Pollen autofluorescence

[viktor j nilsson]

By the way, what is the Nikon objective in your photo? Is it an early CF Plan Apo 40x 1.0 oil, with the black ring worn off?

[Pau]

Hi Viktor,
Interesting links, although the UV transmission of plexiglass is not clear enough for me (and my experience with colorless plexiglass is that it transmits a big portion of UV, filtration improvements will be due to additives)
So, yes, test it

The pictured objective is the one used to take the pollen pictures, it's a dry MPlan Apo 40/0.80 210/0 (CF finite metallurgical series)

[micro_pix]

.
Hi Pau
Great images! I’m hoping to do more fluorescence microscopy and was wondering about UV epifluorescence with no coverslip. My dedicated UV finite objectives (Olympus) have higher than normal NA’s and are not designed for use without a coverslip. I see you used a Nikon MPlan that appears not to have a UV rating and it looks to work OK, is that your experience?

David

[Pau]

David, thanks for the comments.

Not being an expert at all, I've found that most of my objectives do work reasonably well for epifluorescence with the sources I use: UV 365 and 380 and blue LEDs. Of course the transmittance will not be the same, for example I have an old 6.3/0.20 Leitz Plan Apo which is not more luminous than a 4/0.14 or a 10/0.25, and shows unacceptable autofluorescence
As you very likely know objectives with NA up to 0.40 despite being corrected for 0.17 covers work well without cover. What objectives do you plan to use?
I only have used one metallurgical objective, this Nikon 40/0.80 210/0 so I have not a comparison point. The exposure time of the pollen source images is 1/3s-1/4s with a LG 3535 LED powered at a bit less than 700mA.
This also depends a lot of the filters you use, I get very different exposure times with different filter sets, here I've employed the one tan provides the most intense result at this wavelength: EX 380x (Chroma) //BS Dichroic 410DCLP (Omega) //EM ET425lp (Chroma)

[micro_pix]

Thanks for that. I know Olympus produced a no cover UVFL 10x and 20x but their MS plans (which I do have) are infinite so I can’t use them on my fluorescence scope. I was thinking that non-uv rated objectives wouldn’t transmit the UV wavelength but I had a go with my non-uv rated Splanapo 4x and that seemed to work for epi-fluorescing the diamond chips using the UV exciter. My DPlanApo UV 10 (NA 0.4) does seem nice and sharp with no coverslip. Looks like I’ll struggle above 20x with no cover work - I thought about the no cover 60x SPlanApo but it only has a working distance of .4mm so not really practical for pollen etc.

Dave

[jmc]

I've measured UV transmission through a few objectives, and generally they'll let light through in the 365nm to 400nm region. Of course things like the Zeiss Ultrafluars will let UV through down to low 200nm range, but they are not that common.

The bigger issue is whether the objective itself fluoresces while exposed to UV.

[micro_pix]

I've measured UV transmission through a few objectives, and generally they'll let light through in the 365nm to 400nm region. Of course things like the Zeiss Ultrafluars will let UV through down to low 200nm range, but they are not that common.

The bigger issue is whether the objective itself fluoresces while exposed to UV.

Thanks JMC, that's very useful.

[Charles Krebs]

Pau,
These look fantastic! Really nice work.

[Pau]

Thanks so much, Charles

[Pau]

Stereo pair of the Hibiscus stack

[rjlittlefield]

The stereo is a treat!

--Rik

[micro_pix]

.

Excellent! Well worth the effort.

David

[carlos.uruguay]

Wooowww!!! NIce!!!!!!!!!!!11