Here goes:- sorry it's a bit wordy and I won’t include lots of photos as they are easy to find. The manuals for the fluorescence adapters and catalogues for all of the BH2 equipment mentioned are available on a well known web site.Marek Mis wrote:
Could you show your set. I use Olympus BH-2 too and I think to start somewhen fluorescence. I have no experience in fluorescence so every tip is very appreciated.
Marek
I bought my first Olympus BH2 BHS on Ebay a few years ago and it came with a bare BH2 RFL fluorescence attachment which, until recently, was left in a drawer. I didn't get around to using it as I needed more parts to get it working and I was still busy seeking out the parts to explore phase, D.I.C., Pol, epi and darkfield. A while later I bought a job lot from an auction that included the original wooden box for the Olympus RFL fluorescence kit, there was no RFL in there but there was a part set of barrier filters, supplementary exciters, a lamp centering/focusing objective and end caps for the RFL. I started looking at what else I needed to get fluorescence working on the BH2.
What I have learnt (and this is just my experience so it may not all be correct) is:-
There are three attachments that will enable epi-fluorescence on an Olympus BH2. The BH2 UMA, the BH2 RFC and the BH2 RFL. All of them can be used with either a Halogen or Mercury Lamp but the mercury lamp gives more intensity, especially at the shorter wavelengths, you also need the dedicated power supply for the lamp.
The BH2 UMA contains a tube lens so it is designed for use with the metallurgical Neo S/DPlan and M S/DPlan infinity objectives. There are fluorescent cubes available (to go in place of standard brightfield/darkfield cubes) for the UMA, they are for Violet, Blue and Green and contain the exciter and the mirror, there is no UV as the metallurgic objectives aren’t UV rated and do fluoresce in UV, in my experience. You also require a dedicated collector lens and both the cubes and collector lens are very hard to find.
The advantage of the UMA is that it gives you the ability to use the LWD no-cover metallurgical objectives which is great for anything you don’t want to put under a coverslip. The downside of the UMA is that you can’t use UV or the high NA UV objectives made by Olympus – or indeed any finite objectives. You can however put a short ThorLabs tube on to the top and use direct projection from its tube lens to an attached camera. You then need a screen for viewing and, as there is no eyepiece you can use the modern correction free UIS 2 infinity objectives if you can afford them!
The BH2 RFC is a dedicated fluorescence attachment for the Olympus BH2 finite system and replaced the RFL. The RFC uses cubes like the UMA but has a wider range including UV, the cubes also contain the exciter and the dichroic mirror, as in the UMA. One of the big advantages with the RFC over the earlier RFL was that it enabled DIC (Differential Interference Contrast) simultaneously with fluorescence. There is a slot for the D.I.C. prism slider and another for the analyser in the front of the RFC. Of course, the great advantage of combining the techniques is that you can see structures that don’t fluoresce in full D.I.C. glory instead of just a bunch of glowing red dots.
The DIC is transmitted light while the fluorescence is epi illumination but once you adjust the light intensity on the DIC to match the fluorescence the results can be very nice. You will need the D.I.C. universal condenser and the correct prisms to match the high N.A. DPlanAPO UV oil objectives – so good luck with putting that together! The RFC is more flexible than the RFL but it can be hard to find the cubes and they are usually expensive.
The BH2 RFL I believe was the first dedicated fluorescence attachment for the Olympus BH2 finite system. The whole system includes the, RFL body, the dichroic mirror and a separate exciter slider, any supplementary barrier / exciter filters you want to use and of course the Mercury or Halogen Lamphouse with its associated power supply. They are often available on Ebay but the dichroic mirrors and exciters can be hard to find if you don’t get what you need with the RFL. The mirrors and sliders cover two bands each and you select the band by sliding them in and out together. I now have the mirrors and exciters labelled U/V, U/B and B/G. I think that there is a B/0 (blue and full transmission) as well.
Fluorescence microscopy is low light work and high N.A. is important. The dedicated UV objectives are non-fluorescing and high N.A and, if you want to get the most light-capture, the oil objectives are best. For coverslip work I use the Olympus 10x DPlan APO UV, 20x DPlan APO UV oil, 40x DPlan APO UV oil, Leitz 63x Fluoreszenz oil and Olympus 100x D Apo UV oil. You need to use non-fluorescing oil, I use the Cargille LDF immersion oil and it does stink! It has a strong naptha-type smell, it mixes beautifully with the Xylene I use to clean it off! When not using UV the SPlan APO’s work well and the 4X and 10X are good for no coverslip work.
You can’t use the BH2 D.I.C. system with the RFL but I find using transmitted darkfield very effective when combined with fluorescence. I use the Olympus high N.A. oil darkfield condenser, oiled to the bottom of the slide (The photos show the Aplanat condenser not the darkfield condenser).
I wanted look at some subjects with epifluorescence under high magnification with no cover-slip and with a little more working distance. I have the UMA (but no fluorescence cubes) and a set of infinity MSPlans. The MSPlans don’t work with the RFL as it is designed for the finite system, it incorporates a diverging lens to compensate for the extra tube length it adds. I had a close look and the diverging lens is easy enough to remove and leaves you with an empty tube (when there is no exciter and mirror inserted) so basically just an infinity space.
Using the infinity MSPlans and a modified RFL provided an infinity space to the top of the RFL so I just needed a tube lens, some empty tubes and the camera. I found a second RFL on Ebay and took out the lens. I use my CamRanger and iPad for viewing and camera control. The caveat is that you can’t use UV as the MSPlans aren’t UV rated and fluoresce but there’s a lot you can do with V and B excitation. The lack of a final chromatic correction (as there is no photo-eyepiece) for the MS Plans isn’t an issue in fluorescence.
I already had a horizontal rig with a Raynox DCR 5320 some ThorLabs tubes and an RMS adapter so I replaced the RMS adapter with an Olympus BH2 dovetail adapter I got from a Spanish guy on Ebay and mounted it to the top of the RFL.
The nice thing about this set-up is it turns the BH2 body in to an infinity system so you can use any (correction free) infinity objectives, with or without the RFL fluorescence adapter in place. I tried it with the Olymous UIS2 10x UPlanFL and it gave a great image, it also doesn’t show any vignetting on an APS-C DSLR but it doesn’t cover a full frame.
I hope that's useful, any queries please ask and any inaccuracies please point them out.
Standard BH2 RFL set-up with DPlanAPO UV Objectives
Modified BH2 RFL with the MSPlan Objectives
