Hello,
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?
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
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.
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
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.
Hi, Edalat, have you figured out this issue? I am setting up the objective-TIRFM recently and I think I have met similar problems. Do you have some suggestions?
As far as I remember, I have tested two commercial systems, and despite the theory behind TIRF, there are always some elements of scattered light above the objective. However, here are some tips I found that helped me significantly reduce the scattered light:
Try not to use a very large beam that illuminates the whole FOV. If you need to, it’s always better to have an adjustable mirror (a gimbal can be better) at the conjugate plane of the objective’s BFP. This allows you to tweak the direction of the light slightly to avoid hitting the internal objective barrel.
Test your TIRF quality when you have enough biologically matched imaging media on your coverslip.
The thickness of the dichroic and how it is mounted are really important. However, in practice, we do not have many options; it is a real bottleneck. The important point is to displace the beam in a way that the reflection from the secondary surface goes out first, and you can keep the original reflection in the TIRF position.
Did you try switching the face of the dichroic facing light ? If it was put incorrectly, depending on coating side you may get a lot of reflections.
In my experience a large beam at sample plane is in fact better because it yields a smaller gaussian beam at BFP, thus a thiner TIRF.
What is the oil that you use ? Olympus ? What is the medium index of your sample ?
Is it possible in your system to use a Bertrand lens to look at the BFP ? On top of translation of the beam, focusing exactly in the back focal plane is extremely important. Usually i even adjust for this when imaging different sample, at different objective axial position.
Hi Adrien,
Yes! I did try both faces, which for a quad band dichroic, the coated face is quite obvious.
At that point I used a 1.5 oil Olympus objective.
I have no access to the setup anymore, but yes I remember I did check everything multiple times.
