BH-2 RFCA, filtre cubes, filtres and accessories

[Polymerase]

Having had success in asking for help on lubricating the cube slider on my RFCA, I dare post a few more questions, as the epifluorescence modality and its various accessories are new to me - so here are my current inquiries:

1) What is the difference between the BH2-ANF and the analyzer dummy? I only have the dummy, but in all illustrations I've seen, they look exactly the same, but are listed as different units. In my mind, that implies there is a functional difference, although I cannot identify it visually. What have I been missing?

2) In the analyzer dummy, I have a small, removable orange filtre labeled O530. What is its intended use?

3) I've got three filtre cubes, and a BF cube. The B and G cubes are clearly labeled, but the third one has no label at all. Its top filtre is green in appearaance, and provides red light when shining light through it. Is there any way I can identify this cube or its properties? I am not sure whether or not it is a standard configuration, or a custom assembly. What do I need to do in order to identify it? Which information do you need? (I presently haven't quite made out how to post photos here, but I'll be happy to provide photos if this obstacle is overcome).

4) I suppose the UV fluorites aren't required unless I am working in UV light? I have complete sets (4x, 10x, 20x, 40x and 100x) of Dplans, Splans and Splan Apos (in addition to Splan PLs). As I understand, epifluorescence require a lot of light (I have the 100W mercury arc lamp and its ridiculously large power supply!), and large N.A. I believe the Splan Apos would be the best choice. Is this correct? Or do I need the UV/UVFL objectives?

5) UV light conditions is obtained through a UV filtre cube - is that correct? Using my present setup, do I need to take any precautions? Is the orange UV shield required? It looks very fancy anyway, but represents a physical obstacle for operating the objectives, so I'd rather remove it if it is not needed.

I am grateful for any reply. No need to address all the questions at once.

Edit: Corrcted the number of the filtre in the analyzer. It”s 530, not 560.

[Hobbyst46]

Having had success in asking for help on lubricating the cube slider on my RFCA, I dare post a few more questions, as the epifluorescence modality and its various accessories are new to me - so here are my current inquiries:

1) What is the difference between the BH2-ANF and the analyzer dummy? I only have the dummy, but in all illustrations I've seen, they look exactly the same, but are listed as different units. In my mind, that implies there is a functional difference, although I cannot identify it visually. What have I been missing?

2) In the analyzer dummy, I have a small, removable orange filtre labeled O530. What is its intended use?

3) I've got three filtre cubes, and a BF cube. The B and G cubes are clearly labeled, but the third one has no label at all. Its top filtre is green in appearaance, and provides red light when shining light through it. Is there any way I can identify this cube or its properties? I am not sure whether or not it is a standard configuration, or a custom assembly. What do I need to do in order to identify it? Which information do you need? (I presently haven't quite made out how to post photos here, but I'll be happy to provide photos if this obstacle is overcome).

4) I suppose the UV fluorites aren't required unless I am working in UV light? I have complete sets (4x, 10x, 20x, 40x and 100x) of Dplans, Splans and Splan Apos (in addition to Splan PLs). As I understand, epifluorescence require a lot of light (I have the 100W mercury arc lamp and its ridiculously large power supply!), and large N.A. I believe the Splan Apos would be the best choice. Is this correct? Or do I need the UV/UVFL objectives?

5) UV light conditions is obtained through a UV filtre cube - is that correct? Using my present setup, do I need to take any precautions? Is the orange UV shield required? It looks very fancy anyway, but represents a physical obstacle for operating the objectives, so I'd rather remove it if it is not needed.

I am grateful for any reply. No need to address all the questions at once.

Edit: Corrcted the number of the filtre in the analyzer. It”s 530, not 560.

Here is an answer based on other systems, I believe they apply in your case as well.

2) That is an absorption-type long-pass filter. It cuts off light below 530nm (spectra can be found on line) and serves as emission filter. "Downstream" the cube.

3) That filter is an interference-type long-pass filter. It serves as emission filter. Can also function as dichroic mirror (middle component in the cube) if installed at an angle of 45 deg, since it will then reflect the green (excitation) towards the specimen (slide) and pass the red (fluorescence, emission) towards the camera or eyepieces. Some interference filters are enclosed within plastic/aluminum sleeves and the relevant wavelenghts are engraved or printed on the sleeve. But components within a cube might lack the sleeve. I would guess that the cutoff for the filter you describe is around 500nm, which is common, but the only way to verify is with a spectrophotometer.
As for the identity of the cube, try searching Olympus sites , or the internet in general, often standard cube configurations are specified and you can compare with what you actually have in the cubes. I would bet that the cubes fit the description, since most users will not modify a standard cube, which has been optimized by the manufacturer for specific fluorescent dyes.

4) UV objectives are nice to have and provide higher intensity UV than their parallel "ordinary" objectives. Since light gathering is a main challenge of fluorescence, and since the objective doubles as condenser, its UV transmission is significant. For excitation by visible light (violet, blue, green etc etc), UV objectives are not required.
And yes, the higher NA the better, however the other effects of a high NA still exist, especially the short working distance and focusing depth. BUT for fluorescence, a short focusing depth can be an advantage... in addition, a high NA means immersion oil; there are specific low-fluorescence immersion oil (many $$$) that provide dark background.

5) Yes, and with any UV setup, one needs precaution. Including possible leak/spill from the mercury lamp through aeration slots etc, and definitely the light that passes through the objective. The orange filter plate is important in this respect. If it hinders access to anything, find a solution. Maybe wear UV protective goggles, and rely on the camera rather than on the eyepieces.

BTW : very careful handling of interference filters is recommended. Coatings are very thin and sensitive to the elements and VERY sensitive to touching and cleaning.

[Polymerase]

I am most grateful, @Hobbyst46!

However, a few things aren't quite clear, if you would care to elaborate. I will use the original numbers for my questions, for the sake of clarity.

2) I understand what you write - but why is it in the analyzer slider? Any particular reason? What's the benefit of this, in lieu of placing it in the cube itself?

3) Crystal clear, and perfectly understood. Yet, some research remains to identifying the cube in question, but I feel a lot closer.

4) Wonderful! I just found another means of spending money! I want those UV objectives! Happy to know they are optional, but yet.....I know my nature when it comes to buying new objectives....

5) This confuses me slightly - probably because my question was a bit too complex.

I am far too cautious about my eyesight not to ask a few follow-up questions. What you are replying to, is certainly pertaining to the use of UV light. I realize the mercury burner emits light that contains a portion of UV light, and looking directly into its beam or stray light from poorly insulated areas of the construction will cause damage to the eyes. The presence of the UV shield is not really problematic - it just provides a little more clutter when operating the nosepiece turret.

BUT: When using non-UV filter cubes - will the reflected light reaching the eyepieces contain harmful UV light? In that case, looking into the eyepieces could never be done. This confuses me. Under what circumstances would looking into the oculars require protection? Will a non-UV filter cube block out the UV component of the reflected light? And is the function of a UV filter cube to concentrate or focus the UV fraction of the light form the mercury burner? Is it safe to look through the eyepieces under such circumstances?

I am sorry to bother you, but this is important for me to understand. If you do not wish to elaborate, could you point me in the direction of any informative source? (I like reading!)

Thanks for the warning about the interference filtres. I'll be very careful when handling them!

[Polymerase]

Does anybody know the difference between the analyzer dummy and the BH2-ANF analyzer? I am still very curious about this....

[deBult]

Note: on the German forum we have a few cases of Olympus SPlanApo an DPlanApo with fogging, assumption here is the fogging is caused by usage on a UV scope.

[Polymerase]

Note: on the German forum we have a few cases of Olympus SPlanApo an DPlanApo with fogging, assumption here is the fogging is caused by usage on a UV scope.

Now that is interesting. And intimidating.

Is this an association, or a known issue? If this is an established fact, what is the mechanism causing the objectives to fog up?
(Mentioning this, a 40x Dplan coming with this rig was rather foggy....)

[deBult]

English is not my native language so some subtleties in wording .. I stated ”assumption” the UV light damages the lens kit/glue.

I personally have a completely fogged SPlanApo 40* and 2 10* phase Achromats with the effects shown.

https://www.mikroskopie-forum.de/index. ... #msg348691

[Polymerase]

English is not my native language so some subtleties in wording .. I stated ”assumption” the UV light damages the lens kit/glue.

I personally have a completely fogged SPlanApo 40* and 2 10* phase Achromats with the effects shown.

https://www.mikroskopie-forum.de/index. ... #msg348691

English is not my native language either, so I apologize for any inconsistencies. Anyway, you’ve made an assumption on the cause of fogginess. That is fair, andmost likely based on onservations. In a broader sense, I wonder if this phenomenon is associated with fogging up objectives. If that is the case - then maybe someone has an explanation or could suggest a possible mechanism for why objectives fog up by exposure to UV light. You do suggest a mechanism yourself, namely the UV radiation damaging glues in the lens assembly.

If this is a known phenomenon, someone must know something about it. It is likely not something that emerged recently.

Leider ist mein Deutsch nicht so gut, aber ich versuche mal Ihren forum zu besuchen. Ich bedanke mich für Ihren antwort!

[deBult]

Google translate is your friend here

[Hobbyst46]

2) I understand what you write - but why is it in the analyzer slider? Any particular reason? What's the benefit of this, in lieu of placing it in the cube itself?

Are an analyzer and cube installed together, such that the rays pass through both of them in series ? in that case, perhaps the previous microscopist modified the spectral response for his purpose; maybe the cube provided a too low fluorescence cutoff (say, in the yellow region) so adding an O530 shifted the cutoff to the orange region, and made it more selective. A wild guess.

4) Wonderful! I just found another means of spending money! I want those UV objectives! Happy to know they are optional, but yet.....I know my nature when it comes to buying new objectives....

Not sure I understand your opinion here. All I wanted to say is that it might be possible to achieve acceptable UV-excited fluorescence with non-UV objectives. Especially if excitation is in the 380-390nm range.

5) This confuses me slightly - probably because my question was a bit too complex.
I am far too cautious about my eyesight not to ask a few follow-up questions. What you are replying to, is certainly pertaining to the use of UV light. I realize the mercury burner emits light that contains a portion of UV light, and looking directly into its beam or stray light from poorly insulated areas of the construction will cause damage to the eyes. The presence of the UV shield is not really problematic - it just provides a little more clutter when operating the nosepiece turret.

BUT: When using non-UV filter cubes - will the reflected light reaching the eyepieces contain harmful UV light? In that case, looking into the eyepieces could never be done. This confuses me. Under what circumstances would looking into the oculars require protection? Will a non-UV filter cube block out the UV component of the reflected light? And is the function of a UV filter cube to concentrate or focus the UV fraction of the light form the mercury burner? Is it safe to look through the eyepieces under such circumstances?

A detailed explanation of the optical train in epi-fluorescence is available at Microscopy4U and similar sites from Leica, Zeiss etc. Yet the short answer to the question is: A non-UV filter cube is supposed to block all UV to the eyepieces (and camera), due to (a) the dichroic mirror, which directs excitation from the lamp to the slide, AND directs reflected and scattered UV that goes up from the slide sideways, back to the lamp, and (b) the emission long-pass filter, which blocks all excitation light and passes only fluorescence. These two components are installed in series. If they are in good condition and undamaged (for example - the coating is intact), there is no hazard in viewing the slide through the eyepieces.

Happy to be of help.

[/quote]

[Polymerase]

Are an analyzer and cube installed together, such that the rays pass through both of them in series ? in that case, perhaps the previous microscopist modified the spectral response for his purpose; maybe the cube provided a too low fluorescence cutoff (say, in the yellow region) so adding an O530 shifted the cutoff to the orange region, and made it more selective. A wild guess.

This makes sense. The analyzer is inserted into the body of the rfca unitabove the filtre cube, and below the eyepieces.

Not sure I understand your opinion here. All I wanted to say is that it might be possible to achieve acceptable UV-excited fluorescence with non-UV objectives. Especially if excitation is in the 380-390nm range.

Sorry if I confused you. I just wanted to share my excitement for having an excuse to buy more objectives, although they’re not strictly necessary!

A detailed explanation of the optical train in epi-fluorescence is available at Microscopy4U and similar sites from Leica, Zeiss etc. Yet the short answer to the question is: A non-UV filter cube is supposed to block all UV to the eyepieces (and camera), due to (a) the dichroic mirror, which directs excitation from the lamp to the slide, AND directs reflected and scattered UV that goes up from the slide sideways, back to the lamp, and (b) the emission long-pass filter, which blocks all excitation light and passes only fluorescence. These two components are installed in series. If they are in good condition and undamaged (for example - the coating is intact), there is no hazard in viewing the slide through the eyepieces.

Happy to be of help.

This is perfectly clear! Thank you for being patient with me! I’ll have a look at microscopy4U and similar sites. I know where to find them, and should have had a look before posting here. Sorry about that.

I am most grateful for your answers!