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LIMNOLOGY

and

OCEANOGRAPHY: METHODS

Limnol. Oceanogr.: Methods 3, 2005, 499–510 © 2005, by the American Society of Limnology and Oceanography, Inc.

Underwater fluorescence photography in the presence of ambient light

Charles H. Mazel 1

1 Physical Sciences Inc., 20 New England Business Center, Andover, MA 01810, USA

Abstract

Fluorescence photography is traditionally done in dark conditions owing to the relatively low level of the fluorescence emission compared to ambient light levels. Working in the dark can be awkward, especially underwater, and the ability to do the photography during the day would be an advantage. Recent experiments have demonstrated that with the appropriate off-the-shelf camera and lighting technology it is possible to make high-quality fluorescence images in the presence of moderate levels of ambient light. The factors that affect this technique are the ambient light and the specifics of the equipment being used. For the ambient light, the important factors are the inherent optical properties of the water body, depth at which the photography is being done, time of day, cloud cover, and subject orientation and location (insofar as they create shadowing). For the equipment, the important factors are the camera’s flash synchronization speed, flash intensity, flash duration, detector sensitivity, and the spectral characteristics of fluorescence barrier filters. The range of conditions under which this technique can be used was determined by modeling. Results are presented for the relatively clear waters of a tropical reef (Bahamas) and the relatively turbid waters of New England. This method of fluorescence photography allows collection of fluorescence images under daylight conditions, rather than at night or with enclosures to create artificial darkness, and thus is much more practical and applicable in the field.

Introduction

The fluorescence of non-photosynthetic pigments in corals and other marine organisms was of only passing interest for many years (Kawaguti 1944; Limbaugh and North 1956; Catala 1959; Logan et al. 1990). Interest increased with suggestions of a function for the fluorescence (Schlichter et al. 1994; Salih et al. 2000) and exploded with the discovery that many of the instances of fluorescence in cnidarians (Matz et al. 1999; Tsien 1999) and other taxonomic groups (Shagin et al. 2004) arise from proteins of the same family as the green-fluorescent pro- tein (GFP) that is so valuable as a marker in biotechnology (Chalfie et al. 1994). Fluorescence can contribute to the color of corals (Mazel and Fuchs 2003) and has been shown to be of

Acknowledgments

Thanks are due to Dr. Michael Lesser (University of New Hampshire) for the use of his digital camera and underwater housing, to Dave Phinney (Bigelow Laboratory for Ocean Sciences) for the ac-9 data used in the model, and to Dr. Curt Mobley (Sequoia Scientific) for advice on the use of Hydrolight. Drs. David Zawada and Eran Fuchs provided valu- able suggestions to improve the manuscript. This research was support- ed by the Environmental Optics Program of the Office of Naval Research. The author is the founder and proprietor of NightSea, which supplied the filters used for the photography.

functional value as a signal in stomatopods (Mazel et al. 2004). Additional directions of investigation and applications of fluo- rescence are currently in development. Fluorescence photography is a tool that can play a signifi- cant role in a wide variety of areas of research for documenta- tion, quantification, and communication. Part of the investi- gation into the role of fluorescent pigments in corals (Salih et al. 2000; Mazel et al. 2003a) relates to their distribution within the animal, at both the microscopic and macroscopic level, and imagery communicates these relationships better than words. Similarly, documentation of fluorescence patterns in other organisms (Mazel et al. 2004) aids in the understanding of their function. Whatever the function of the fluorescence for the animal, it can have value for the researcher. The fluo- rescent pigments are being used as a means to explore the evo- lution of color diversity in corals (Kelmanson and Matz 2003; Ugalde et al. 2004). Investigation of fluorescence led directly to the discovery of cyanobacteria in corals (Lesser et al. 2004). Fluorescence is proving to be a useful tool for locating coral recruits on natural surfaces (Mazel, unpublished data), and flu- orescence photography can be used to explore for specimens and to perform repetitive observations of juvenile develop- ment on a surface being monitored over time. A preliminary investigation suggests that it might be possible to use the

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Mazel

information in fluorescence imagery to perform automated bottom classification to some level of taxonomic specificity (Mazel et al. 2003b). Fluorescent dye is sometimes used to study flow in and around sponges (Ilan and Abelson 1995) and other organisms, and photography can be used to docu- ment the flow patterns. The use of fluorescent tracer particles combined with fluorescence imaging has been used to study sediment turnover by benthic organisms (Solan et al. 2004). Applications for fluorescence are not limited to marine research. For example, the same GFP that is found in cnidari- ans is used widely in biomedical research to create transgenic organisms, where the occurrence of fluorescence is an easily accessible marker for gene expression (Chalfie et al. 1994). Flu- orescence coloration can play a functional role in terrestrial organisms (Arnold et al. 2002). Fluorescence finds application in many other fields, including forensics, leak detection, non- destructive testing, surface contamination detection, mineral- ogy, botany, plant health, palynology, technology of food- stuffs, textiles, paper, laundering, dentistry, and medicine, to name just a few. Fluorescence is fundamentally a technique for making features of interest visible, whether using naturally occurring or artificially introduced fluorophores. As such, photography is almost universally used to perform, docu- ment, and communicate research. Fluorescence tends to be a weak signal. A fluorescent mate- rial absorbs only a fraction of the light that reaches it, and only a fraction of the light that is absorbed is re-emitted as flu- orescence. Fluorescence imaging is normally carried out in darkness due to the relatively low level of the fluorescence emission compared to the ambient light levels encountered underwater in the daytime or in a laboratory with the lights on. In situ underwater fluorescence photography must there- fore be done either by night diving or using a custom-made opaque enclosure that holds the camera and strobe. Night div- ing is difficult and presents more safety considerations than daytime diving, both for the dive itself and for the access to the dive site, whether by shore or boat. Enclosures can work, but are cumbersome and create their own challenges of prop- erly framing the subject, focusing the camera, and achieving satisfactory exclusion of ambient light. It is much easier to work with some level of ambient light rather than requiring complete darkness, both in the field and in the laboratory, and the approach described here makes that possible. If a fluorescence photograph is taken in the presence of ambient light, there will be two sources of light reaching the detector: ambient light reflected from the scene and fluores- cence stimulated by the electronic flash (and to some degree by the ambient light). For the resulting image to faithfully record the fluorescence alone, we need the relative light levels from the two sources to be such that the fluorescence is prop- erly exposed and the reflected ambient light is underexposed to the point of not appearing in the image. We determined through experiment that with the appropriate off-the-shelf camera and lighting technology it is possible to achieve this.

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Daylight fluorescence photography

Once the technique was demonstrated by experiment for several operating conditions, modeling was used to under- stand the range of conditions under which it could be applied. The main factors that affect this technique from the point of view of ambient light are the inherent optical properties of the water body, depth, time of day, cloud cover, and subject ori- entation and location (shadowing). The important factors from the point of view of the imaging technology are flash synchronization speed, flash intensity and duration, detector sensitivity, and the use of fluorescence barrier filters. Results are presented for the relatively clear waters of a tropical reef (Bahamas) and the relatively turbid waters of New England.

Materials and procedures

Photography—A fluorescence photograph can be taken in the presence of ambient light if the conditions are such that the fluorescence contribution is properly exposed and the ambient light portion is underexposed to the point of not contributing to the image. An underexposure of at least three f-stops (a fac- tor of eight) is enough to achieve this. (Because the technique described here is to be applied to camera systems in the field, the analysis refers to f-stop settings for best utility.) A photographic system set up for fluorescence will consist of a camera, an electronic flash fitted with a filter that restricts its output to the wavelengths needed to stimulate fluores- cence, and a barrier filter in front of the camera lens. The bar- rier filter blocks any excitation light reflected from the subject and transmits the fluorescence. The important point is that the fluorescence emission you want to photograph occurs only when the flash is fired, and typical flash durations are on the order of 2 milliseconds. The key to applying the technique described here is a cam- era that can synchronize with the flash at very fast shutter speeds, ideally 1/500th of a second. As the exposure time is decreased, the ambient light contribution is decreased propor- tionately, whereas the fluorescence contribution is unaffected. Conventional focal plane shutter film cameras do not syn- chronize with the flash at exposures this short, but leaf shut- ter cameras can, as can many of the digital cameras available today. (A digital camera does not have a mechanical shutter, and the “shutter” speed setting is actually an integration time set by the camera electronics.) Many such cameras can syn- chronize at 1/1000th of a second, but there is no benefit because the flash is generally longer than this, and you would be eliminating some of the fluoresced light. Photography was done with a Nikon Coolpix 990 digital camera. For underwater use, the camera was enclosed in an Ikelite underwater housing. The electronic flash was an Ikelite Substrobe 300 (nominal 300 watt-seconds rating) fitted with a Model BE5 exciter filter (NightSea). This restricted the flash output to wavelengths shorter than approximately 470 nm. A yellow barrier filter (also supplied by NightSea) was used in front of the camera port on the housing. The transmittance curve of the barrier filter is shown in Fig. 1. The camera was

Mazel

Daylight fluorescence photography

Mazel Daylight fluorescence photography Fig. 1. T ransmittance curve of yellow barrier filter used for fluorescence

Fig. 1. Transmittance curve of yellow barrier filter used for fluorescence photography.

operated in manual mode and the proper settings for fluores- cence imaging were determined by trial and error. With this configuration it was found that high-quality close-up images of chlorophyll fluorescence, a relatively weak fluorescence emission in the far red part of the visible spectrum (peak emis- sion at about 685 nm), required a setting of approximately f3.4 when the camera was set to its ISO 400 equivalent. Modeling—The fluorescence exposure was fixed by the cam- era setup. To model the conditions under which these settings would produce at least a three-stop underexposure of the ambient light, a reference point was required. Photographers have a time-tested rule of thumb for exposure (which we ver- ified experimentally) called the “Sunny 16 Rule.” It states that in air under a clear sky around midday, the proper shutter speed to be used with an aperture setting of f16 is the recipro- cal of the film speed, or the ISO equivalent for a digital cam- era. Thus if you were operating at ISO 400 you would set your shutter at 1/400th of a second and your aperture at f16 to cap- ture a good image. Because 1/400 is not a standard setting on cameras, you would set it to the closest value, which for this example would be 1/500. For underwater photography, some light is always lost at the air/sea interface, and the Sunny 16 Rule becomes the Sunny 11 Rule just below the water surface. The light level just below the water surface was used as the reference for the modeling. The ambient light level just below the surface under full sun, assuming the Sunny 11 rule is cor- rect, would require an exposure of 1/400 and f11 for an ISO 400 setting. This is 3.1 f-stops, or a factor of 8.6 (2 3.1 , because each f-stop represents a factor of 2 increase or decrease in

light reaching the detector), more light than that which pro- duces the proper fluorescence exposure (1/500, f3.4, ISO 400). If we were to configure the camera at the exposure settings dictated for fluorescence, the ambient light would severely overexpose the image. Conversely, if we exposed the ambient light properly, the fluorescence would be underexposed to the point where it would not contribute to the image. To get from 3 f-stops overexposed to 3 stops underexposed is a reduction in ambient light of 6 f-stops, or a factor of 64. Therefore, to have a fluorescence-dominated exposure using the system we are working with, we must find conditions in which the ambient light level is 6 f-stops lower than that just below the surface. Modeling of downwelling light was performed using Hydrolight 4.1 (Sequoia Scientific). Model runs were made for clear tropical waters (Lee Stocking Island, Bahamas, 23 46.5N, 76 05.5W) and for coastal New England waters (Isles of Shoals, New Hampshire, 43 N, 70.5W). In both cases the Hydrolight computations incorporated water optical property data that had been measured at the two locations with a WET Labs ac- 9, an instrument that simultaneously determines the optical absorption and attenuation in nine wavelength bands. For the tropical case, downwelling irradiance was computed for depths from 0 to 30 m at 3-m intervals. For the New England case, downwelling irradiance was computed for depths from 0 to 21 m at 3-m intervals. In both cases runs were made for 0%, 50%, 80%, and 100% cloud cover, and at 1-hour intervals from 0700 to 1200 local time. The date used was 1 June. Wind speed was 0 m/s for the runs described here, but test runs were

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Mazel

Daylight fluorescence photography

Mazel Daylight fluorescence photography Fig. 2. (A) Graphs of downwelling light reduction, expressed in terms of

Fig. 2. (A) Graphs of downwelling light reduction, expressed in terms of f-stops relative to the irradiance just below the surface at local noon, as a function of depth and time of day for the Bahamas. In B, the differences between the values at the times from 0700h to 1100h and the values at noon are plotted.

also made with wind speed set at 5 m/s and 10 m/s (10 to 20 knots), with no significant difference in results. One of the outputs of a Hydrolight simulation is the down- welling irradiance as a function of wavelength. The integral of the downwelling irradiance from 400 to 700 nm was com- puted to represent total ambient light available for photogra- phy at each depth under each of the modeled conditions. To factor in the impact of the yellow barrier filter, the down- welling irradiance spectrum was multiplied by the filter trans- mittance spectrum before the integral was performed. The

light level just below the surface at local noon with 0% cloud was taken as the reference. The light level relative to this ref- erence, expressed in f-stops below the reference, was com- puted as log 2 (sample/reference).

Assessment

Fig. 2A shows the reduction of the ambient light, expressed in terms of f-stops, as a function of depth and time of day for the Bahamas case. It is evident that as depth increases and as time is further from noon there is less light. To isolate the

is further from noon there is less light. To isolate the Fig. 3. (A) Graphs of

Fig. 3. (A) Graphs of downwelling light reduction, expressed in terms of f-stops relative to the irradiance just below the surface at local noon, as a function of depth and time of day for New England. In B, the differences between the values at the times from 0700h to 1100h and the values at noon are plotted.

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Mazel

Daylight fluorescence photography

Mazel Daylight fluorescence photography Fig. 4. Impact of 100% cloud cover on the ambient light, expressed

Fig. 4. Impact of 100% cloud cover on the ambient light, expressed in f-stops, as a function of depth and time of day for the Bahamas and New England.

importance of time of day, for each depth and time the expo- sure difference between the light reduction at that time and the corresponding value at noon was computed (Fig. 2B). From Fig. 2B we see that at 1000 h and 1100 h there is only a small reduction in ambient light relative to local noon. At 0900 h we see a reduction of 0.5 to 1 f-stop, depending on depth. At 0800h this reduction increases to a range of 1 to 2 f-stops, and at 0700h from 2 to 3 f-stops. The Hydrolight solution is the same for times equally spaced from local noon,

is the same for times equally spaced from local noon, Fig. 5. Graphs of the additional

Fig. 5. Graphs of the additional reduction in light available for imaging, expressed in f-stops, due to the yellow barrier filter, for Bahamas and New England. The dashed lines indicate the range of variation for cloud cover from 0% to 100% and time of day from 0700 h to 1200 h, and the solid red lines are the means of those values.

so the solution can be interpreted in terms of hours before or after noon (eg, we would get the same results for 1500h as for 0900h, 3 hours away from noon). Figure 3 shows the same graphs as Fig. 2, but for the New England case. The overall reduction in light level occurs much more rapidly, as would be expected for the more strongly attenuating water column. Note, however, that the added decrease due to time of day (Fig. 3B) is less than for the Bahamas case. The impact of cloud cover was computed as the difference between the f-stop reduction with and without clouds for each condition of depth and time of day. Fig. 4 shows the addi- tional f-stop reduction due to 100% cloud cover as a function of depth, for time of day ranging from 0700 h to 1200 h. The impact of cloud cover shows a similar pattern for both the Bahamas and New England cases. Note that the f-stop reduc- tion shown in this figure would be added to that due to depth and time of day. The impact of the yellow barrier filter (Fig. 1) was com- puted as the difference between the f-stop reduction with and without the filter for each condition of depth, time of day, and cloud cover. Figure 5 shows the added f-stop reduction provided by the filter as a function of depth for cloud cover from 0% to 100% and time of day from 0700 to 1200 (dashed lines), and the mean of those values (red lines). The graphs show that the barrier filter had a depth-dependent effect on the exposure that was nearly independent of time of day and cloud cover. The error associated with using the mean value instead of the actual computed value for each case is a few tenths of an f-stop at most. The clear waters of the Bahamas have maximum water transmission in the blue, so the use of a yellow (minus blue) filter has significant impact on the light that reaches the detector. This impact increases with depth as

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Daylight fluorescence photography

Table 1. Summary of reductions in ambient light, expressed in terms of f-stops, for the Bahamas case

Depth (m)

 

0

3

6

9

12

15

18

21

24

27

30

Reduction in ambient light (f-stops) Depth Filter Time of day 1200 h 1100/1300 h 1000/1400 h 0900/1500 h 0800/1600 h 0700/1700 h 50% cloud

0

0.8

1.3

1.8

2.3

2.7

3.2

3.6

4.0

4.4

4.8

0.8

1.1

1.3

1.4

1.5

1.6

1.7

1.8

1.8

1.9

1.9

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.2

0.3

0.3

0.3

0.3

0.4

0.4

0.4

0.4

0.4

0.5

0.5

0.6

0.7

0.7

0.8

0.8

0.9

0.9

1.0

1.0

1.0

1.1

1.2

1.3

1.4

1.5

1.6

1.7

1.8

1.8

1.9

2.0

2.0

2.2

2.4

2.5

2.6

2.7

2.7

2.8

2.9

3.0

3.1

1200h

0.1

0.1

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

1100/1300 h

0.1

0.1

0.1

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0.2

1000/1400 h

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.2

0.2

0.2

0900/1500 h

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0800/1600 h

0.1

0.1

0.1

0.1

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0700/1700 h

0.1

0.1

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

80% cloud

1200 h

0.6

0.7

0.7

0.8

0.8

0.8

0.8

0.8

0.9

0.9

0.9

1100/1300 h

0.6

0.7

0.7

0.7

0.8

0.8

0.8

0.8

0.8

0.9

0.9

1000/1400 h

0.6

0.7

0.7

0.7

0.7

0.7

0.7

0.7

0.8

0.8

0.8

0900/1500 h

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0800/1600 h

0.6

0.6

0.5

0.5

0.5

0.5

0.4

0.4

0.4

0.4

0.3

0700/1700 h

0.6

0.5

0.5

0.4

0.4

0.4

0.3

0.3

0.3

0.2

0.2

100% cloud

1200h

2.0

2.1

2.2

2.2

2.2

2.3

2.3

2.4

2.4

2.4

2.5

1100/1300 h

2.0

2.1

2.2

2.2

2.2

2.3

2.3

2.3

2.4

2.4

2.4

1000/1400 h

2.0

2.1

2.1

2.1

2.1

2.2

2.2

2.2

2.2

2.2

2.2

0900/1500 h

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

0800/1600 h

2.0

1.9

1.9

1.8

1.8

1.7

1.7

1.7

1.6

1.6

1.6

0700/1700 h

1.9

1.8

1.7

1.7

1.6

1.6

1.5

1.5

1.4

1.4

1.4

To determine the total light reduction for a given depth, time of day, and cloud cover, add the values for the various factors in the appropriate depth column. Example: depth: 18 m; time of day: 1500 h; cloud cover: 80%. Total light reduction = Depth + Filter + Time-of-Day + Cloud = 3.2 + 1.7 + 0.9 + 0.6 = 6.4 f-stops.

the downwelling light becomes more dominated by blue wave- lengths. In contrast, in the greener waters of New England, the transmission band of the filter corresponds fairly well with the transmission band of the water, so the impact of the filter is smaller overall and decreases with depth. The results presented in Figs. 2 through 5 are summarized in Tables 1 and 2 for the Bahamas and New England cases, respectively. The tables enable computation of the total light reduction for any combination of depth, time of day, and cloud cover. The results are summarized in the contour plots shown in Figs. 6 (Bahamas) and 7 (New England). For the particular photographic setup described earlier, it was determined that the camera settings for a fluorescence exposure would reject essentially all ambient light when the ambient light was 6 f-stops reduced from the level just below

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the surface at midday with no cloud. The depth/time/cloud space in which the 6–f-stop reduction is satisfied is summa- rized in the contour plots in Fig. 8. In that figure the 6–f-stop contour is shown for the cloud covers modeled here. All of the shaded space to the right of each red contour line in the fig- ure represents combinations of depth and time that should allow the fluorescence image to dominate the exposure. From this presentation format we can see that choice of time of day to dive makes a significant difference in being able to take flu- orescence images at shallower depths in tropical waters. Cloud cover also makes a difference, but only when it is heavy (>80%) cover. In the more turbid New England waters, the choice of time of day makes less of a difference, and only com- plete overcast brings the required reduction in ambient light to significantly shallower depths.

Mazel

Daylight fluorescence photography

Table 2. Summary of reductions in ambient light, expressed in terms of f-stops, for the New England case

Depth (m)

 

0

3

6

9

12

15

18

21

Reduction in ambient light (f-stops) Depth Filter Time of day

0

2.1

4.1

5.7

7.1

8.2

9.1

10.0

0.8

0.6

0.6

0.5

0.5

0.5

0.5

0.5

1200h

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

1100/1300 h

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

1000/1400 h

0.1

0.2

0.2

0.2

0.2

0.2

0.2

0.2

0900/1500 h

0.3

0.5

0.6

0.6

0.6

0.6

0.6

0.6

0800/1600 h

0.7

1.0

1.1

1.1

1.1

1.1

1.1

1.1

0700/1700 h

1.2

1.6

1.8

1.8

1.8

1.8

1.8

1.8

50% cloud

1200h

0.1

0.2

0.2

0.2

0.2

0.2

0.2

0.2

1100/1300 h

0.1

0.2

0.2

0.2

0.2

0.2

0.2

0.2

1000/1400 h

0.1

0.1

0.2

0.2

0.2

0.2

0.2

0.2

0900/1500 h

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0800/1600 h

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0.1

0700/1700 h

0.1

0.0

0.0

0.0

0.0

0.0

0.0

0.0

80% cloud

1200

h

0.6

0.8

0.8

0.8

0.8

0.8

0.8

0.8

1100/1300 h

0.6

0.8

0.8

0.8

0.8

0.8

0.8

0.8

1000/1400 h

0.6

0.7

0.7

0.7

0.7

0.7

0.7

0.7

0900/1500 h

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0.6

0800/1600 h

0.6

0.6

0.5

0.5

0.5

0.5

0.5

0.5

0700/1700 h

0.6

0.5

0.4

0.4

0.4

0.4

0.4

0.4

100% cloud

1200

h

2.0

2.2

2.3

2.3

2.3

2.3

2.3

2.3

1100/1300 h

2.0

2.2

2.3

2.3

2.3

2.3

2.3

2.3

1000/1400 h

2.0

2.2

2.2

2.2

2.2

2.2

2.2

2.2

0900/1500 h

2.0

2.0

2.0

2.0

2.0

2.0

2.0

2.0

0800/1600 h

2.0

1.9

1.9

1.9

1.9

1.8

1.8

1.9

0700/1700 h

2.0

1.8

1.7

1.7

1.7

1.7

1.7

1.7

To determine the total light reduction for a given depth, time of day, and cloud cover, add the values for the various factors in the appropriate depth column. Example: depth: 12 m; time of day: 1500 h; cloud cover: 0%. Total light reduction = Depth + Filter + Time-of-Day + Cloud = 4.1 + 0.6 + 0.6 + 0.0 = 5.3 f-stops.

In situ photography verified that the technique can work under true field conditions. The digital images in Fig. 9 were made at 1130 local time on 22 May 2002 at a depth of approx- imately 9 m at White Horse Reef, Lee Stocking Island, Bahamas, on a day with 100% overcast. From Fig. 8 and Table 1 we expect the light level to be approximately 5.5 f-stops less than the light just below the surface at noon on a clear day, or just about at the right level for rejection of the ambient light in the fluorescence exposure. The photograph on the left was made using ambient light, without the yellow barrier filter in place. The exposure for the photograph was 1/10 second at f3.6 at ISO 400. This is equivalent to 8.5 f-stops below the just-below- the-surface reference of 1/400 second at f11 at ISO 400 (Sunny 11 Rule). From Table 1 we would expect the yellow filter to fur- ther reduce the light level by 1.4 f-stops. Note, though, that the

modeling was for downwelling light on a horizontal plane, and the coral in Fig. 9 was on a vertical surface in a bowl-like fea- ture that created additional shadowing. The fluorescence image (Fig. 9B) was made with settings 1/500 s, f3.6, ISO 400. At this setting the ambient light is underexposed by between 5 and 6 f-stops, and there is no ambient light at all evident in the image. The red fluorescence of chlorophyll in the macroalgae in the background and in the symbiotic algae in the coral can be seen clearly. The green in the image results from GFP in the coral tissues (Mazel et al. 2003a). In some cases the correct ambient light exposure approxi- mates that of the fluorescence and the resulting image shows a mixture of the two. The images of an anemone in Fig. 10 were made at the same dive site and approximate time as those in Fig. 9, on the following day. Cloud cover was still

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Daylight fluorescence photography

Mazel Daylight fluorescence photography Fig. 6. Contour plots of light level reduction in Bahamas waters, expressed

Fig. 6. Contour plots of light level reduction in Bahamas waters, expressed in f-stops, as a function of depth and time of day for cloud covers of 0%, 50%, 80%, and 100%.

100%. The anemone was on an exposed horizontal rock sur- face. The photograph on the left was made using a white-light flash. In the fluorescence image (Fig. 10B), ambient light can be seen as the dark green areas. This low level of ambient light does not interfere with our ability to see the chlorophyll fluo- rescence from symbiotic algae in the anemone, and to some degree from surrounding benthic algae. The image also shows that this anemone does not contain the shorter-wavelength fluorescing pigments (eg, GFP).

Discussion

The work presented here demonstrates the practicality of taking fluorescence photographs in the presence of moderate levels of ambient light and describes the equipment and

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photographic settings needed to achieve this. The modeling results provide guidance as to the range of real-world condi- tions under which the technique can be applied underwater, and suggests strategies for increasing chances of success (e.g., making early morning or late afternoon dives to reduce the ambient light contribution, or using a more powerful flash to increase the acceptable f-stop for a good fluorescence exposure). There are pros and cons to imaging fluorescence in day- time, and one should not consider it a complete substitute for night operations. Daytime operations have numerous advan- tages for the diver, including ease of navigation, viewing large areas of the substrate, spotting subjects at a distance, keeping track of one’s buddy, and reduced fatigue. Daytime boat oper-

Mazel

Daylight fluorescence photography

Mazel Daylight fluorescence photography Fig. 7. Contour plots of light level reduction in New England waters,

Fig. 7. Contour plots of light level reduction in New England waters, expressed in f-stops, as a function of depth and time of day for cloud covers of 0%, 50%, 80%, and 100%.

ations are also easier and safer than night operations. The drawback of daytime work is that the diver does not see the fluorescence directly—it only appears in the image. With dig- ital photography, the image is displayed immediately in situ, but in a small, low-resolution format. By diving at night with the appropriate excitation light source, the diver can find very small (~1 mm) or cryptic fluorescing subjects that stand out because of their contrast, but which would easily be missed or hard to photograph by day. In addition, we know from prior experience that two specimens of the same species can look very similar in visible light but have radically different fluo- rescence responses. This circumstance is obvious at night, but searching by day would require photographing multiple spec-

imens of each species to find out if this kind of variability was present. The potential exists to produce high-quality fluores- cence images in the presence of ambient light, but the choice to use the technique must be balanced against the goals of the fluorescence research project as a whole. Whereas the modeling was carried out for underwater ambient light conditions, the method presented here is com- pletely applicable to above-water fluorescence photography, whether in the laboratory or outdoors. No formal analysis was done, but our experience shows that this technique works well in conventionally illuminated laboratory spaces with no need to provide any shading. In the laboratory we have found it necessary to turn the lights off or provide an enclosure to view

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Daylight fluorescence photography

Mazel Daylight fluorescence photography Fig. 8. Contour plots illustrating the combinations of conditions (depth and

Fig. 8. Contour plots illustrating the combinations of conditions (depth and hours before/after local noon) that provide the reduction in ambient light level of 6 f-stops (factor of 64 reduction) needed for fluorescence photography, for cloud covers of 0, 50, 80, and 100%, for the Bahamas and New England. Daytime fluorescence photography should be possible over the range of conditions to the right of each contour line.

fluorescence by eye, but then we turn the lights back on to do the photography.

Comments and recommendations

The results should be applied with caution, since there are many factors that can cause deviations from the conditions modeled here. The model assumed a flat bottom with no obstructions, whereas actual benthic surfaces are often highly three-dimensional, with blockage of light from various direc-

tions. At a given depth one area might be in full sun while another is in strong shade under an overhang. The computa- tions were performed for one set of inherent optical properties (IOPs), whereas actual IOPs can vary over various temporal and spatial scales. Furthermore, whereas most of the results are presented in general terms of light reduction relative to a reference condi- tion, the summary analyses presented in Fig. 8 relate to the particular camera/strobe/filter combination that we used.

particular camera/strobe/filter combination that we used. Fig. 9. Ambient light (A) and fluorescence (B) photographs

Fig. 9. Ambient light (A) and fluorescence (B) photographs of a coral on a reef wall at a depth of 9 m, Lee Stocking Island, Bahamas. Both images were made at approximately 1130 h.

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Daylight fluorescence photography

Mazel Daylight fluorescence photography Fig. 10. White light flash (A) and fluorescence (B) images of anemones

Fig. 10. White light flash (A) and fluorescence (B) images of anemones on a horizontal surface at a depth of 9 m, Lee Stocking Island, Bahamas. Both images were made at approximately 1130 h.

Any other setup might have different exposure requirements for fluorescence imaging, demanding either a greater or lesser exposure. A different exposure level for fluorescence would lead to a different requirement for f-stop reduction from sur- face levels to achieve underexposure of the ambient light. A different barrier filter would have its own interaction with the downwelling light and might require a different correc- tion factor. The study here was related specifically to digital imaging, but the concepts apply equally well to any kind of fluores- cence imaging or detection. Any system will have some expo- sure level that produces a high-quality fluorescence response, and it is this level that must be compared to the background illumination.

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Submitted 26 May 2005 Revised 13 September 2005 Accepted 14 September 2005