Coupling lasers to a widefield microscope?

Hardware
1

Dear μforum -

I have a Zeiss Axioobserver microscope, and a EMCCD camera, and wish to purchase a laser light source, and to set up a single molecule imaging system in my lab (I am a PhD student). However, I am ignorant of a good way to couple the lasers to the microscope.

In particular, I want to use the 100x, 1.4 numerical aperture objective, and to achieve a maximum power of 30 kW/cm^2 with a far-red laser of ~640nm, but to achieve much lower powers of 30 W/cm^2 with a 405nm, 488nm and 549nm laser. The illumination field should be at least 30 microns by 30 microns. I would like a Widefield, homogenous, square illumination field.

What do you recommend regarding laser engines and couplers, for a non-expert in optics, who is comfortable with reading the manual? A single component or very few components that I might purchase and easily assemble, in order to connect the high/low power lasers (uv,blue,red, and far-red), to the microscope, and to get Widefield illumination, would be ideal. Because the lab is an experimental biology lab, a coupling solution/part that couples the laser while completely encasing the laser beam is ideal.

I would be happy to hear also recommendations about which companies to consult with.

Thank you for your time -
Yossi

Weizmann Institute of Science

2

It sounds like you are interested in doing single molecule localization microscopy (SMLM)? If you haven’t already, probably the first person to ask is your Zeiss representative. They likely sell attachment(s) that would do what you are looking for and that would be easier to install and safe for most users.
Lumencor (https://lumencor.com/) is one company that sells laser engines who might be worth contacting.

3

I know the folks who created DNA PAINT had a super simple setup that included some laser pointers that they put together after the initial publication. Check out some of their papers–not the first, but something early.

4

Purely from optics point of view:
-if you illuminate the objective with a large collimated beam, you get a focused beam out
-on the contrary, if you want to get a (roughly) collimated beam out, you need to focus your laser in the back focal plane of the objective. This means for instance placing a 20 cm lens something like 19-20 cm from the objective. The illumination will not be homogenous, but will rather have a roughly Gaussian profile.If you want to do better than that, you’ll have to use additional tricks.

Moreover, if you now have separate lasers, you’ll need a beam combiner.