Thorlabs Fluorescence standards - UV wavelength dependence

[JMC]

Not been able to post much for a while (mix of work, computer and broadband issues and family) but wanted to shared this. I have enjoyed reading the posts though, so thank you all for that.

 

Finding reliable fluorescence standards which don't cost the earth has been something that was lurking at the back of my mind for a while. Browsing the Thorlabs site I came across some microscope slide shaped fluorescence standards which were surprisingly cheap - 5 different colours for about 20USD. In fact shipping cost me more than the filters did. Here's the link to the slides - https://www.thorlabs...partnumber=FSK5

 

Thorlabs do give fluorescence graphs for these, but they did not always use UV as the illuminant.

 

I've been wondering whether there is a wavelength shift in the emission spectra from these types of standards if different wavelengths of UV were used to illuminate them, or whether they were relatively consistent. Manufacturers of fluorescence standards often say things like - to be used with 365nm illumination - but of course we have different light sources with different spectral characteristics.

 

Below are some results of initial testing to look at this.

 

Basic setup for the experiments - fluorescence standards placed in a cardboard box painted with Semple Black 3.0 paint (low fluorescence and reflectance). Experiments run in a dark room. Light sources - Hamamatsu LC8 200W Xe lamp with a UV collimator lens and a Nemo 365nm torch. Filters for the light sources - Baader U, Edmund Optics 310nm, 320nm, 330nm (BP 10nm) and Thorlabs 340nm, 350nm, 360nm, 370nm, 380nm and 390nm (bp 10nm) filters.

 

Camera - unmodified Canon EOS 5DSR with Rayfact 105mm UV lens (at f8) with a 420nm longpass filter from Klaus which has very low fluorescence. ISO 400. Images captured as JPEG in the camera - with a Daylight Whitebalance setting (no auto whitebalance, and everything done with the same setting). Exposure time either 1/4s or 1s as shown in the images. I used the Rayfact lens as it is nice and sharp and has a 52mm filter thread on the front to match my 420nm long pass filter.

 

Quick image of the setup.

 

Fluorescence of the slides using the 200W Xe lamp filtered with a Baader U filter (f8, ISO400).

 

With the broad UV source, as expected a nice range of fluorescence colours from the slides.

 

Next a Nemo 365nm torch filtered with a Baader U, shown with 1/4s and 1s exposure time.

 

 

The 365nm torch also gave a good range of fluorescence, although the second slide from the left (the Green FSK2 one) seems to be much more fluorescent than the others when the Nemo with its strong 365nm line was used.

 

As the final test it was back to the Hamamatsu Xe lamp again in combination with 310nm to 390nm bandpass filters. This time exposure was 1s for the images.

 

 

 

 

 

 

 

 

 

Now obviously there is a variation in the intensity of the light source between 310nm and 390nm and the filters have different max transmission values, which will have an impact on the degree of fluorescence occuring. It is interesting to note that there is a wavelength dependence on the degree of fluorescence from each filter - the relative brightness of each one is the not the same for all illuminant wavelengths. But, visually at least, there does not look to be much of a variation in the fluorescence colour of each filter at the different UV wavelengths. Ideally I'd like the measure the fluorescence emission spectra of each slide at each wavelength, but I'm not really setup for that.

 

Overall, very impressed with the Thorlabs fluorescence slides, especially given their price, and they should be a good options when looking for standards to help when imaging UV induced fluorescence.

[colinbm]

I would be interested in seeing the fluorescence they give in UVC & UVB light too please.

[JMC]

For now 310nm is as low as I can check Colin (combination of light source and available filters).

[colinbm]

Ok thanks Jonathan, some minerals show different colours in UVA, B & C.

[JMC]

Ok thanks Jonathan, some minerals show different colours in UVA, B & C.

Actually perhaps I spoke too soon. I've had a thought and am going to try something. Will post again in a bit.

[JMC]

So Colin, just for you (and anyone else who may well be interested of course).

 

Two more wavelength ranges done with the same setup as above.

 

Firstly, an Edmund Optics 300nm (10nm bp) filter in combination with a Hoya U-340 4mm filter. This was done like that as the EO 300nm doesn't have great blocking about about 500nm, and while my light source doesn't emit much about 450nm, I wanted to make sure I was blocking unwanted wavelengths. Therefore I added in the U-340 4mm.

 

The second one uses the 254nm band pass filter from the Sirchie forensics camera (the filter transmission was given here - https://www.ultravioletphotography.com/content/index.php/topic/3402-60mm-f35-c-mount-uv-lenses-on-ebay/page__view__findpost__p__31367).

 

This was a bit of an unknown as I wasn't sure if my collimator would still be good below about 280nm as it looks to have some form of coating on it, however a test within and without the collimator gave similar results (with a bit of variation in intensity). Two exposure times, 30s and 15s (note the much longer exposures than at 300nm and above, as my light source is not giving out much light that far down).

 

 

The 254nm images I am less sure about - the 254nm has quite a broad transmission curve and the light source intensity drops rapidly below 300nm, so this is probably more of a 260-290nm fluorescence response, although that is a guess from me. It is however as good as I can do.

[Stefano]

A few questions and observations:

- the 300 nm bandpass filter needed additional blocking, but are all the other bandpass filters blocked to a high enough OD?

- there's a noticeable difference between the 300 nm and the 310 nm images, the blue standard is significantly darker;

- some standards seem to have a double exitation peak, this is interesting as I think (I can be wrong) that this isn't very common.

[JMC]

Stefano,

Yes, the other filters have good blocking from 200-1200nm in the regions where they don't transmit.

The blue seems to drop in intensity of fluorescence compared to the other filters below 330nm. No idea why that is but is presumably to do with the chemical structure of the dye.

My big concern was whether the wavelength of illumination had a big impact on the wavelength of fluorescence (as it does with some minerals). While there are changes in the degree of fluorescence from the slides when using different illuminant wavelegths, the colours with which they fluoresce seems fairly stable.

[Andrea B.]

Is there any information which comes with the fluorescent slides about induction wavelength or emission wavelengths? Just curious.

[colinbm]

Thanks Jonathan, so the standards are pretty stable over a range of UV excitations.

[Stefano]

Another curiosity, is there a particular reason you would like to have stable fluorescence standards?

[colinbm]

Another curiosity, is there a particular reason you would like to have stable fluorescence standards?

 

I wonder how they could be useful for us in UV photography.... ?

[JMC]

Is there any information which comes with the fluorescent slides about induction wavelength or emission wavelengths? Just curious.

Not with the slides, but there is some information on the website - https://www.thorlabs.com/newgrouppage9.cfm?objectgroup_id=12142 Note that not all the graphs use UV as the illuminant though.

[JMC]

I wonder how they could be useful for us in UV photography.... ?

As far as I can remember on this board there has been discussion around white balancing fluorescence images. I'm lazy - I just use Daylight White balance in the camera - but not everyone does that. One of the questions around these types of standards though is 'does the colour of the fluorescence depend on the wavelength they are illuminated with?'. Hence sharing my findings. At the end of the day it was a few hours work and it may be of interest to a few of our members.

[colinbm]

With the fluorescent mineral people, yes it is important to see if there is any difference in UVA, B & C.

For UVC excitation they use the quartz UVC fluorescent tubes & block the visible light with Hoya 325c.

UVB excitation is with a UVB tube that is phosphor coated & block the visible with Hoya U360.

UVA excitation is with a 365nm Black Light or a 365nm LED & the visible blocked with ZWB3.

[colinbm]

Underwater photographers use a 455nm LED torch to fluoresce the marine life.

[Stefano]

If you want to mix them up to get white, they won't stay white at different exitation wavelengths. You can have a mix that gives you white at 365 nm for example.