Light sheet characterisation

openspim

#1

Hi all,

I’m looking for some advice on characterising an adapted OpenSPIM and i’m using a Thor Labs beam profiler to measure the size of the beam at different points of the OpenSPIM illumination light path. In the end I am expecting a sheet waist thickness (2Wo) of 9-10µm (@488nm, through a 4x 0.1NA objective, in air at this stage) given by the following formula Wo = wavelength/numerical aperture (0.488µm/0.1). However what i’m finding is that the beam is never narrower than 100µm beyond the illumination objective. Below i have listed the size of the beam at various points to see if this can explain my findings.
-At output of Versalase 680µm
-After beam expander (25mm and 100mm) 2800µm (roughly 4x expansion)
-Immediately after the corner mirror with cylindrical lens and slit in place 400µm x, 1800µm y
The beam diverges beyond the mirror
-Immediately after telescope (50mm and 25mm) 160µm x 1400µm y
-Moving away from the second telescope (25mm) lens (towards sample chamber) the beam is sheet like then converges to more of a spot size 135µm x 90µm y then starts to diverge to a line again beyond that. I’m not sure i expected this as the output from the telescope should be parallel given the light is coming from the focus on the other side?
-With the 4x objective then mounted i measure the sheet size and that’s when i’m getting anywhere between 100-200µm thickness according to the beam profiler depending how far the profiler is from the front of the objective lens. To calculate the position of the back focal plane I pointed the front of the objective at a ceiling light then moved the lens towards the ground until a sharp image of the light was formed and measured the distance.

I have assumed I can use the profiler instead of using the camera and detection path with a plane mirror perpendicular to the illumination axis for these tests. I don’t know if anyone see’s anything odd about the above observed numbers. I suspect there is an issue with the distance of the telescope lenses and the objective as i don’t see what else could be the issue. I had assumed what you want is a line formed on the back FP of the obj lens which is at least as long as the back aperture to use the full NA of the lens.

Thanks for any advice.

Matt


#2

Realised my estimation of predicted light sheet width (2Wo) based on NA 0.1 was wrong, should be around 3.1µm.