Sample chamber
One of the key elements in SPIM/DSLM microscopy is the design of the sample chamber. Samples are usually embedded in agarose. In order to keep the humidity conditions we need to submerge them into a water like environment. The chamber is typically square having lateral windows made of coverslips glued with, silicone glue, pluriuretane or dental liquid silicone.
Nevertheless, chamber design might change according to sample. Here we present a few examples. The 60x and 16x chambers have a similar design only changing the dimensions of the objective hole. The sample chamber is held with the objective with a common O-ring. For air objectives the chamber is fixed by a mirror holder whereas the chamber with temperature control it is temporary glued using dental liquid silicone into a dipping objective. In this case the temperature is measured via a thermocouple and controlled with a PID device West Instruments N2300 that drives an SSR. This SSR switches at 2Hz 12V that powers 4x32 ohm resistors giving maximum power of 18W.
NEW: below you can download the STL files of two of our chambers ready for 3D printing (if available)!
Types
We have specially designed four chambers:
One for the x16 and one for the x60 waterdipping objective
How to build a water dipping
objective sample chamber
Sample mounting
Depending on the sample size we use two different ways of mounting the sample on low melting-point agarose gel.
In order to perform multiple view stack fusion we just need to add 1:10000 diluted TetraSpeck 0.5um fluorescent beads (Invitrogen) togegher with the1% low melting-point agarose (LMA)
For small samples (zebrafish embryos, drosophila embryos, cell aggregates, ...) we use a especially design sample holder
Click on the image to enlarge
Common 60x and 16x chamber and how it is fixed in the objective with an O'ring. This is the most used setup for being so versatility and stable.
One for air objectives:
Click on the images to enlarge
It is made upon a pipetting tip (Sarstedt Biospheres 200 microliters) commonly used in biological labs and two 5mm mounting hubs to attack the tip to the stepper motor:
(http://ptrobotics.com/product.php?id_product=1032).
These hubs are connected with screws that allow to make the tip complete perpendicular for sample rotation.
For bigger samples (adults flies or insects) we use another system, based on a glass cut with a diamond knife, and the bottom part of a pipetting tip. The glass will be used to shape an agarose cylinder and the tip to attach it to the stepper motor, since it has the same diameter.
Pictures of the procedure are shown in the following video.
Click on the image to enlarge
The most simple one is the air objective chamber. Basically only an acrylic cube with glued coverslips
One for any dipping objective with temperature control
Images
Temperature controlled chamber. Resistors are imbibed in thermal grease witch is in contact with an aluminium plate at the chamber base.
NEW!
Download below the STL files of our new chamber ready for 3D printing.
FUTURE PLANS!
We are developing a new design of sample chamber with a perfusion system with the temperature control for being able to apply drug and change temperature more rapidly.
Different sample chambers built with plexiglass