|
|
| Home | Fluorescent Protein Flashlights | Marine Science | Sport Diving | Reef Aquarium | Order | Contact |
|
Fluorescence photography with the lights on! You don't have to do fluorescence photography in the dark. Fluorescence photography is conventionally done in the dark because fluorescence emission tends to be weak and is easily overwhelmed by ambient light. But this isn't always necessary - with the right equipment and techniques it is straightforward to take fluorescence photographs with moderate levels of ambient light, such as might be found in a normally lit laboratory environment, or under the right set of conditions underwater. And these photographs will look as though they were taken in pitch darkness! In the laboratory, darkness is still needed to look at fluorescing subjects by eye, but when it came time to take pictures on go the lights, making it much easier to set up and work. Underwater, selection of a site to monitor over time needs to be made in the dark, but periodic photography can be done in the daytime. The technique was originally developed for underwater fluorescence photography, in order to remove the necessity for night diving. A scientific paper on this application, titled Underwater fluorescence photography in the presence of ambient light, was published in the journal Limnology and Oceanography: Methods. The paper describes the technique and analyzes the range of conditions in which it can be applied underwater, using a combination of modeling and experiment. The paper is available for free download from the L&O: Methods web site. This page describes the principles and application of the technique for above-water photography. The beauty is that it does not require any particularly specialized or expensive equipment. How the method works The trick lies in relative exposures. When you take a flash fluorescence photograph with ambient light (room lights or sunlight) present there are two sources of light that can contribute to the photographic exposure: fluorescence induced by your flash, and ambient light reflected from the subject and the surrounding scene. Think of these as completely separate images, each with its own exposure requirements. If you can arrange it - and you can - so that the fluorescence part of the image is properly exposed and the ambient light part is severely underexposed, the result will look as though the ambient light wasn't even there. How to do it Just a few photographic facts you need to know: If you underexpose a photograph by at least three f-stops the image will appear essentially black. The duration of a typical electronic flash is about 2 milliseconds (1/500 second). And one fluorescence fact: The fluorescence only exists when the flash is going off. (Yes, there will be some trivial amount of fluorescence excited by the ambient light, but we can ignore that here.) To take fluorescence photographs with the lights on you need: An electronic flash with appropriate filter as the fluorescence excitation source - the more power the better. A camera that synchs with the flash at a fast shutter speed - 1/250 or 1/500 second preferred - either a digital camera with manual exposure controls or a leaf shutter camera like a Mamiya or Hasselblad. From here on in we'll assume a digital camera. There will also be a fluorescence barrier filter over the camera lens. That's it. Since you only really care about the fluorescence image, you want to set your camera to expose that properly. And since the fluorescence only occurs during the time that the flash is going off you can use a very fast shutter speed as long as the camera can synchronize with the flash at that speed. Focal plane shutter cameras can't synchronize at very fast shutter speeds, but the electronic 'shutters' of many digital cameras can. As an illustration, I took fluorescence photographs of a moderately fluorescent subject in a well-lit laboratory. Some of the shutter speeds and f-stops may look odd because the camera set these automatically. Equipment used for the following photographs: Nikon Coolpix 990 digital camera operated in manual mode
From Figure 4 we see that a good fluorescence exposure was obtained at 1/500 sec and f5.8, while the good ambient light exposure with the barrier filter in place (Figure 3) was at 1/63 sec and f3.4. So with the settings for the fluorescence photograph the ambient light is underexposed by about 5.4 f-stops (f3.4 to f5.8 is 2.4 stops, and going from 1/63 sec to 1/500 is equivalent to about 3 f-stops). This is more than enough to severely underexpose the ambient light portion of the exposure so that it appears almost black (Figure 5). This technique has been used successfully with many different subjects. If the ambient light is too strong or if the fluorescence is too weak this technique won't work, but it certainly works well in most laboratory or home lighting conditions. The benefit of not having to turn the lights out is tremendous. There is no benefit in flash synching at faster speeds (e.g., 1/1000 second). Why not use that as the shutter speed and get even more ambient light rejection? Because at 1/1000 second the exposure time is shorter than the flash duration and the fluorescence exposure is reduced. So to summarize, here's what you need to take fluorescence photos with the lights on: Digital (or leaf shutter) camera that can flash synch at 1/250 or 1/500 second What do you do if you don't get sufficient reduction of the ambient light? There are a few things you can try: Reduce the ambient light in any of a number of ways - turn down the overhead lights, provide some shade with your body, or whatever. This technique can even work outdoors! Get a more powerful flash to move your fluorescence exposure further from your ambient light exposure. If any of this isn't clear enough, or if you want additional information, please feel free to e-mail. Acknowledgment: The research work that led to developing this technique was supported by the Environmental Optics Biology Program of the Office of Naval Research.
|
