Heterochromatic Flicker Photometry for Objective Lens Density Quantification

Abstract

Purpose: Although several methods have been proposed to evaluate lens transmittance, to date there is no consensual in vivo approach in clinical practice. The aim of this study was to compare ocular lens density and transmittance measurements obtained by an improved psychophysical scotopic heterochromatic flicker photometry (sHFP) technique to the results obtained by three other measures: a psychophysical threshold technique, a Scheimpflug imaging technique, and a clinical assessment using a validated subjective scale.

Methods: Forty-three subjects (18 young, 9 middle aged, and 16 older) were included in the study. Individual lens densities were measured and transmittance curves were derived from sHFP indexes. Ocular lens densities were compared across methods by using linear regression analysis.

Results: The four approaches showed a quadratic increase in lens opacification with age. The sHFP technique revealed that transmittance decreased with age over the entire visual spectrum. This decrease was particularly pronounced between young and older participants in the short (53.03% decrease in the 400–500 nm range) wavelength regions of the light spectrum. Lens density derived from sHFP highly correlated with the values obtained with the other approaches. Compared to other objective measures, sHFP also showed the lowest variability and the best fit with a quadratic trend (r² = 0.71) of lens density increase as a function of age.

Conclusions: The sHFP technique offers a practical, reliable, and accurate method to measure lens density in vivo and predict lens transmittance over the visible spectrum. An accurate quantification of lens transmittance should be obtained in clinical practice, but also in research in visual and nonvisual photoreception.

During the process of normal aging, the eye undergoes numerous optical and neuronal transformations that impact retinal sensitivity and response to light. In addition to a reduction in its elasticity with age, the ocular lens undergoes a gradual decrease in light transmittance.^1–5 The mechanisms responsible for this age-related process involve denaturation, oxidation, and cross-linking of the crystalline proteins, leading to aggregates and pigment formation in the different structures of the lens.⁶ Throughout the lifespan these altered proteins are not evacuated and their accumulation leads to coloration and opacification of the lens.⁷ This phenomenon, also known as lens yellowing, leads to cataract, the leading cause of blindness.⁸ Ultraviolet light exposure along with smoking, trauma, and metabolic pathologies such as diabetes have been shown to exacerbate lens yellowing.^9–11 Different types of cataract can be diagnosed (e.g., cortical, nuclear, subcapsular), each affecting lens density measurements differently. It has even been reported in the literature that a lens classified as cataractous could have a low optical density.³ To date, the only treatment for cataract is surgical removal of the natural lens, yet, in the absence of an objective measure of lens density, the therapeutic decision for a cataract surgery relies on patients' visual acuity and a subjective estimate of lens opacification by the clinician. 

In research on visual and nonvisual photoreception,^12–15 estimates of lens density often rely on templates or age-dependent models.^2,16 Although these templates provide relatively good estimates of lens density in young subjects, they offer a limited predictive value in the older population where interindividual variability in lens density is increased.^17,18 Recent studies^19,20 have attempted to develop alternative means to objectively measure lens density. These methods, however, have not gained popularity owing to the high component cost or the lack of available tools. Therefore, there is a scientific and clinical need for an objective quantification of lens transmittance in vivo. 

A refined scotopic heterochromatic flicker photometry (sHFP) technique has recently been proposed to accurately assess lens density and transmittance in vivo.¹⁷ The aim of this study was to use this approach to evaluate changes in lens transmittance over age, and to compare the results with those obtained with three other techniques: (1) a threshold detection method, (2) a Scheimpflug imaging technique, and (3) a subjective clinical assessment using a slit-lamp. 

Methods

Subjects

A total of 43 subjects participated in this study (23 males and 20 females). They all underwent the sHFP procedure. Subjects were assigned to one of three groups by their age. The young group included 18 subjects (20–35 years; 26.3 ± 1.0 years), the middle-aged group included 9 subjects (36–55 years; 45.9 ± 1.4 years), and the older group included 16 subjects (55–70 years; 63.2 ± 0.9 years). A subgroup of 25 subjects (11 younger [25.8 ± 0.9 years], 6 middle aged [43.8 ± 1.5 years], and 8 older [65.5 ± 1.1 years]) underwent all four lens density measurement procedures. Subjects were screened for medical health including medical history, physical examination, and ophthalmologic examination. The protocol was approved by the National Ethical Committee and all subjects gave written informed consent. Institutional Review Board/Ethics Committee approval was obtained and all procedures were in compliance with the Declaration of Helsinki. 

Principles of the Psychophysical Tests

Within the ocular media, the lens and the macular pigment are the principal contributors to changes occurring with age in the amount and spectral composition of retinal light exposure.²¹ The macular pigment is located mainly in the central retina, therefore, psychophysically testing peripheral spectral sensitivity to scotopic light is thought to provide a nearly pure measure of changes in lens density as a function of age.³ The action spectrum of the rods' photopigment rhodopsin is genetically determined and unvarying with age.²² In this study, we selected a pair of wavelengths (410 nm [violet] and 560 nm [green]) with equal absolute psychophysical scotopic thresholds (L1 and L2), based on a recent nomogram for rhodopsin sensitivity (λ_max = 495 nm) (Fig. 1).²³ Without any filtering by the ocular lens, scotopic thresholds for L1 and L2 would theoretically be obtained at the same quantal intensity (I1 = I2). A different threshold would be obtained (I1 ≠ I2) whenever a nonhomogeneous lens filtering of the light spectrum occurs (Fig. 1), as in normal aging. 

Figure 1

 

Scotopic sensitivity. Two wavelengths (L1, L2) with equal scotopic threshold (I1 = I2) are chosen on the basis of this rhodopsin nomogram. After nonhomogeneous alteration of scotopic sensitivity due to lens yellowing, these two wavelengths are no longer detected at same scotopic threshold (I1 ≠ I2). The difference in scotopic light intensity required to detect L1 and L2 is an indicator of the degree of lens density (I_index).

A lens density index can be calculated by using the following equation:  

  .

 

The lens transmittance spectrum is then derived from I_index by using the ocular media model proposed by van de Kraats and van Norren.¹⁶ 

Scotopic Threshold Comparison Technique

The absolute threshold technique is a classical approach that estimates lens density by directly comparing absolute scotopic thresholds to the rhodopsin absorption curve, based on the principle described above. In our study, subjects entered the darkroom 1 hour before testing. They were seated in front of a full visual field covering box with a chin rest and a joystick, and were explained the testing procedure. After 45 minutes of dark adaptation (DA), the subject controlled the intensity of an annular light stimulus until he/she could detect it flickering in his/her peripheral vision field. The subject gaze stability was maintained by fixating a central dim red light-emitting diode (LED) (10′ diameter) with the dominant eye. The flicker frequency was chosen to be 1 Hz (500 ms of light, 500 ms of dark) on the basis of pilot results and published temporal summation characteristics for scotopic vision.²⁴ Once the annulus was detected, the subject confirmed his/her decision by pressing the button. The intensity at which the annulus was detected represented the absolute scotopic threshold of the subject for the wavelength used. This procedure was repeated five times with both 410- and 560-nm lights. Both lights were perceived as gray under scotopic conditions. Difference of quantal light intensities required for detection was used for calculation of the lens density index (I_index) as described above. 

Scotopic Heterochromatic Flicker Photometry Technique

Following the afore-described threshold detection procedure, subjects were instructed to use the two-way (up/down) joystick to adjust light intensity of a flickering 410-nm light stimulus (annulus, 3° wide, 15°–18° off-center), until it fused with the 560-nm reference light stimulus (of fixed intensity). The 560- and 410-nm LEDs were square wave-modulated in counterphase (500 ms on/500 ms off, 1 Hz flickering). Since both lights are perceived as gray (uncolored) in scotopic conditions, total fusion is achieved once both lights are perceived to be equiluminous. Subjects' fixation was aided by a small central 10′ red fixation light (7–10 cd/m²). Subjects were given five trials for each eye. Acquisitions lasted 10 minutes after DA. For more technical details see Teikari et al.¹⁷ In addition, we tested the sHFP technique with a 5-minute and then 0-minute DA period in a subgroup of 14 subjects (8 young, 2 middle aged, and 4 older). Results were compared to those with the 45-minute DA protocol. 

Scheimpflug Imaging

The Scheimpflug principle, first described in 1906 by Theodor Scheimpflug,²⁵ defines an optical imaging position at which the parameters of an obliquely tilted object can be assessed with optimal depth of focus and minimal image distortion. For more technical description see Wegener and Laser-Junga.²⁶ For this measure we used the Pentacam (Oculus Optikgeräte GmbH, Wetzlar, Germany), a commercially available device containing an automatically rotating Scheimpflug camera for anterior segment tomography. This device shows lens density measurements that are correlated with clinical assessments.²⁷ 

After pupil dilation using antimuscarinic eye drops (tropicamide, 2 mg/0.4 mL; Novartis Ophthalmics, Rueil Malmaison, France), participants were instructed to sit in front of the camera with their head stabilized on a chin rest. After automatic calibration of the device at start-up, the image of the eye was focused and centered on the screen by the clinician. Subsequently, 50 single-slit images were obtained in 2 seconds during rotation of the camera (0°–180°) around the eye by using a blue LED to image the anterior eye segment. The 25,000 raw data points collected (500 data points per slit image) were then processed to generate a three-dimensional representation of the anterior segment of the eye comprising the lens. Lens density was then assessed on a range from 0 to 100 by using the device's built-in lens-density module. Estimates of average nuclear and cortical opacifications were computed for each eye. 

Subjective Clinical Assessment of Lens Opacification

A full ophthalmologic assessment was scheduled the day subjects underwent the Scheimpflug imaging. After pupil dilation, participants' anterior eye segment was examined with a slit-lamp by an ophthalmologist. Lens opacity was visualized (nuclear, cortical, or capsular) and subjectively quantified on a seven-step validated clinical scale (0, 0.5, 1, 1.5, 2, 2.5, 3+; 0 = no opacity, 1 = minor opacification, 2 = moderate opacification and 3 = major opacification). This subjective scale is the recommended scale in clinical practice and is similar to the Lens Opacities Classification System III (LOCS III) scale.²⁸ 

Statistical Analyses

Statistical analyses were performed with Statistica v10 (StatSoft, Inc., Tulsa, OK, USA) and regression analyses with SigmaPlot v12 (Systat Software, Chicago, IL, USA). Psychophysical results are presented in values relative to a standard observer (25 years old) based on an ocular media model (van de Kraats and van Norren¹⁶). Lens density and opacity data of the preferred (dominant) eye were fitted as a function of age by using a quadratic trend that ignored the linear term (f = y0 + bx²) as suggested by van de Kraats and van Norren.¹⁶ For the analyses of the effect of age on transmittance, lens densities and areas under the curve (AUCs) were compared between age groups by using a Kruskal-Wallis ANOVA. Post hoc analyses were done by using multiple comparisons of mean ranks of all groups.²⁹ The Wilcoxon matched pairs test was used to compare intertrial variability between the sHFP and the threshold techniques. Comparisons between lens density measurements were evaluated by using general linear regression models. To make Scheimpflug imaging measures comparable to psychophysical measures, values were log transformed. Squared Pearson correlation coefficients (r²) were calculated as indicators of relationships between measures, not as indicators of agreement between methods. Because lens density measures are not in the same units, and therefore are not measuring the same quantities, the limits of agreement approach proposed by Bland and Altman's agreement test could not be applied between different measurement strategies.³⁰ The Bland-Altman agreement test was used, however, to compare measurements obtained with sHFP after different DA durations (45, 5, and 0 minutes). Data are represented as mean ± standard error of the mean (SEM) unless indicated otherwise. 

Results

As expected, using both objective and subjective measurement techniques, lens density increased with age (Kruskal-Wallis test: sHFP, H (2, n = 43) = 31.2, P < 0.001; threshold, H (2, n = 43) = 22.4, P < 0.001; Scheimpflug imaging of the preferred eye, H (2, n = 25) = 15.9, P < 0.001; cortical clinical scale, H (2, n = 28) = 24.3, P < 0.001) (Fig. 2). Lens densities obtained with sHFP were more highly correlated with age (quadratic trend, n = 43, r² = 0.71, P < 0.001; Fig. 2A) than were the densities obtained with the threshold detection technique (n = 43, r² = 0.38, P < 0.001; Fig. 2B), and the Scheimpflug imaging technique (n = 25, r² = 0.46, P < 0.001; Fig. 2C). Clinical slit-lamp assessment of cortical and nuclear lens densities were also very highly correlated with age (cortical, n = 28, r² = 0.85, P < 0.001 [Fig. 2D]; nuclear, n = 28, r² = 0.88, P < 0.0001). By restricting our statistical analysis to the 25 individuals who underwent all four measures, we found that the strength of the quadratic relationship between age and quantitative lens density remained the lowest with the threshold method (r² = 0.31, P < 0.01), and the highest with the sHFP technique (r² = 0.75, P < 0.001). 

Figure 2

 

Individual lens density (opacification) as a function of age. Lens density of the preferred eye was fitted by using a quadratic trend that ignores the linear term. Subjects were divided into three age groups: young, middle aged, and older. Lens density was assessed by using (A) sHFP (n = 43), (B) threshold comparison (n = 43), (C) Scheimpflug imaging (n = 25), and (D) a clinical ophthalmologic slit beam procedure (n = 28). Among objective measures, the best fit was obtained between sHFP and age (r² = 0.71). Average lens density/opacification (mean ± SE) of each age group was represented as colored filled circles on top of the individual scatter plot. The young group is shown in blue, middle-aged group in gray, and older group in red. All techniques showed a lens density increase with age. Compared to psychophysical techniques (sHFP and threshold) that showed no difference in lens density between young and middle-aged groups, Scheimpflug imaging yielded a difference between these two age groups (P = 0.009).

With the sHFP and threshold methods, post hoc analyses revealed that lens density in the older group was significantly higher than in the young (sHFP: n = 43, P < 0.001, threshold: n = 43, P < 0.001) and middle-aged groups (sHFP: n = 43, P = 0.008; threshold: n = 43, P = 0.009) (Figs. 2A, 2B). Lens density of the young and middle-aged groups were not significantly different when using sHFP (P = 0.33) and threshold methods (P = 1). Using Scheimpflug imaging with the preferred eye, lens densities of the young and middle-aged groups were significantly different (n = 25, P = 0.009), and lens density of the older group was significantly increased compared to young (n = 25, P < 0.001) but not to middle-aged subjects (P = 1) (Fig. 2C). When lens density of the nonpreferred eye was tested by using Scheimpflug imaging, however, the middle-aged group was not significantly different from both young and older groups (P = 0.33, P = 0.21; data not shown here), and the older group had a significantly increased lens opacity compared to young group (n = 25, P < 0.001). Clinical subjective assessment results showed a significant increase in cortical and nuclear lens opacification between young and older groups only (n = 28, P < 0.001; Fig. 2D). 

Individual trials using sHFP were more reproducible and 71% less variable (P < 0.001) (average trials standard deviation [SD] = 0.04 ± 0.03; Fig. 3A) than the threshold detection technique (average trials SD = 0.14 ± 0.17; Fig. 3B). 

Figure 3

 

Intertrial variability of lens density estimates in 43 subjects. (A) Scotopic heterochromatic flicker photometry technique and (B) threshold comparison. Data are presented as mean ± SD for five trials. Note the high variability in the repeated trials with the threshold comparison method (B). Although sHFP is also a psychophysical technique, variability between trials using sHFP (average trials SD = 0.04 ± 0.03) is significantly reduced by 70.6% (P < 0.001) compared to the threshold method (average trials SD = 0.14 ± 0.17).

Individual lens transmittance spectra were derived from the sHFP lens density index. Transmittance spectra were compared between groups of subjects (Fig. 4). The AUC decreased as a function of age (Kruskal-Wallis test: H (2, N = 43) = 32.1, P < 0.001). Transmittance was lower in the older group than in the young (P < 0.001) and middle-aged (P = 0.013) groups, but there was no difference in AUC between young and middle-aged groups (P = 0.19) (Fig. 4). Specifically, transmittance was mostly affected in the short wavelength region of the visible spectrum (400–500 nm) and decreased on average by 53% (P < 0.001) between the young and older group and by 45.7% (P = 0.014) between middle-aged and older group. Transmittance was less affected in the middle wavelength region of the spectrum (500–600 nm) and decreased by 16.9% (P < 0.001) between young and older participants and by 14.1% (P = 0.014) between middle-aged and older participants. A 5.1% (P < 0.001) and 4.2% (P = 0.014) decrease in transmittance was noted between young and older groups and between middle-aged and older groups, respectively, in the longer wavelength region of the spectrum (600–700 nm). 

Figure 4

 

Light transmittance spectra of the ocular lens estimated with the sHFP technique. Results show a decreased AUC and lens transmittance over the entire visible spectrum in the older subject group (red line) compared to young (blue line) (16.3%, P < 0.001) and middle-aged group (gray line) (13.1%, P = 0.013). The decrease in transmittance is more pronounced for short (400–500, 53%, P < 0.001) than for middle (400–500, 16.9%, P < 0.001) and long wavelength lights (400–500, 5.1%, P < 0.001). The AUC was not different in middle-aged compared to young participants (P = 0.19).

The sHFP (n = 43) and Scheimpflug imaging (n = 25) techniques both showed a very high correlation in lens density estimates between right and left eyes (respectively r² = 0.94, P < 0.001; r² = 0.75, P < 0.001) (Figs. 5A, 5B). Clinical slit-lamp assessment of lens opacification (n = 28) yielded a perfect correlation between eyes (r² = 1, P < 0.001) (see Supplementary Fig. S1). 

Figure 5

 

Correlation between left and right eye lens density. (A) Data from sHFP show a significant high correlation in lens density between eyes (r² = 0.94). (B) Data from the Scheimpflug imaging system show a high, yet lower correlation between eyes (r² = 0.75).

A relatively good correlation was found between sHFP and Scheimpflug imaging for both eyes (n = 25; preferred eye, r² = 0.56, P < 0.001; nonpreferred eye, r² = 0.59, P < 0.001; Fig. 6A). Data from sHFP were also well correlated with the data from threshold detection (r² = 0.61, P < 0.001; Fig. 6B). Scheimpflug imaging and threshold detection data were less comparable (r² = 0.31, P = 0.004; Fig. 6C). 

Figure 6

 

Comparison of the lens density measurement techniques. Results are from the preferred dominant eye. (A) The sHFP results correlated well with the Scheimpflug imaging results (r² = 0.56, P < 0.001). (B) Correlation was even stronger between the two psychophysical measures (r² = 0.61, P < 0.001) but (C) lower between Scheimpflug and threshold technique (r² = 0.31, P = 0.004). (D) The psychophysical sHFP yielded closely similar results to the clinical assessment of cortical lens opacity (r² = 0.71, P < 0.001).

The sHFP and Scheimpflug imaging yielded a high correlation with the clinical scale. The sHFP results, however, were generally better correlated with clinical assessed cortical opacifications (r² = 0.71, P < 0.001; Fig. 6D) than the nuclear opacifications (r² = 0.64, P < 0.001). It is worth noting that, compared to sHFP, Scheimpflug imaging results showed a lower correlation with clinical estimates of cortical opacification (r² = 0.55, P < 0.001) and with clinical estimates of nuclear opacification (r² = 0.49, P < 0.001). 

To test the impact of reducing the period of DA before assessing lens density with the sHFP technique, we conducted a final analysis. Our results (n = 14) showed that a reduced DA period of 5 minutes, or even no DA before testing, provides results that are highly correlated with the ones obtained with a 45-minute DA (0-minute DA – 45-minute DA, r² = 0.90, P < 0.001, Fig. 7A; 5-minute DA – 45-minute DA, r² = 0.83, P < 0.001, Fig. 7B). The Bland-Altman agreement test comparing lens densities obtained at the different DA durations showed that the average bias between the procedures is close to zero (0-minute DA – 45-minute DA: −0.02 log, Fig. 8A; 5-minute DA – 45-minute DA: −0.03 log, Fig. 8B) and 93% of the measurements fall within narrow limits of agreement (0-minute DA – 45-minute DA: −0.12 to 0.1 log, Fig. 8A; 5-minute DA – 45-minute DA: −0.17 to 0.15 log, Fig. 8B). 

Figure 7

 

Impact of reducing dark adaptation duration in the sHFP protocol. Preliminary results in 14 subjects show that the dark adaptation period in the sHFP can be eliminated (A) or reduced to 5 minutes (B) without a significant impact on the results. Repeated measures with an elimination or reduction of the dark adaptation period correlate well with the original 45-minute dark adaptation protocol (r² = 0.90, P < 0.001 and r² = 0.83, P < 0.001, respectively).

Figure 8

 

Bland-Altman plots showing the agreement between (A) 45-minute and 0-minute DA and (B) 45-minute and 5-minute DA procedures. The black horizontal full line represents the average bias or the average of the differences between measures obtained by the two procedures ([A]: −0.02 log; [B]: −0.03 log). Dashed horizontal black lines represent the limits of agreement between the two procedures ([A]: −0.21 to 0.16 log; [B]: −0.25 to 0.19 log). Preliminary results in 14 participants show that the average bias between the procedures is close to zero, and 93% of the measurements fall within the narrower limits of agreement ([A]: −0.12 to 0.1 log; [B]: −0.17 to 0.15 log).

Discussion

Our results demonstrated that the sHFP technique provides accurate measures of lens density in healthy individuals. These results are in agreement with previous in vivo and in vitro findings, suggesting that lens density increases gradually with age, and at a higher rate after the age of 60 years.^1,2,4 Lens transmittance results are also in accordance with in vivo,⁵ and donor studies,³¹ showing a relative decrease with age over the entire visible spectrum. This decrease in transmittance is particularly pronounced for short wavelength light. 

Measures of lens opacification obtained with the sHFP method were in agreement with the subjective clinical assessment, the psychophysical threshold comparison, and the Scheimpflug imaging technique. Moreover, sHFP measures were better predicted by the proposed quadratic model of increase in lens density with age.¹⁶ There was no statistical difference in lens opacification within the same age group between the preferred or nonpreferred eye when using sHFP. 

Clinical assessment of lens yellowing with a slit-lamp is an economic cataract grading approach that is widely used by ophthalmologists. This subjective assessment was shown in the current and previous studies to provide results that are largely in agreement with Scheimpflug imaging.²⁷ In the present study, we found that sHFP measures of lens density are more coherent with clinical lens opacification estimates than with the Scheimpflug imaging measures. Similarly to all subjective evaluations, however, clinical assessment of lens yellowing has limitations rooted in operator bias that might lead to increased variability and systematic bias.³² Clinical lens opacification estimates showed a perfect linear regression score (r² = 1) between the two eyes. On the other hand, objective techniques showed a highly, but not perfectly, correlated lens opacification degree between eyes, as shown previously.³ This implies that minor to moderate opacification differences between both lenses may not be detected or adequately scored by clinicians, owing to the discrete clinical scale used, but may be appropriately quantified with the sHFP technique. 

To justify lens replacement surgery, cataract should be associated with a significant visual impairment. With the growing interest in refraction corrective surgery, patients may want to exaggerate their visual cataract-related impairment in order to get health insurance coverage for an exclusively esthetic corrective surgery. The need for an objective measure of lens density is thus clinically and economically relevant. Our preliminary finding that DA, before assessing lens density with the sHFP approach, can be reduced from 45 to 5 minutes, or even be eliminated, would render this method more practical for a quantification of lens transmittance in clinical settings. 

It is of importance to note here that the eye does not solely serve for perceptual vision but is also involved in “nonvisual” functions that involve intrinsically photosensitive ganglion cells containing the photopigment melanopsin.³³ Together with the conventional visual photoreceptors (rods, cones), this system integrates and processes photic information for non-image forming functions such as the pupillary light reflex and circadian photoentrainment.³⁴ The lack of objective measurements of lens opacity is a serious impediment for research in the domains of vision and chronobiology,³⁵ as quantifying the amount and spectral composition of retinal light is crucial for understanding photoreceptor contribution in nonvisual photoreception.^12,13,18,36 The objective estimate of lens transmittance provided by the sHFP approach could easily be implemented in research. 

The visual system tends to compensate for multiple age-related visual impairments.^37–39 Even though we have recently published evidence for nonvisual compensation to precataractous lens-related spectral alterations,¹⁸ it remains critical to assess the impact of cataract and cataract replacement surgery on nonimage-forming responses to light. Indeed, despite the fact that a number of studies using questionnaires claim that cataract might be responsible for poor sleep,⁴⁰ the relationship between lens transmittance and circadian sensitivity to light remains unevaluated. Therefore, knowledge of the spectral composition of light reaching the retina would help understand the etiology of circadian disruptions. 

The sHFP approach described in this study is not devoid of limitations. First, since the light stimulation we used involves the peripheral retina, we believe that sHFP reflects mainly cortical lens density. This is supported by the finding that sHFP values showed a higher correlation with cortical lens opacification measures in our current study. Second, because sHFP relies on visual detection, it cannot be used in visually impaired individuals (e.g., patients with altered peripheral visual field due to glaucoma). Third, although participants are likely to have displayed their largest pupils for the psychophysical measurements (after DA), and for the Scheimpflug measures (after tropicamide administration), measurements may have occurred at slightly different pupil sizes. Additionally, the Beer-Lambert law results in less incident light reaching the deeper parts of the lens, and in turn could influence the assessment of the opacity of the lens nucleus with the imaging technique. Therefore, it cannot be excluded that part of the differential variability between the imaging and the psychophysical techniques could be due to the different methodologies. 

In conclusion, our results suggest that the sHFP technique gives an accurate and objective estimate of lens density and transmittance. The densities obtained with this approach are compatible with those obtained with the Scheimpflug technique, clinical subjective scales, and psychophysical threshold methods, but with a lower intra- and interindividual variability, and a more accurate description of the effects of age on lens density. This approach is practical, cost-effective,⁴¹ and can easily be implemented for use in clinical settings, and photoreception and nonvisual photobiology research. 

Acknowledgments

Supported by Grants from FP6-EUCLOCK, ANR 12-TECS-0013-01, Fédération des Aveugles et Handicapés Visuels de France, Société Française de Recherche et Médecine du Sommeil, and Ministère de l'Enseignement Supérieur et de la Recherche Français. K.K. is supported by ANR-11-LABX-0042. The sponsor or funding organization had no role in the design or conduct of this research 

Disclosure: R.P. Najjar, None; P. Teikari, None; P.-L. Cornut, None; K. Knoblauch, None; H.M. Cooper, None; C. Gronfier, None