Flower colours through the lens: quantitative measurement with visible and ultraviolet digital photography

Abstract

Background: The study of the signal-receiver relationship between flowering plants and pollinators requires a capacity to accurately map both the spectral and spatial components of a signal in relation to the perceptual abilities of potential pollinators. Spectrophotometers can typically recover high resolution spectral data, but the spatial component is difficult to record simultaneously. A technique allowing for an accurate measurement of the spatial component in addition to the spectral factor of the signal is highly desirable.

Methodology/principal findings: Consumer-level digital cameras potentially provide access to both colour and spatial information, but they are constrained by their non-linear response. We present a robust methodology for recovering linear values from two different camera models: one sensitive to ultraviolet (UV) radiation and another to visible wavelengths. We test responses by imaging eight different plant species varying in shape, size and in the amount of energy reflected across the UV and visible regions of the spectrum, and compare the recovery of spectral data to spectrophotometer measurements. There is often a good agreement of spectral data, although when the pattern on a flower surface is complex a spectrophotometer may underestimate the variability of the signal as would be viewed by an animal visual system.

Conclusion: Digital imaging presents a significant new opportunity to reliably map flower colours to understand the complexity of these signals as perceived by potential pollinators. Compared to spectrophotometer measurements, digital images can better represent the spatio-chromatic signal variability that would likely be perceived by the visual system of an animal, and should expand the possibilities for data collection in complex, natural conditions. However, and in spite of its advantages, the accuracy of the spectral information recovered from camera responses is subject to variations in the uncertainty levels, with larger uncertainties associated with low radiance levels.

Figure 1. Spectral sensitivities corresponding to the red, green and blue colour channels of a Canon D40 camera (solid lines) and the UV-sensitive red channel of a Fuji S3 UVIR digital camera (magenta solid line), along with the long (red dashed line), medium (green dashed line) and short (blue dashed line) human photoreceptors .

Spectral sensitivities were normalised by dividing the sensitivity at each by the total area under each channel/photoreceptor curve.

Figure 2. Standard, non-linear, digital images of flowers belonging to the species Oxalis pes-caprae, Goodenia ovata, Gazania rigens and Geranium sp. in the visible region of the electromagnetic spectrum (first column); reconstructed images representing the linear camera response in the visible region of the electromagnetic spectrum as recorded by the red, green and blue colour channels of a Canon 40D camera (second column); and, pseudo-colour representations of reconstructed images representing the linear camera response in the UV region of the spectrum (third column) as recorded by the ‘red’ UV-sensitive channel of a Fuji S3 UVIR camera.

Second column insert depicts the mean spectral reflectance of three readings taken at the tip, middle and bottom of a single petal to account for spatio-chromatic variability within a single flower . Included on each image is a white reflectance standard for spectrophotometry (large circle) and a grey achromatic standard reflecting about 33% of incident radiation. Error bars on the reflectance spectra represent one standard deviation in all cases.

Figure 3. Standard, non-linear, digital images of flowers belonging to the species Malus domestivus, Freesia laxa, Eremophila macculata and Sonchus oleraceus in the visible region of the electromagnetic spectrum (first column); reconstructed images representing the linear camera response in the visible region of the electromagnetic spectrum as recorded by the red, green and blue colour channels of a Canon 40D camera (second column); and, pseudo-colour representations of reconstructed images representing the linear camera response in the UV region of the spectrum (third column) as recorded by the ‘red’ UV-sensitive channel of a Fuji S3 UVIR camera.

Second column insert depicts the mean spectral reflectance of three readings taken at the tip, middle and bottom of a single petal to account for spatio-chromatic variability within a single flower . Included on each image are a white reflectance standard for spectrophotometry (large circle) and a grey achromatic standard reflecting about 33% of incident radiation. Error bars as per Figure 4. Note: Images in row 2 column 1–2 are rotated relative to the image in row 2 column 3.

Figure 4. Images representing the recovered linear response of Goodenia ovata as recorded by a Canon 40D camera sensitive to visible radiation (panel a) and a Fuji S3 UVIR camera sensitive to UV radiation (panel b).

Panel c) summarises the total reflectance recorded by the red, green, blue and ‘red’ UV-sensitive channels of the two cameras (hatched bars), and the predicted total reflectance recorded by each colour channel (white bars) along with the results of their statistical comparison. Predicted camera responses were calculated by applying Equation 1 to 15 independent spectrophotometric readings taken across the lower petals of the floral sample (graphically summarised in panel II). Panel I depicts mean reflectance spectrum, predicted and actual camera responses (Panel I insert) for an achromatic grey sample used as exposure control. Error bars represent standard deviation in all cases. P-value significant at ; NS P-value not significant at . Refer to text for details.

Figure 5. Examples of the two different grid schemes employed to measure local chromatic variability within a petal.

a) Oxalis pes-caprae 43 sampling scheme, grid size 40 pixels. b) Geranium sp. 62 sampling scheme, grid size 30 pixels.

Figure 6. Linear (panel a) and non-linear (panel b) camera responses to a set of six achromatic samples from an X-Rite Colour Checker Passport corresponding to the green channel of a Canon 40D camera.

The six achromatic samples uniformly reflect, from bottom to top, 3.10%, 9.11%, 19.5%, 37.2%, 60.9% and 94.8% of incident visible irradiation . Camera responses correspond to an area of 900 pixels² located at the centre of each grey swatch. Error bars along the x-axis represent pixel intensity variation within the sampling square, whilst error bars along the y-axis represent variation within recovered linear values arising from the uncertainty associated with the use of a biexponential linearisation equation . Error bars represent standard deviation in both cases. P-value significant at ; NS P-value not significant at .

Figure 7. Total reflectance recorded from flowers belonging to the genera Oxalis pes-caprae, Goodenia ovata, Gazania rigens, Geranium sp., Malus domesticus, Fressia laxa, Eremophila macculata, and Sonchus olereaceus by the different colour channels of a Canon 40D and a Fuji S3 UVIR.

Expected camera responses, in reflectance values, calculated from spectrophotometric data and the spectral sensitivity curves of each colour channel (Figure 1) (white bars), camera responses for three 225 pixel² areas located at the tip, middle and bottom of the petals (cross-hatched bars), and camera responses from sampling areas wider than those covered by a 400 µm standard spectrophotometer probe (diagonal-hatched bars). Figure inserts represent measured and predicted camera responses for a spectrally flat, achromatic standard reflecting about 33% of incident irradiation, which was included as an internal control on each image. Error bars represent standard deviation in all cases.

Figure 8. Graphical summary of the mean total reflectance recovered from fifteen 900 pixel² square sampling areas covering the entire lower petals of a floral specimen of Goodenia ovata by the red, green and blue channels of a Canon 40D camera sensitive to visible radiation and the red UV-sensitive channel of a Fuji S3 UVIR camera.