Hello all,
I am setting up a Zeiss Standard for epifluorescence using a Zeiss Standard and a mercury vapor lamp.
There is a Blue filter slider in the epic-illuminator. The filter block has a mirror-like excitation filter, a dichroic mirror, and an orange colored emission filter. The slider above the illuminator is blank. This is an Ebay filter block- no specifications were included with the purchase. Is gives a purple glow on the slide, and the view through the oculars is green.
Question: Is there a way to determine the characteristics of the filter cube? I'm not so concerned about the which fluorophores I'm exciting right now, but I am concerned that I not putting dangerous levels of UV out through the eyeballs!
Any help appreciated!
New Epi-Fluorescence
New Epi-Fluorescence
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William
Astoria, Oregon
Zeiss Axiomat
Zeiss Stereomikroskop
Zeiss Tessovar
Astoria, Oregon
Zeiss Axiomat
Zeiss Stereomikroskop
Zeiss Tessovar
Re: New Epi-Fluorescence
wstenberg wrote:Question: Is there a way to determine the characteristics of the filter cube? I'm not so concerned about the which fluorophores I'm exciting right now, but I am concerned that I not putting dangerous levels of UV out through the eyeballs!
That's very wise of you : Sorry, I have no real expertise in this area ... But I am interested in following your adventure.I have a small collection of secondhand filters [Nikon and others] which I am attempting to characterise, and I have found a few links which may be of interest to you:
https://www.microscopyu.com/techniques/ ... ilter-sets
https://www.chroma.com/products/complete-filter-sets
https://www.chroma.com/products/single- ... ts_display
http://www.nikon.com/products/microscop ... /index.htm
https://www.thermofisher.com/uk/en/home ... iewer.html
I have also found that a 405nm laser pointer [cheap from ebay] is a handy reference to have.
https://www.ebay.co.uk/p/Powerful-Stron ... 1392477074
Enjoy the journey, and stay safe.
MichaelG.
Too many 'projects'
Re: New Epi-Fluorescence
Hi,
My experience is not with this specific Zeiss but the principles are similar for most microscopes. If you have the manual for the epifluorescence accessory to post, it can help. Also, I recommend the manual for the HBO lamp-house.
I would suggest that you read the details about the light path in epi-fluorecsense microscopes.
Besides, I add some points.
1. The lamp housing appears to be HBO 50W or 100W (I think it is 100W). The 50W is fine for this microscope and the 100W is more than adequate. Replacing a bulb must be done gently and carefully, step by step. These mercury lamps should only be held by means of gloves or (better) a piece of tissue paper, not with bare hands.
NEVER touch a lamp when it is not absolutely at room temperature.
The light that comes out from the openings in the collimating optics near the HBO itself is not filtered and contains UV, along with other wavelengths. On the microscope, it is controlled with the filters (later below). However, sometimes you need to center and align the lamp, especially after bulb replacement. This is often done when the HBO is not installed on the scope, and the light beam points at a remote (2m away) white surface (say, a white wall). Whenever you handle the HBO in such operations, never look directly at the lamp. And wear UV-safety goggles. The alignment can be perfected after the lamp is fitted on the scope, however then you look at a piece of paper on the stage as described below.
2. Mercury lamps emits a lot of heat, that with time is liable to cause cracking of other optical elements that are near the lamp housing. You may want to place a heat filter between the scope and the HBO, as close to the lamp as possible. A Schott KG5 3mm thick (or 5mm thick) heat filter, that blocks IR, works fine, at least for many months. A neutral density filter is important but should follow the heat filter, not precede it.
3. Regarding the cube and other filters and UV. The excitation filter and dichroic mirror are designed and arranged to direct excitation light to the specimen, through the objective, to the specimen. (the objective is also the condenser in your setup!). This optical path suits UV light as well, since 365nm UV is very often used for excitation. However, The amount of reflected UV from the specimen unto the surrounding is very small. The remedy, if concerned about safety of the observer when he/she looks at the stage, is to place a UV-filter rectangular place on the microscope head. The plate protrudes forward, towards the user, at an angle, and the stage, specimen and nosepiece are viewed through the protective filter.
4. The above mentioned ($ 3) refers to the case that the cube is meant to excite at UV. Otherwise, the excitation filter and dichroic mirror in the cube prevent any UV (and other unwanted wavelengths) from reaching the specimen.
Note - and I apologize for saying the obvious: if you do not shine any UV light on the glass and/or specimen, they will not emit any UV.
5. Some UV excitation filters transmit in a wide range, covering from 360 to over 400nm. When you turn the lamp on, and place a piece of white paper on the stage, you see a bluish-violet spot light on the specimen (not through the eyepieces!), if the excitation filter may be a UV (365nm) transmission filter. HOWEVER, that does not mean you are exposed to much UV.
6. When looking through the eyepieces, and for most practical work, the dichroic mirror and emission filter further block UV so you are not exposed to UV (and neither is your camera).
7. The cube excitation and emission filters may be interference filters or just colored glass filters. I believe they are interference filters even on this old scope. They should not be touched with bare hands. Cube disassembly is not recommended by manufacturers (they prefer to sell whole cubes to order). However, if you do take the cube apart, place the components in order and mark the sides as well. They are not symmetrical. On quality filters, on the periphery, the specifications are printed, they tell you the bandwidth, middle wavelength and more. You can easily identify that a filter is an interference filter by holding it against natural or room light: the reflected and transmitted colors are different. For more detailed evaluation you need to inspect them with a spectrophotometer. If the excitation and/or emission filters of the cube are colored-glass, i.e. absorption rather than interference filters, then probably there are no designations and they must be tested with a spectrophotometer (but see $ 9 below).
8. If the cube is taken apart, you can almost certainly identify a filter as a UV-specific filter when it is opaque to natural light, or transmits a very very weak violet.
9. From the photo you posted, I do not see any UV component in the cubes, apparently these two cubes are for visible light. The most popular cubes have been for fluorescein and similar fluorophores, excitation 490-495nm, emission 515-520nm, but could be others. Some other cubes that I have used could be taken apart and modified by filter replacement, including the dichroic mirror. With the cube outside the scope, try to shine light from bright color LEDs into and through the excitation filter, and inspect what passes through the objective to the specimen and what passes to the eyes.
9. Ordinary glass (and objectives) block most of the UV. For UV-excitation in epifluorescence, specific UV-transmitting objectives are recommended (and cost a fortune). Phase objectives, such as the 10x one in the photo, are not optimal.
Good luck
My experience is not with this specific Zeiss but the principles are similar for most microscopes. If you have the manual for the epifluorescence accessory to post, it can help. Also, I recommend the manual for the HBO lamp-house.
I would suggest that you read the details about the light path in epi-fluorecsense microscopes.
Besides, I add some points.
1. The lamp housing appears to be HBO 50W or 100W (I think it is 100W). The 50W is fine for this microscope and the 100W is more than adequate. Replacing a bulb must be done gently and carefully, step by step. These mercury lamps should only be held by means of gloves or (better) a piece of tissue paper, not with bare hands.
NEVER touch a lamp when it is not absolutely at room temperature.
The light that comes out from the openings in the collimating optics near the HBO itself is not filtered and contains UV, along with other wavelengths. On the microscope, it is controlled with the filters (later below). However, sometimes you need to center and align the lamp, especially after bulb replacement. This is often done when the HBO is not installed on the scope, and the light beam points at a remote (2m away) white surface (say, a white wall). Whenever you handle the HBO in such operations, never look directly at the lamp. And wear UV-safety goggles. The alignment can be perfected after the lamp is fitted on the scope, however then you look at a piece of paper on the stage as described below.
2. Mercury lamps emits a lot of heat, that with time is liable to cause cracking of other optical elements that are near the lamp housing. You may want to place a heat filter between the scope and the HBO, as close to the lamp as possible. A Schott KG5 3mm thick (or 5mm thick) heat filter, that blocks IR, works fine, at least for many months. A neutral density filter is important but should follow the heat filter, not precede it.
3. Regarding the cube and other filters and UV. The excitation filter and dichroic mirror are designed and arranged to direct excitation light to the specimen, through the objective, to the specimen. (the objective is also the condenser in your setup!). This optical path suits UV light as well, since 365nm UV is very often used for excitation. However, The amount of reflected UV from the specimen unto the surrounding is very small. The remedy, if concerned about safety of the observer when he/she looks at the stage, is to place a UV-filter rectangular place on the microscope head. The plate protrudes forward, towards the user, at an angle, and the stage, specimen and nosepiece are viewed through the protective filter.
4. The above mentioned ($ 3) refers to the case that the cube is meant to excite at UV. Otherwise, the excitation filter and dichroic mirror in the cube prevent any UV (and other unwanted wavelengths) from reaching the specimen.
Note - and I apologize for saying the obvious: if you do not shine any UV light on the glass and/or specimen, they will not emit any UV.
5. Some UV excitation filters transmit in a wide range, covering from 360 to over 400nm. When you turn the lamp on, and place a piece of white paper on the stage, you see a bluish-violet spot light on the specimen (not through the eyepieces!), if the excitation filter may be a UV (365nm) transmission filter. HOWEVER, that does not mean you are exposed to much UV.
6. When looking through the eyepieces, and for most practical work, the dichroic mirror and emission filter further block UV so you are not exposed to UV (and neither is your camera).
7. The cube excitation and emission filters may be interference filters or just colored glass filters. I believe they are interference filters even on this old scope. They should not be touched with bare hands. Cube disassembly is not recommended by manufacturers (they prefer to sell whole cubes to order). However, if you do take the cube apart, place the components in order and mark the sides as well. They are not symmetrical. On quality filters, on the periphery, the specifications are printed, they tell you the bandwidth, middle wavelength and more. You can easily identify that a filter is an interference filter by holding it against natural or room light: the reflected and transmitted colors are different. For more detailed evaluation you need to inspect them with a spectrophotometer. If the excitation and/or emission filters of the cube are colored-glass, i.e. absorption rather than interference filters, then probably there are no designations and they must be tested with a spectrophotometer (but see $ 9 below).
8. If the cube is taken apart, you can almost certainly identify a filter as a UV-specific filter when it is opaque to natural light, or transmits a very very weak violet.
9. From the photo you posted, I do not see any UV component in the cubes, apparently these two cubes are for visible light. The most popular cubes have been for fluorescein and similar fluorophores, excitation 490-495nm, emission 515-520nm, but could be others. Some other cubes that I have used could be taken apart and modified by filter replacement, including the dichroic mirror. With the cube outside the scope, try to shine light from bright color LEDs into and through the excitation filter, and inspect what passes through the objective to the specimen and what passes to the eyes.
9. Ordinary glass (and objectives) block most of the UV. For UV-excitation in epifluorescence, specific UV-transmitting objectives are recommended (and cost a fortune). Phase objectives, such as the 10x one in the photo, are not optimal.
Good luck
Re: New Epi-Fluorescence
Thank you both for the helpful advice. I have two filter cubes; I probably need to get them set up with appropriate excitation-emission ranges once I have the scope and illuminator working properly.
It looks like this will be a long term project. The filters and blocks seem to be quite pricey, even on eBay. I may have to spread this out to afford it. I'll do the best I can with the filter blocks that I have now.
Once I get some photos, I'll post them.
Thanks again!
It looks like this will be a long term project. The filters and blocks seem to be quite pricey, even on eBay. I may have to spread this out to afford it. I'll do the best I can with the filter blocks that I have now.
Once I get some photos, I'll post them.
Thanks again!
William
Astoria, Oregon
Zeiss Axiomat
Zeiss Stereomikroskop
Zeiss Tessovar
Astoria, Oregon
Zeiss Axiomat
Zeiss Stereomikroskop
Zeiss Tessovar
Re: New Epi-Fluorescence
Since fluorescence is much more specific than most other modes of microscopy, it is important to define, even very roughly, the wavelengths that are relevant. In fact, your equipment right now is fine. The major investment is done already. So you can proabably enjoy it already. For example, to watch red fluorescence from green plants, you do not need uv but violet light (400-430nm).