TIRF illumination

When I try to use TIRF illumination, I still have a lot of scattering light which generate sort of epi illumination as well. My set up includes high power laser, 10X beam expander, f=250mm achromatic doublet, four bands Semrock super-resolution laser filter set, 100X Olympus NA=1.5 objective. The alignment looks good (at least in the epi mode) and I can see the TIR beam returning through the objective.
I am not sure if this due to the 3 mm thickness of dichroic or not but I can see secondary scattered spots and a lot of interference patterns on the ceiling.

Also can anyone let me know the importance of circularly polarized light in TIRF microscope? Should I use it?

these are the light patterns on the ceiling for epi and tirf; I do not understand why I have the tirf one!!!


when you are at the critical angle you should not see the TIRF beam at all. If you change the angle the beam should go from center (~widefield) to the side walls (HiLo) and then not get out anymore from the objective.

You surely know it, but be aware of laser safety when you are doing such adjustments.



Thank you for the reply. Yes that is exactly what I can’t understand. It seems that there is a reflective surface which returned a part of light to the coverslip. I have some epi lights contamination in TIRF

Is this a home built microscope?
Is the emission filter at an angle? Or is it basically perpendicular to the optics path?

It’s a home build. Actually the emission filter is placed perpendicularly but in front of camera and far away while that unknown light looks somehow brighter than you might expect, however I will double check it.

I have removed the emission filter and I can confirm this is not the reason. Perhaps something in the objective lens which I don’t know what and why!

Maybe, but that doesn’t seem that likely to me. It is painful, but probably you just continue removing parts from the emission path until the spots go away… Or if you have a different objective you could see if it shows the same thing. It doesn’t need to be a TIRF objective, you should only see one spot that moves as you move the beam.

How are you controlling the beam? Are you translating it across the back aperture of the objective? Do you have a optical diagram of your setup?

very good suggestions. The only component in the emission path is the tube lens, and I can remove it easily. Also I can put other TIRF objective on it.
About the beam, yes, I am using a manual stage to adjust the position of focusing lens.
Olympus believes that I should work on dichroic alignment and I know that its holder is not good enough; so I am going to buy a new one as well. However I am not sure that’s the case

When I have done this I include a mirror on the manual stage, as shown in the attached diagram.

As I understand your design, I think that you may also be changing the direction of propagation of the beam at the same time that you are translating it.

Actually I have already tried both and I didn’t notice any significant changes, especially due to the large diameter of both laser beam (12mm) and lens (50mm, f=250) in my current setup. So I finally decided to leave it simple. You think that it is still important?

I’m not sure. I have only ever built TIRF setups using the mirror/lens/stage configuration, I don’t have any hands on experience with the lens/stage only configuration.