Fluorescence microscopy

Nezza · Post by **Nezza** » Thu May 05, 2016 3:02 am

I briefly used a fluorescence microscope at work an thought it had the potential to make some interesting images. Here are a couple I created at home using acridine orange, a 545nm laser as the light source and a 500nm blocking filter.

René · Post by **René** » Thu May 05, 2016 3:06 am

Cheek cells? It works well!

Best wishes, René

Nezza · Post by **Nezza** » Thu May 05, 2016 3:43 am

Yup. Nice bacteria laden cheek cells. The bacteria fluoresce bright orange and human cells fluoresce green.

Jacek · Post by **Jacek** » Thu May 05, 2016 4:19 am

Very nice

carlos.uruguay · Post by **carlos.uruguay** » Thu May 05, 2016 4:46 am

Interesting

Pau · Post by **Pau** » Thu May 05, 2016 5:00 am

Very interesting, could you post more details of your home setup?

545nm is green and the human cells also show green, could it be possible that they are just being illuminated by the laser (some kind of dark field illumination?) and not fluorescing like the bacteria?

Nezza · Post by **Nezza** » Thu May 05, 2016 5:37 am

Sorry. my mistake. It was a 445nm laser I used as the light source. The laser was used to illuminate the slide from the side. I will post a picture of the setup I used. My next strategy will be to use a beam expander and try dark ground illumination.

Nezza · Post by **Nezza** » Fri May 13, 2016 12:44 pm

Here are a couple of pictures of the setup I used. The laser is on a tripod to the side of the microscope just above the level of the slide giving a very oblique light. I have tried using a beam expander and dark ground condenser but the light level achieved is too low to work well. The laser is a 445nm blue laser running at about 30-50mW with a lens as a beam expander and I use a 500nm blocking filter. The reason for using a laser is that the beam is monochromatic and intense and the blocking filter gets rid of all of it. a 450nm LED would have some light overspill above 500nm.

zzffnn · Post by **zzffnn** » Fri May 13, 2016 5:13 pm

Nice photos, Nezza. Thank you for sharing.

I am surprised to hear that your laser and darkfield condenser did not produce enough fluorescence light. With a normal light source about as wide as field aperture (open all the way up), darkfield condenser should produce sufficient light (more so than incident oblique light). If you reduce light source width by closing down field aperture (or shrinking light beam diameter), darkfield light will eventually disappear (when it falls inside darkfield condenser's internal stop size).

Was you laser beam wide enough for your darkfield condenser to work? Is your darkfield condenser of cardioid type or paraboloid type (paraboloid type may need wider light cone, though even cardioid darkfield condenser need a certain beam width that goes beyond its internal stop size).

bernhardinho · Post by **bernhardinho** » Sat May 14, 2016 8:40 am

Hi Nezza

please don`t take it amiss and forget my sermon if you are experienced with this stuff, but with this kind of set up you are very prone to damaging your eyes!! Beware of reflections from the slide. I would seriously suggest the use of a protection shield and do remove the condensor as well (reflections!)

Kind regards

Bernhard