melting the parafilm and thus sealing the sides of the chamber. This nicely solved the problem of leakage.
Version 3B: Fancy scissor-work with a twist
Version 3A still had some issues with evaporation, especially if the chamber width was larger than about 5mm. At this point we decided to literally add a twist to the construction by making a longitudinal channel:
The rate of evaporation is not necessarily decreased but as the chamber volume is increased we can image for longer before the evaporative effects become restrictive.
If it’s not one thing…
The remaining (and as yet unmentioned) problem with loading the chambers is that you load them by capillary action. This means that the surface tension in the aqueous medium drags it in between the two glass surfaces. Unfortunately, what tends to happen is that the liquid (below in red) will seal off the chamber entrance and then stop progressing. This happens because the air bubble in the chamber resists the force of the capillary action in the liquid (pink vs yellow arrow respectively).
This can be resolved in two ways. Firstly the Manchester Screwdriver method involves finding the smallest pointiest thing you have to hand and cracking the coverglass towards the back of the chamber. This method lacks daintyness but works and provides very little extra evaporative surface area.
With that in mind, it’s worth using the middle of the chamber for measurements to be as far away from evaporative surfaces as possible.
If you don’t like the idea of such a barbaric technique, we did find an alternative, which is to add an “escape channel” to the back of the chamber.
The downside of this of course, is that you now have two surfaces for evaporation. A very good reason to keep the escape channel as thin as possible.