I am designing a single-molecule microscope using a Nikon Eclipse Ti-E inverted microscope. My setup involves focusing a collimated laser beam onto the back focal plane of a 100×/1.4 NA objective via a ~200mm external lens mounted just outside the microscope’s back port (photo of the Thorlabs adapter attached).
Does anyone know the optical path distance from the microscope’s back port to the objective’s back focal plane? I haven’t found this specification in the product documentation.
Hello. I don’t know much about Nikon specifically but it is highly unusual for objectives to have their BFP outside the objective at all (except for some very low magnification objectives like some x4 and below - but even then not always depending on the exact model).
There have been some microscopes specifically made in the past that allow access to a conjugate of the objectives’ BFP in a standardised position up the tube but those are very specific models of scope which were designed for direct access Fourier filtration with a SLM.
I note from Nikon’s brochure that the Ti2-E has the ability to do ‘external’ phase contrast with any (non-phase) objective by means of a phase ring in the eyepiece base unit which implies that that model provides access to a conjugate of the BFP in a standardised position in the eyepiece base unit. Not sure about the ordinary Ti model though.
Thanks for the reply—and I realize my original question may not have been clear.
I’m not trying to access the back focal plane (BFP) outside the objective. I’m using the rear port of a Nikon Eclipse Ti (non‑Ti2) and plan to place a focusing lens just outside that port to focus a collimated laser onto the objective’s BFP (Nikon 100×/1.4 NA oil, HP Plan Apo VC).
What I’m looking for is the nominal optical distance from the rear‑port reference surface to the objective’s BFP along the epi‑illumination path. If this depends on configuration (e.g., filter cube, magnification changer), a typical value, a Nikon drawing/spec, or a practical way to measure it would be extremely helpful.
In a previous post of mine, the value 200mm was suggested, but not with certainty.
Hopefully someone of specific knowledge of your microscope can answer this. In the mean time, again speaking generically, it is unusual for the BFP of a set of objectives to lie in a single plane unless the objectives are specially designed as a set to do this for some reason. So, if you plan to use more than one objective for your experiment you will need to know the position for each objective separately and adjust the distance whenever you change objectives.
To measure the distance there are a few methods. You can shine a parallel beam up from the front of the objective and measure where it comes to a point out the back - although that would be impractical because most objectives have their BFP inside their barrel, esp. high power ones.
Another method is to focus a tiny spot of light onto the centre of the back of the objective and move the spot’s position (in length) till a parallel beam comes out the front of the objective - i.e. empirical trial and error. There is no need to use a laser for this but the spot must be tiny or you will never get a parallel beam. Practically, because of the difficulty in getting a tiny spot and because of the dangers of using lasers, it might actually be more practical to just focus an image of something (like an EM grid mask) on the back of the objective and place a converging lens of known focal length in front of the objective. Then measure the light distribution at the focal length of the lens you placed in front of the objective. When that light distribution is in the form of a focussed image of your mask you know that the primary mask image is focussed at the BFP of the objective.
Make sure that any mag changers or filter cubes you intend to use in your experiment are also in place when you do this. This is probably the most practical approach for you - unless someone else here can give you more specific guidance to your model.
I’ve done exactly what you did on a Nikon Eclipse Ti2, and to my surprise the tube lens (your 200mm lens) had to be quite inside the microscope body. Its not exactly like this anymore, but this is a screenshot from my original design.
But keep in mind that I got there by trial and error. I couldn’t find the actual information. Therefore, I used the rods be able to translate the lens along the optical axis and properly align it.
The next question is how should you align the lens then? Because this alignment wasn’t crucial for me, this is how I approached this problem.
DISCLAIMER: this is a dangerous procedure and should use laser safety equipment, I take no responsibility for the content of this post and if you are following the instructions below you will do so at your own risk.
You know that if your beam is focused in the back focal plane of the objective lens, it should come out collimated after the objective lens. You kind of want to avoid using the immersion lens because the following measurement would be harder to setup. Change to a low magnification objective (or at least one without immersion) and assume the parfocality is preserved by changing the lens. This assumption holds better if the lenses are from the same manufacturer. Then, shine your laser through the back port. Tilt the condenser column to get it out of the way and let the laser beam reach the ceiling. Move the 200-mm lens along the optical axis until you make the smallest possible spot on the ceiling. In doing so, you’ll focus the beam at “infinity” (the higher the ceiling the better), thereby collimating it.
As a side note, a free space collimated laser beam is quite dangerous because the light intensity remains approximately constant as it travels.
Next time I realign my setup, I’ll take some pictures and measurements for you. But I hope this still helps.
