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I'm working with luciferase which has a wavelength of blue 480 nm and I want to be able to take a photo of it. The trouble is, I can see the luciferase glowing in all of its glory in front of me but no matter how hard I try, I can't take a photo of the luciferase with my DSLR (Nikon D80) using a Nikon 50 mm lens and a Nikon 35 mm lens. I've tried removing the filters (Nikon L37 and Hoya UV).

I'm curious if I'm missing a certain lens/filter or if I should be shooting using a different lighting setup. I'm already exposing for 30" at f/22 on ISO 1600. Perhaps longer?

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What is your lighting setup? –  MikeW Mar 7 '12 at 9:36
    
I suspect much, much longer exposing times are needed (. And in absolute darkness Same as photographing stars by night for example. I guess an EV -5/-6, so you need much more stops to correct that. So set the ISO at 400 for example and start at 1 minute exposure time at a F/4.0 Diafragma. Something visible? Decrease time by 1 stop, nothing visible? Increase to 800 iso, and so on, play with ISO and time to maintain quality. –  Caspar Kleijne Mar 7 '12 at 12:20
    
In a darkened room. how does brightness compare to luminous hour markers on a wristwatch dial? See my image below. I suspect your luciferase should be brighter than this. –  Russell McMahon Mar 7 '12 at 13:46
    
The luciferase will actually be duller than a wristwatch (depending on the concentrations). I'm using a completely dark room. –  bobthejoe Mar 7 '12 at 14:33
1  
No, not an idiot, you were smart enough to find us for help lol! Glad it's working now. –  MikeW Mar 7 '12 at 17:16

2 Answers 2

up vote 8 down vote accepted

That wavelength is certainly within the spectrum you can capture with any lens, with or without filters. Digital sensors capture between 350-1000nm If it is glowing, then you'd want that to be your main light source. Any additional light you throw onto it is going to dilute the glow from your subject and make it harder to see.

What aperture are you using? Unless this is incredibly dim, I can't imagine 30" not capturing something unless you're using a very small aperture.

Are you doing this in a dark room with a dark background? What do you see in your 30" exposures?

Through trial and error, you could do a range of shutter speeds (and ISO) to try to hone in on a good exposure value. If you can see it with your eyes, you should be able to photograph it without special equipment.

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I'm using f22, ISO 1600. Confuses me too. –  bobthejoe Mar 7 '12 at 14:35
4  
Oh gosh, f/22 - there's your problem. f/22 with 30 seconds definitely isn't going to get it. Open as wide (lower f numbers) as you can - at least to start off with. f/22 basically means there's a pinhole size of light getting through. –  rfusca Mar 7 '12 at 15:28

If you have other light present it can easily "wash out" low level luminescence. You should have NO other light to start.

Focus with a light on. Either use manual focus or, if your camera allows, set to manual focus, toggle temporarily into AF, focus and then drop back to MF so the camera is focused.

Then turn other lighting off so that the luciferase is the only lighting source, expose from there and see what you get.

If that does not work, manual exposure as well as manual focusing allows you to control what the camera does and to expose across a range of likely exposures.

I just tried photographing my not especially bright luminous wrist watch dial in total darkness. At 250 mm and watch about 500 mm away so the watch filled 1/3 of width of frame exposure was 6 seconds at f5.6 at 800 ISO. Individual hour markers, which are not very bright to the eye in roomlight, glow brightly in the photo when they are the sole light source.

Thusly. Focusing "could have been vastly better [tm] :-)

6S, f5.6, ISO800.

enter image description here

Key questions are - are you using other lighting and, what ARE you seeing in your images.

These many Luciferase images suggest a reasonable light level and do not look like they should be hard to photograph.

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