AU2014208236B2 - Improved photobleaching method - Google Patents

Abstract

The present disclosure provides an improvCd method for photobleaching an Cye of a subject. The disclosed method may be used in a number of psychophysical test methods, including, but not limited to, measurement of dark adaptation. The improved method for 5 photobleaching involves at least one of the following improvements: (i) the use of a bleaching light emitting a particular wavdength of light or a tailored spectrum of wavelengths; (ii) restricting or otherwise spatially tailoring the region of the retina that is subject to photobleaching; and (iii) utilizing a bleaching light having an intensity that is at or below the intensity of ambient daylight. The present disclosure additionally provides a 10 combination of a photobleaching light and an apparatus to administer a psychophysical test suitable for use in practicing the disclosed mehods HA.9774-A

Description

2014208236 31 Μ 2014 AUSTRALIA PATENTS ACT 1990 REGULATION 3.2

Name of Applicant· THE UAB RESEARCH FOUNDATION John G. Edwards; and Gregory R, Jackson. Address for Service; E. F. WELLINGTON & CO., Patent and Trade Mark Attorneys, 312 St. Jidda Road, Melbourne, Southbank, Victoria, 3006. Invention Title: “IMPROVED PHOTOBLEACHING METHOO”

Actual inventor/s:

Details of Associated Provisional Applications Nos:

The following statement is a foil description of this invention including the best method of performing it known to us, 1 2014208236 31 Μ 2014 5

CM0S$-:EEFE:R®NC':E;:TO RELATED APPLICATIONS

This application is a 'divisiooaF application -Patent

Application Ho, 200121668¾ which is the national phase oCinternahonal Application No. PCT©S2008i00209Sy clahvhng priority ofU.S. Provisional Application No. 60/890,13 L filed 15 February 2007. the entire text of which are hereby iincorporated herein by reference.

FIELD OFTHE DISCLOSURE

The present disclosure relatesto improved methods for photobleaching an eye, or a desired portion thereof, ofa subject.

BACKGROUND 10 The retina is comprised of a thin layer of neural cells that lines the back of the eyeball of vertebrates. In vertebrate '-embryonic development, the retina and: the optic nerve Originate as outgrowths of the developing brain. Hence, the retina is part of the centra! nervous system. The vertebrate retina contains photoreceptor cells (both rods and cones) that respond to light; the resulting neural signals then pfiOcessihg 15 the retina. The retinal output takes the form of notion gshgKoo cells whose axons form the optic nerve.

Due coffipOnent of tlie retina is the macula,; The macula of the;human eye, which is aboiit h min in diameter and covers the central 21,5 degrees of visual angle, is designed for detailed vision. The macula itsel f comprises a smail cone-dojrunated fovea dUiToonded by a 20 rod-dominated parafovea (Curcio 1990,1. Clomp.: Neurol· :292:497), Rods: are responsible for vision in dim light (septopie visidnfwhile; cones are responsive to bright light andfoolors (photopic; vision};. In young adults, the number of rods outnumbers cones by approximately 9tl. nnitS; proportion; of tods: to cones changes: as individual's age.

The function of the rod and cone photoreceptors is impacted by the health of the rod 25 and cone photoreceptors themselves. Thu health arid function* bf the rod and cone photoreceptors are maintained by the retinal pigment epithelium fRPE}, the Bruch's membrane arid the chpridcapillaris {collectively referred to as the RPE/Bruch/s membrane complex)· The RPl is a dedicated layer of nurse cells behind the neural retina. The EPE sustains photoreceptor health in a number of ways, including, but not limited to^ maintaining .proper ionic .balance.,, transporting and filtering nutrients, providing retinoid intepnediaios to replenish photopigment bleachedby light exposure and absorbing shay photons, The/RFE and the photorepeptfifs are sepasatedhy the chorioeapillarts, which provides blood flow to the neural retina. Further separating the RPE and: :tfee\;;c]bcm^p*;Hari8 is the Bruch’s 5' fiieritbrane, a depate vessel Atall only 2··6 μι» thick;,.. 2014208236 31Jul2014

The impairment:: of the rod and/or mm photoreceptors may lead to impairment in dark adaptation and other visual processes. Bark adaptation is defined as the recovery of light sensitivity by the retina in the.-dWk\al|er. expose:to -a conditioning light In this regard, dink adaptation and other visual processes can essentially be viewed as a bioassay of 10 the health of the rod photoreceptors, the RPE, the Bruch’s membrane and the ehprloeapillarls, and impaired dark adaptation and the impairment <$ip&amp;er >ds&amp;4'li»ifo«s. may be used as a chnicai marker of disease states that impair one or more of the rod and/or cone photoreceptors, the ERE, the Bruch's membrane and the ehoriocapillaris. For impairments in dark adaptation such disease states include, but am not; limited to age-related 15 macular degeneration (AMD;: which is also: known as age-related maeulopathy ARM), vitamin A deficiency, Sorsby’s Fundus Dystrophy, late autosomal dominant retinal degeneration, retinal impainnent related to diabetes and diabetic retinopathy, A subjeetT abiity to dark adapt can be characterized by measuring septopie sensitivity meoyery (i;e,, rod function) after photobleaebing using psyehophysieaF testing 20 methods known in die art. hi such psychophysical tests, t^iclUy a test eye of the subject is first pre-conditioned to a state of relative seotopic insensitivity by exposing the eye to a conditioning light (a procedure referred to as photobleaefiing or bleaching),: After this pre-; conditioning (or bleaching) step, the: subject's scotopie; sensitivity: (the minimum light: intensity drat can be detected. in a dark environment) is measured, at One or more successive 25 times. The measurement is made by exposing the bleached region; of the test eye to a Series of·'Stimnhts: lightstof yarjangfintensmes.: Based on subject Feedback as to which stimulus intensities: can be def ected, a sensitivity,.or threshold, is determined for each successive time, The subject is kept in a dark environment throughout the test. The absolute levels and/or kinetics of the resulting threshold curve indicate the subject’s ability to dark adapt, 30 Impairment: in the subject's dark adaptation parameters may indicate tite:subject is currently 3 suffering from. and/or at :;fisk;fof a disease states that impairs one or more of the rod and/or cone photoreceptors, the RFE, ihe Brueh's membrane and' the eharipcapiilaris, 2014208236 31 M2014

The bleaching proeedufe is a critical clement in the usefulness and utility of methods used to measure dark adaptation: and in other psychophysical tests,. Although it is well 5 known that edneS: (the photoreceptors in the retina primarily responsible for photopic sensitivity) and, rods (the photoreceptors in the retina primarily responsible: for subtopic sensitivity) have different spectral response curves, existing plmioWeaching protocols used: in psychophysical tests ,such as dark adaptation and;dark adaptoraeters and other instruments used in such psychophysical tests invariably :rely on white, (achromatic) or very broadband 10 light to achieve the desired photob teaching. Furthermore, all or a major portion of the retina area is photobleached, and the bleaching MghtTntcnsity is set above anrhient daylight (i.e., it has an intensity above the intensity of ambient daylight). The use of achromatic light, bleaching of all or a majority of the retina during the photob!caching process and the use of higher Intensity bleaching lights can increase the duration of the psychophysical test, such as IS dark adaptation, can increase patient burden and discomfort during testing and can lead to greater iesi-tb'test variation and/or measurement bias caused by variable lens opacity or other iuctors, wi th corresponding pmblerns in inforpmtation of the psychophysical tests. The chromatic composition of the bleaching light, the portion of the retina area that is photohleached andlfoeihle^hingiafonsHy;.cim' all have profound affoets on the duration of 20 the test, patient burden, test-to-test variability and measuremeitt bias.

Therefore, the art is lacking an improved method of photobleaching for use with psychophysical tests, such as but not limited to, dark Captation, and for use with instruments Used in implementing such psychophysical tests. The present disclosure provides such an improved method of phofohleaching, almig with bleaching Hghts for use in 25 the disclosed methods, and exemplary devices incorporating such bleaching lights and suitahle for use in practicing the disclosed methods. Such disclosures were not heretofore appreciated in the art.

BRIEF DESC RIPTION OF THE FICORES FIO: 1 shows various peak absprptfon profiles for the S, M and L cones and rod 30 photoreceptors. 4 FIG, 2 sho ws a theoretical iilustmtiopof accelerating the rod-c<me break in a dark adaptation 2014208236 31 Μ 2014 curve by oriug a phombleaehing light emitting a light consisting esseniiaily of a range of wavelengths that prefeentially bleaches the rod photoreceptors. FIG, 3 shows a theoretieal illnstfation oF.a^hl^{ihf#ie;^d^oafehre^k:tn a dark adaptation 5 carve fey using a photobleaehlhg light emitting a light consisting essentially of a range wavelengths that complement a target stimulus having a specific wavelength of light. FIGS* 4A-D show a comparison between dark adaptation Curves generated using a photobleaching light emitting an achromatic white light comprising a broad range of wavelengths from about 400 to about 700 nm and dark adaptation curves generated rising a 10 pbotob 1 eaching tight emi tting a tailored spectrum of wavelengths centered on only the blue, green and red portions of the achromatic white bleachinglight. FIG. 5 shows the results of a: preference test conducted comparing a photobleaching light emitting an achromatic white tight comprising a broad wavelength spectrum of about 400 nm to about 700 nm and a hlcacMng light emitting a tailored spectrum of light consisting I S essentially of wavelengths of about 400 nm to about 510 not (green spectrum). FIGS. 6A-0 show a comparison between dark adaptation curves generated using a photobleaching light emitting a tailored spectrum of light consisting essentially of wavelengths of about 490 nm to about 510 nm (green spectrum) and ahhotohleaehihg light, emittihga tailored spectrum of light consisting essentially of wavelengths of:about 440 nm 20 to about 460 nm:;(blue specfrumh both with and without a blue absorption filter in from of the test subject's eye to simulate lens opacity, FIGS, 7A and B show· a comparison between dark adaptation curves: generated using: a photobleaching light having an;: intensity above the intensity of ambient: daylight and a photobleaching light having an intensity below the intensity of ambient daylight. 25 FIGS. SA and 8 show a comparison between dark adaptation curves generated using a photobleaching light emitting an achromatic white light comprising a broad wavelength spectrum of about 4()0 to about 700 nm and a photobleaching light emitting a tailored spectrum of light consisting essentially of wavelengths of about 490 mo to about 510 nib 5 (greeo speetraii!), for both normal test splpeets and lest subjects having age-related mactdopathy (ARM), 2014208236 31 Μ 2014

DETAILED DESCRIPTION

General Description : 5 Rhodopsin and cdiie pigments are the visual piginepSiebiitained in the outer portions of the rod and cone photoreceptors of the retina, respectively, As the visual, pigment absorbs light, it breaks down irsto intern icdiate moleeuia r forms and initiates a signal that proceeds down a tract of nerve tissue to the brain, allowing for the sensation of sight. The outer Segments of the rods and' cones: contain: large amounts ofThese pigments, stacked in layers 10 lying perpendicular to the light incoming through the pupil. There are live typ-ek of visual pigment in the retina, with slight differences thatallow for dlfierenees in; visual perception, Rhodopsin is the visual pigment in the rods and allows for scotopic vision, Rhodopsin in the rods absorbs light energy in a broad band of the electromagnetic specteUiplfeaitmg1 at 505 urn. There arc throe types of visual pigments in the cones, each with a slightly: different peak 15 ablorpilbn: short wavelength (S) cpnedliave atspeeirdl response peaking around 419 nth (the blue spectrum)*, medium wavelength (M) cones have a spectral response peaking around 531 nrn (the green spectrum), and long wavelength (L) Cones have a spectral response peaking around 550 pm (the red spectrum). The visual pigments in the cones allows for photopic visiim The various peak absorption profiles for the S, M and L cones and rod 2p photoreceptors are shown ip FIG. 1. Furthermore, about 1% of human retinal ganglion cells are photoreceptors. These light sensitive ganglion cells contain melanopsin photopigraenh which have a spectral response peaking; around 4S0 nrn. These ceils are thought to help regulate circadian phofoeritrainment. Depriving these ganglions pells of 460-n.m light is hypothesized ro disturb sleep/wake cycles ip humans; 25 The following is a. description of the biochemistry of rhodopsin, although the biochemistry of the code pigments mid melanppsionis thought to be Very similar. Rhodopsin consists of 11 -ciswetmal and the protein opsin, and Is tightly hound ip the outer segment of tire rods, 11- cls-retinal is the photOreaetive portion of rhodopsin, which is converted to dll·· trans-retipal when a photon of light in the active absorption band strikes The molecule. This 30 process goes through a sequence of chemical reactions as 11-eispetihal, isonrerizes to all· 6 trans-retinal. Paring ^s^ri^sirif/ch^Eiic-aJ steps, thenerve fiber, whi^ to that 2014208236 31 Μ 2014 particular t&amp;dfor .cobs; * stimulus that: is ultimately perceived in the brain as a.

MsMal;s|gna1/Fo1l0>vihg''th©breakdown of31-cis·retinal 11-eis-retmai is regenerated by a series of steps that result an llrcis-retinei being recombined with: opsin 5 protein, The isomerization: to ali-trmis-reiinaiis the reaetion that ocetirsriuring: the bleaching process.

The present disclosure provides an improved method for photobi caching an. eye: ;of'a, subject, The improved photoblegching process is achieved by using; at least One of the. following modifications to prior art bleaching protocols; (i) the rise of a bleaching light :10 emitting: a particular wavelength of light or a tailored spectrum of wavelengths; (ii) restricting1 or otherwise spatially tailoring the region of thp: reiina that is subject to phofobleaehmg; and (hi) utilizing: ahleaching light having anrintcnsity that is:at or below the intensity of ambient daylight. Stich improvements; to the prior ari bleaching protocols for use ip psychophysical tests, whether alone .or in various combinations, have not been 15 previously appreciated in the art. iLik^$eC'Hi§tt!Ments':::Using'.:th&amp; improved photobleaching methods and light source disclosed herein and improved instruments for administering psychophysical tests are: also provided.

Using the improved photpbleaehing methods described herein, certain disadvantages associated with the prior art phpfobleaching methods are reduced or eliminated, resulting in a .20 psychophysical test that is more efficient to administer and is shorter in duration. Furthermore, the patient burden and patient discomfort using the unproved photohieaehing method described herein is significantly reduced. Finally, the impr^ described herein increases the accuracy and reproducibility of psychophysical tests by reducing test-to-test variation' add tneasurenient bias paused by pre-existing conditions. '25 Therefore, such psychophysical tests are more appnr^e:nnd,^^br:::^:'^terpret.

The improved bleaching method; deseribedlhereiu. can be used in. any psychophysical: test or other testing: procedure whCro photobleac©^ subjecTs eye is required. The present disclosure describes the: use of the bleaching methodidiselosedin conjunction; with measurements of dark adaptation as;;one example of application. However, the teachings of :3P the present: disclosure slmtdd hnfbelimited to the Use of;iheihIbaChinglnteth.odS described to 7 measurements of dark, adaptation or any oilier siqgle psychophysical test. The teachings of the present disclosure may be used: in confomatipn with any visual function: test or any psyehophysieai; test known in the art that requires bleaching the eye of the subject, or- a< portion thereof. 2014208236 31 Μ 2014 5 Psychophysical tests measure a subject's sensation arid perception: of physical stimuli. The sitruuK can be visual·, auditory, oifaetdry, tactile or gustatory, hhfual stimuli ? include, for example, varying intensities of light, diSejiflg;co&amp;^ of text!

Psychophysical tests using visual stimuli include, Bt example, dark adaptometry, visual sensitivity tests, acuity tests, contrast: sens ittvify tests, flicker ptoipmeiiy, ΊΟ photostress tests. Vernier acuity tests, colorimetry, motion detection tests, object recognition, and perimetry. Psychophysical tests can be used to assess the status of visual funetions including, for example, dark adaptation, photepie sensitivity, seotopic sensitivity, visual acuity, color sensitivity, con sensitivity, color discrimination, and visual field. Psychophysical tests can be used to diagnosis the risk, presence or severity of eve diseases is including, for example^ age-related maenlar degeneration, vitamin A deficiency, Sorsby's fimdus dyslrophy, autosomal dominant latsfonset degeneration, rod-eons dystrophies, color blindness, ocular tumors, cataract, diabetic retihopathy, and glaucoma.

The improved photohldaching methods described may be used iu:: a: variety of protocols, as would be ob vious to pile of ordinary skill in the art. As shell, fiis 'exact protocol. TO : used with the described photobleacliing methods may be varied as is: known in the art.. The goal: of the photobleaching procedure in a psychophysical test (such as, but hot limited to, dark adaptation) is to precondition the test eye of a subject, or portions thereof by desensitizing at feast a portion of the visual pigments of the test eye through exposure to a photobleaching light. In dark adaptation, for example, visual recovery of seotopic vision is 25 then measured as ifte test eye adapts to a second light (often referred to as the target or target sthnulns). Thereft>m, the phoiohleaching light serves as a standardised baseline ifotn which visual recovery is measured. Therefore, the photobleaching step Is of importance to psychophysical: tests since it plays a role in establishing a baseline for the tests, Funhennore, depending: on the nature of thcrphotobieacfdng: method used, the Time required :30 to complete the psychophysical test, the patient: burden and patient discomfort, and the mproducibihiy,and/or aceiuacy of the psychophysical test may' "he' .impacted, 8

In one embodiment for dark adaptation, the phbfobleactnng light has a greater intensity than the target stimulus but the absolute intensity values^ of the phoidbleaching light and the target stimulus may be varied as desired. Generally, th# greater the ahsbliito value of the intensity of' the photobieaching light, the shorter the period of exposure of the;: (5 'test eye to the photobieaching light to achieve the baseline· Ifor bvdfopie,/thO'phptfbleaching: light ruay be an intense Sight, such as that provided by an electronic strobe or flash, and Che; light of: the intensity of the target stimulus mav be at or close to 0 edfof ,sueh as ννούίίί oecur in a dark; room, Alternatively, the photonleaching light may he a: light produced by an ordinary light: bulb or:by the ambient :light in a room, and' the intensity of the target stimulus: 10 may be at or plosefo 0;cdM:2ysuch as;: would occur in a dark room, However, in general, the: greater foe intensity of thephotobleaehmg light, the longer the psychophysical test takes to administer. 2014208236 31 Jul2014

The wavelength of light emitted by the phoiohleachlng light may also be varied. While the prior art methods utilised; sh achromatic bleaching; light having a broad band 15 speeirntn of wavelengths, the present disclosure describes photobieaching methods that .utiWise -aipho'tQbJeaehing :.H||ltt":tai1ored:to emit alight of a particular wavelength or a range of wavelengths of the visible specif tup so that light o f only a particular wavelength of range of wavelengths is used in the bleaching process, in one embodiment, the -particular wavelength or range of wavelengths is selected to match the specific absorption: spectra of the rod, cone 20. apdfof retihai ganglion cell photoreceptors. As discussed abdv0, rdds: absorb light in a broad band of:foe; spectrum peaking; at a; wavelength of 505 m%;while the three types of;cone photoreceptors have:spectral responses peaking around 419;nni: (S eOnes), around: S31 nm (M cones), and around 558 nm (I, cones) and the retinal ganglion; ceils; absorb light having: a, spectral response; peaking around; -460 nm. Therefore, In ;one embodimeitt, the 25 .photobieaching light may bo selected to stimulate one or more of the rod, cone; and/bf retinal ganglion cell photoreceptors by utilizing a pliotobleachingfight emitting a wavelength Or a range: of wavel engths: based; on the Spectral responses of the photoreceptors.

3Q

The; photObiehchiiig light emitting ;a particular wavelength or range of wavelengths: of light may be generated by an achromatic light source eqaipped with ayuttable filter, such, as, but not limited to, a nafoow-band pass filter, a high pass filter (eliminating; lower wavelengths) or a low pass filter (eliminating thehighwavelengths). A variety of harfow- 9 band pass filters, high pass filters and low pass fillers are commercially available and one of ordinary skill in the art would be well versed in the selection of the appropriate biter based on the test conducted and the results desired, Alternatively, the photobieaching light of a particular wavelength or range of wavelengths may he generated directly by a source 5 generating the desired wavelength or wavelengths (such as, but not limbed to, light emitting diodes, IdSDs, or organ i e light- emi tting di odes, DL EDs). 2014208236 31 Μ 2014

Many Sight delivery methods can be used to generate and/or deliver the photohS caching light In one embodiment, the photpbieaehing ligbt is generated by a xenon lamp, ah are: lamp, a: tungsten: bulb, a photographic flash, a LED or DLED light source. Other io possibilities include the use of display technologies such as cathode ray tubes (CRTs), plasma displays and LED bisplap. Other; sources may also bo used to generate the photObledchiugi.lights As discussed above, the light sources may be equipped with filters or Other· devices to emit and/or generate light of a specific wavelength Or range of Maveiengths. The photobleachihg light may be deli vered using a variety of techniques as welf such as but IS not Sipiidd. id, adapting Helds, illuminated backgrounds, direct projection into the eye, exposure to ambient light, or staring into a light bulb. Classically, subjects viewed an adapting field In photobleachihg methods. This bleaching method causes discomfort to the subject, and it is difficult to reliably deliver bleaches in psyehophysicaliy inexperienced subjects; Another method of bleaching is to project light into the eye using a Maxwellian 20 view system. Ibis: method cahSes less imfatibn, but requires the subjects to fixate very Steadilyarm notblink far 31)m 60 seconds. Many inexperienced subjects find this to be a difficult task. If the subject changes fixation or blinks, it is necessary to wait itp to two hours before the bleaeb is repeated to avoid the cumulative effects of bleaching. Bleaching light delivered by art electronic strobeor flash delivers the phmpbleaeMng light in a short period 25 of time. In addition, the intensity and/orwavelengthor range of wavelengths emitted by the bleaching light may beeasily mpdUlafed. In addition, the use of masks or similar devices allows the bleaehing light to be of a desirable size and positioned at a desired location. Because the light exposure is brie^ the intensity and/or wavelength(s) of the photobieaching light can be emitrolled and can be localized to a deSired area, the photobieaching light is not 30 irritating do not need to maintain fixation for a long period of time. With proper patient instructions blinking is tipi an issue. 10

The photoMeaohing ..light may be delivered to a desired portion of the retina. Using the delivery methods described above, it is possible to deliver i%rphotoi?leacKtng:'Il^lt to a single discrete area of the retina or to more than one discrete area of the retina during; a single test. By selecting a particular area or areas of the retina: to be bleached by the 5 photobleaching light, patient discomfort can be minimized by avoiding sensitive areas of the eye such as the fovea, in addition, depending on the goal of the test to be administered, a specific region of the retina may be selected for pliQiobleaching. For example, when administering a pssohophysical test for dark adaptation of other rod mediated fonefion, it is mot; required to bleach the fovea since there are no rod photoreceptors in the fovea, ,10 Therefore, photobleaching a desired area or areas of the retina outside the fovea is: advantageous, Finafly:, by pbotobleaching more than one discrete area of the retina; not only are the above mentioned advantages obtained, but in addition, different areas of the retina may be tested foofotpr disease progression and/or to: get differential 2014208236 31 Μ 2014 measurements Ifotn areas havihg or suspected: of having greater or lesser dTsfoneion or to IS increase the statistical accuracy of the test results by providing more than one reading.

As discussed above; the photobleaehing; protocol desensitizes the desifod amonnt of visual pigment in the rod:, cone and/br retinal ganglion cell photoreceptors by exposure te a photobleaching light and pfovidds a standardized baseline to measure visual recovery. The intensity of the photobleaching: light, the time of exposure to foe photobleaching light and/or 20 the waveiebgth(s) Of the pbOtobleaching light caiT be rhoduiated fo produce the desired amount of d^ensidzatioi*. one embodiment, an equivalent ofaboi.it 50% to 10b%: of the visual pigment/m/foe: area subj'eettO'Bhotdbis^tblliftg is desensitized. Fhe intensity of the photobleaching lightcan be athusted tp desensitize the appropriate amount of visual pigment in the area subject to photobleaching. For example, a photobleaching light 25 intensity of 7.48 log seot Td sec's will bleach apppximately 08% of the rhodopsin molecules, while a phmpbleacMng light intensity of 5.36 log seot Td see* will bleach approximately 50% of the rhodopsin molecules. Alternate phohfofeaching fight intensities which desensitize less than 50% or more than 50% of foe rhodopsin (or other visual pigment) mpfeeulespay also bd used i f desired. 30

After the bleaching protocol, visual recovery is monitored. In dark adaptation, for example, this recovery is mediated primarily by the mrina and measures predominately rod- 11 mediated scotopie .sensitivity,; Although many: methods to monitor rod-mediated .scolppid sensitivity are known, generally, the- subedit ·ρΓρν$^8,··8.,«^ρβ§ of responses to a target stimulus (winch is varied in intensity, location and/or'Wavelength as described herein),, in one method, the response of the subject is rtsed to determine a threshold measurement. 2014208236 31 Jul2014 5 Doring threshold measurements, the subject is presented with a target stimulus. The target stimulus may be a spot of light, including a light spot on a darker background or a d|tk spot on a lighter background. Subjects may view the target stimulus with or without their best optical correction lor the test distance, A variety of classical methods can be used to determine the threshold measurement, including hut not limited to method of limits, just to. noticeable difterenee^ and method of adjustment, These techniques are well known in the art Thmsholds measurements can be sampled in such a way as to provide mil St models of darii; adaptation. In one embodiment, threshold measurements are sampled dnee every I to 5 minutes. Another embodiment would be tp sample threshold measurements twice every minute. Yet another embodiment would be to sample 2 threshold: IS measurements pet minute early during the test then Sample l threshold measurement, every 2 minutes thereafter. Higher or lower sampling rates may be used as desired to balance the need of producing an adequate dark adaptation function lor model litting agairist sulpect burden, As an example of lower sampling rates, a small number bf threshold measnrmnents may be sampled based on predictions of rod photQu@^^::fdpP!%Il: Arc normal individuals, 20 For example, a threshold measurement may be obtained at 3-5 minutes fwhieh using classical pboiobleuch|ng and targpt; stimulus parameters in nonna! individuals would be before the rod-edne break) and at 5-11) minutes and 10-15 minutes. If these threshold measurements ho hot correlate with the rod photoreceptor function in uonnal individuals, the sulpect is likely to have impaired dark adaptation. Such a sampling schedule would ftufhpr 25 reduce subject burden. Additional description of methods and apparatus used in phoiobleaching methods and methods Of analysis for determining the dark adaptation status of a patient are described in U.S, Patent Application Horil)/5? 1,230, which is hereby incorporated by relererice. 30 In one embodiment of the phefobleaching method described herein, the phoiobleaching light is tailored to emit a spectrum consisting essentially .of a selected 12 2014208236 31 M2014 25 wavelength or range of wa^elengihs of light rather than an adnomatie phpfebieaehing light having a bread range of waveienglhs^ to-naany- j^yohopftyskai teste., such as, but not limited to, dark adaptation, it mag be advatitageoua to choose a phptobleadtirig light tailored to emit a spectrum consisting essentially of a desired wavelength or a range of wavelengths that reveal the rod-mediated feotopic sensitivity as quickly as possible. Alternatively, it may be advantageous to choose a photobleaehiog light tailored to emit a spectrum consisting essentially of a desired wavelength or a range of wavelengths that provides a clearly visible red-eone break as a characteristic benchmark for dark adaptation.

In a particular embodiment, the photobleaching light tailored re emit a spectrum 10 consisting essentially of a desired wavelength or a range of wavelengths selected to preferentially photohleach the rod photoreceptors, the cone photoreceptors and/or retinal ganglion cells. For example, the phofeb leaching light may be selected to preferentially bleach the rod photoreceptors. In such an example, the photobleaching light Would emit a spectrum consisting a.waveiep^lfipf %Μό£..5.05 nm or a range of wavelengths IS of light centered on 505 am. As used herein, the term “centered” on a particular wavelength means the photobleaching light contains the particular wavelength of light and a range of other wavelengths -of 1ight|r$m^fbnm on either side of the particular wavelength; the term centered should not be interpreted to mean the range of wavelengths is symmetrical about the particular wavelength. In the above example, light consisting essentially of arange 2d of wavelengths centered on 505 nm could include, tor example; wavelengths of light from pm: fiS.-iuto on..either side of 505 nm), from 490 to 510 m or irem 490 to 525 nnu In another example, the photobleaching light may be selected to preferentially bleach the S cones photoreceptors. In such an example, the photobleaching light would emit a spectrum consisting essentially of a wavelength of light of 419 nm or a range of wavelengths of light centered on 419 nm. In yet another example;, the photobleaching light may he selected to preferentially bleach the M and: h cone photoreceptors while leaving the rod photoreceptors relatively unaffected. In such an example, the photobleaching light would emit a spectrum consisting essentially of a wavelength- of light of 650 nm or a range of wavelengths of light centered on .:650 nm, dr aiicvnately afrroadnmge of wavdiengths of light from about 60O nm: to about 700 lira.

Other embodiments may also ho envisioned. For example, when desired to prefereniiaily bleach the visual pigment in the retinal gang! ion eehsrfephotobieaehmg light may be tailored to .emit a spectrtun consisting essentially of a wavelength of light of 460 nm or a range Of wavelengths of light center eel oil 460 run, such as but not limited do, about 450 S to about 470ηιη. 2014208236 31 Μ 2014

In a further example,;the photobleaehing light may "be tailored to emit a spectrum consisting essentially of a wavelength of light or a, range of wavelengths of light; over about 480 nm. Siieb a spectrum of photohle&amp;chiiig: light may be . used to exclude wavelengths of light in the biue: spectra to reduce variability and confounding effects introduced by lens 10 opacity.

In vet another example, the: phoiobleaching light may be tailored to emit a spectrum, consisting essentially of a Wavelength of light of about 41:0 nm or centered on 410 nm, such:: as but not limited to a range of about 400 fi about 420 hilt. Such a spectrum: of photobleaehing light may be used to maximize; absorption due to:le»s opacity. 15 In still a further example, the photobleaehing light may be tailored to emit a spectrum consistmg essentially of a wavelengtlr of light of about 570 nm or centered on 570 nm, such hs but not limited to a range of about 560 to about 580 nm. Such a spectrum of photobleaehing light may he used to minimize absorption dueto lens opacity.

In yet another example, when a target stimulus is used, the photobleaehing light may 20 be tailored to emit a spectrum that matches the spectrum of the target stimulus. When it is desired to accentuate the rod response, the spectrum of the photobleaehing light and the target stimulus may he tailored to emit a spectrum consisting essentially of a wavelength of light of about 500 nm or centered on 500 nm, such as but not limited to a range of about 490 to about 510 nm. When it is desired to accentuate the cone response, the spectrum of the 25 photobleaehing light and the target stimulus may he tailored to emit a spectrum consisting essentially of a wavelength of light of about 650 urn or centered on 650 nm. such as but not limited to a range of about 640 to about 060 nm, in a forther yanation, the photobleaehing light may be tailored to emit a spectrum that does not match the spectrum of the target stimulus. 14 |ρ one version of this embodiment, rather than mi Sizing an achromatic or broadband bleaching Sight, a dark adaptometer can. be configured: to preferentially photobteacir the:rods^ This coaid be accompli shed, for example, bv placing a band pass filter narrowly centered on 505 nm over a broadband xenon arc flash or other fighf source -^diijSiDg-^'resuibhg-iE^jbw 5 spectrum emitted light as the bleaching source, Alternatively, the bleaching light could he eonfi giued to preferantiaily phOtobleaeh rods by eohstructing a Mnk of one or more light-emitting diodes (LEDs), organic light-emitting diodes (DLEDs) or other light source of a single type having a characteristic emission spectrum cSose id 505 nm. Other possibilities include the: use of display technologies such as cathode my tubes (CRTs), plasma displays 10 and LEO displays. Utilizing a bleaching spectrum; that: is tailored to prefemntially photobleach the rods oilers several advantages. Therefore, lihe:1 phptobleaehidg light is taifored to emit a light consisting essentially of a desired wavelength or range::of wavelengths oElighf 2014208236 31 Μ 2014

As discussed above, the photohleaching: light emittmg a desired wavelength or 15 spectrum of wavelengths may be generated using a variety of methods^ For example, a light source equipped with a suitable filter, such as, but not limited fey a narrow-band pass filter, a high pass filter (eliminating lower wavelengths) or a low pass filter (eliminating the high wavelengths). A variety of narrow -band pass filters, high pass filters arid low pass filters are eommereially available and one of ordinary skill in the art would be well versed in the 20 selection of the appropriate filter based bit the test conducted and the results desired. ^ernafively»-'-tH.e ''photobfoaehing light of a particular wavelength or range of wavelengths may be generated directly by a source generating the desired: Wavelength or wavelengths (such as, hut not limited fo, light emitting diodes, LEDs, or organic light-emittihg diodes, DLEDs). 25' Using a photohleaching method incorporating a photohleaching light tailored to emit a desired wavelength or range 6f Wavelengths hafi:Several adfaniages. A first advantage is the ability to administer a psyehophysical test, such as, but not linfited to, dark adaptation, in a decreased amount of time, thereby increasing the efficiency of the test operator and minimizing patient burden. When an achromatic or broadband bleaching light is utilized in a :38.: photohleaelung method, all of the photoreceptors, both rod and cone, are: strongly bleached. Cones recover more rapidly than rods. Nevertheless, daring the initial post-bleach period the

IS sensitivity threshold is still dominated by the cotie recovery, and the important rod seotopic sensitivity recovery mfonpation is obscured. However, Using a phoiohleaching method incorporating a. photobleaching light tailored to emit a desired wavelength or range of wavelengths can miuimife the bleaching of photoreceptors whose fonction is not being 5 tested. For example, using a bleaching light consisting essentially of a wavelength of light of 505 nm or photobleaching of foe foree tone photoreceptors is 2014208236 31 Μ 2014 minimized and they are only weakly photobleached. As a result, the cones recover more rapidly, and the important rddvmediated scofopie sensitivity recovery infbfmaion is more quickly revealed fsce FIG. 2.f TIG^ 2A is a theoretical illustration of a dark adaptation 10 curve for a normal individual obtained using abroad, achromatic phmobleaebing light and a 505 lira stiMuMs target. Cone recovery and rod recovery axe bofo exponential. Sedtopie sensitivity is cone-mediated until the none recovery plateaus to reveal the ultimately more sensitive rod-mediated response. PIG. 2B: is a theoretical illustration of a dark adaptation curve for a normal individual obtained using a photobleaching light emitting W range of IS wavelengths centered on 505 run and a target stimulus:»f 505» The cone recovery reaches its plateau essentially instantaneously and the rod recovery is more rapid foan for the conditions of FIG. 2 A, inore t|uickly reaching the rod-cone break and revealing the subsequent rod-raefoutedyecdyery'.' A second advantage is reduced patient burden during the test. Visual discomfort 20 from bright lights is mainly associated With the short wavelength portion of the visible spectrum. As illustrated in Example 2, using a photobleaching light having a wavelength of 505 nm or a spectrum of wavelengths centered on 505 nm reduces patient burden by eliminating the most imtaiiug short w&amp;velCngdi components of the light Furthermore, the cone photobleaching associated with ifo achromatic or broadbMd phofobleaching light 25 creates a more persistent after image,which in turn causes the light of the target stimulus to be less salient and makes die test more:difficult for the patient» A third advantage is reduced measurement Mas due to variation, iii foe lens opacity of the patient. Wi th aging or in the event of cataracts, foe lens in foe eye becomes more opaque and preferentially· absorbs light af short wavelengths (i.e., 480 nm and below). With ah 30 achromatic or broadband bleaching light: that contains a signibcant short wavelength component, Variable lens density between otherwise similar sufoects Causes variability in the .16 phoiobleaching achieved, and in turn an artificial variability in the measured dark adaptation. By using a photohleaehing: light tailored to .omit a desired wavelength or spectrum of wavelengths that do not contain the shorter wavelengths, such variability is reduced. 2014208236 31 Μ 2014

Using a photobleachiitg metlhtd incorporating a naoowdband pass photohleaching light other than 505 nm or a range of wavelengths centered oft 505 panΑ$Ιί'm»amizd:or maximize the degree of the above described advantages, depending on the wavelength or range of wavelengths chosen. In additidn, at least some of the advantages described above (such as lowered patient burden and wd ueed bias due to lens opacity) can be obtained by use of a high pass filter to eliminate spectrum to rather than a narrow-band pass filter, aitbpngh a narrowband pass filter ipay also be used. fir yet another embodiment, further advantage may also be obtained using a fthotobieaobft^it^thM'::fttilizinga ^^#|l^ii^fii^ftaibr^.|b emit a desired wavelength or spectrum of wavelengths selected to compfement a target stimulus of a specific wavelengthfs) of light. For example, by combining a photohleaching light consisting 15 essentially of a wavelength of light of 560 nmpr a range of wavelengths ©flight centered on 5.6fi:«m:'Virith:.a-:tai^^':Mmu.his..i6onsiSti«g essentially of a wavelength of light of 450 nm or a range of wavelengths oflight centered on 450 nm, it k possible to obtaina rapid assessment of the rod-mediated scotopic sensitivity recovery fi.e.. dark adaptation). Such a photohleaching light will: only weakly photoble.ich the S cones, but: will strongly 20 pliotobleach the M and L copes as well as the rods. Conversely, all of the $, M and L cones as: well the rods are-strongly responsive to such a target stimulus. Given5this combination,: during (he initial: portion Of the rod-mediated scotopic Sensitivity recovery the S cone: response will dominate the.: "M and L cone responses, ; apd rapidly saturate at the short wavelength cone plateau until the ultimately more sensitive rods take:over. This provides a 25 clear rod-cone break (the point at which sensitivity recovery transitions from being; cone dominated to feeing rod dominated) in the threshold curve, Ap :illustration is provided in FIGS. M; :ahd B, FIG, 3A 'is afheomical iliustration of a dark adaptation curve lor a: normal; individual obtained, bsingAbroad, achromatic bleacbingtllght htid a 505 nm target stimulus. Gone recovery and rod recovery are both exponential. SeoiopieAensftmty is eone-ftiedi&amp;ted 30 until the cone recovery plateaus to reveal the ultimately more sensitive rod-mediated response. FIG* 3B is a theoretical ilhistratiOn of a dark adaptation curve for a normal 1/ individual obtained ;bsiog;:a p h o tob 1 e aching light epiitting a range of Wavelengths centered on $0 nm and a stimulus target centered on ;4S0 nm, The cone recovery plateaus at a higher level and the rod recovery i$ more rapid: than, for the conditions of FIG. 3A, more quickly r^ch|pgdhe-rod-co^o::feak::ap|^yi^feg,^::^l!^!?P4tttll^«^dld^d^ovory» 2014208236 31 M2014 5 in an alternate embodiment of tire photobleaching method described herein, the bleaching light is restricted,tp; a portion of the retina so that only a portion of the retina is phoiobleached. The area of the retina to fee photoy^Aedimtay^l^iected based on the particular test to be -administered, the results desired^ Or the nature of the photoreceptors desired to be photobfeaehed; furthermore, the area of the mtina to be phoiobleached may fee 10 selected in order to maximize diagnostic sensitivity for a particular disease andfor to minimize patient burden. A combination of the above factors may also suggest ci?rtain portions of the retina to fee phoiobleached. The postiiobing of the bleaching light to a desired area of the retina can be aecppiplished, for example, by an. appropriately located and sized mask over the bleaching light or the bleaching light could be projected onto only the IS desired region of the retina. In addition, a fixation light or other element and/or a restrairih such as, but not limited to, a chin rest or bite bar, couM be used in combination with foe ffoegoihg fororient the patient's retina to allow precise placement offoe bleaebing light on a desired portion of the retina.

In a particular embodiment, ip a psychophysical: test for dark adaptation, it may be 20 benefieiai to festribt ppplicatjbhof thq: bleaching light to an area of the parafovea and avoid application of foe bleaching; light to foe- fovea, application of the bleaching light to the fovea causes greater irritation than light: directed at regions of the retina outside the fovea*, such: as, butfoof-hfolted to, the patefoy-tsfo· Ftfoherinpre, there are no rod photoreceptors in the fovea, so bleaching foe fovea will not; contributeTfe1 Assessment of rod-mediated function. 25 In addition, some diseases that are associated with impaired dark .adaptation· exhibit": greater or lesser impairment depending op the region Of the retina tested. In the ease of age-related macular degeneration, for example, AlyiD-reMed impairment of the rods is greatest near the fovea and: decreases as: a function: of eccentricity towards the peripheral retina. It is therefore possible to monitor disease progression; by detenfohmg the patient’s dark; :30 adaptation status at several points of the retina,as a function of eccentricity towards the 18 peripheral retina. Therefore, byvbefeMveh photobleachmg only desired 'areas of the retina withdiffeteot degrees of eccentricity, the proyresMon of certain diseases can be monitored, Iii: such embodiments, several arbas bf the retina with different degrees of eccentricity can be photobleaehed si one time, with the patient’s dark adaptation status being determined for 5 each region of the retina that is photobleaehed, for example by interleaving threshold measures at the-multiple regions. As is obvious, the di fferent regions of the fotipa could, also be studied independently in completely separate tests, 2014208236 31 M2014

In a particular embodiment suitable for the testing of dark adaptati0ii»;:.®e-.f6igdh of the retina that is photobleaehed is restricted to a small focal area equal to of visual field 10 centered at 5° in the inferior visual field (in the macnia but outside the fovea), with this beaching region being only moderately larger than the target stimulus tight spot This choice of bleaching region offers: several advantages. For one, patient burden is minimized, both because the fraction of the retina being photobleaehed is small and because the region selected excludes the fovea,which is the portion of the retina most susceptible to irritation, IS Avoiding the fovea also allows the patient to maintain fixation easier during the test, which is critical for test reliability. For another, diagnostic sensitivity for-AMD; is optimized, because AMD-related impairment Ofdarkadaptation is greatest intlns region of the mtina.

In other embod iments, the pbotohl caching light photobleaehes a portion of the retina as set forth below. 20 In one example, the portion of the retina exposed to the photobleaching light is an area of the fovea, an area of the parafovea or acombination of the foregoing. The portion of the retina exposed to the photobleachmg. li|hTi»ay he located entirely inside the; fovea tat about 0° to abohf 0;5S eccentricity). SuehlqeabzaiiOn would allow photobleaching primarily of the cone photoreceptors and may be useful in such psychophysicSl; tests :as Color 25 sensitivity or color discrimination,. The portion of the retina exposed to the phoiobieaehmg light may he located entirely inside the macula (at about T* to about ItF eccentricity or at about 3V> to about 10° eccentricity). Such localization; would allow photobledchihg primarily of the rod photoreceptors and may he USefifi insuch psychophysical tests; as dark: adaptation. Furthermore, the portion of the retina exposed to the photobis-achmg light may be located in

Ft the peripheral retina(atabout1Θμ to about 30!? eeceotneity). Such localization may be useful in such psychophysical tests Us visual field of perimetry. 2014208236 31 Μ 2014

In another exanrpfe, the portion of the retina exposed totile photobleae^ be an annular region completely excluding the fovea. In a specific example* the anrmiar 5 region may have an Inner edge located at or outside about T eccentricity and an outer edge located at or inside about 10'* eccentricity, ;Sneh: localization; would allow primarily bleaching of the rod photoreceptors ns di scussed abpvc. in. a further examples the portion of the retina exposed to; the, photobleaehing;;: light covers an area of about 4'’ of visual field to about of visual field. Such an area allows a 10 minimum ettectiye. area of the retina to be exposed to photobleaehing while providing a phetobleaehed area that can be effectively exposed to the target stimulus. In another example, the portion of the retina exposed to the photobleacbing light is eo-loeated with the portion of the retina exposed to the target stimulus and the portion of the retina exposed to the bleaoMhg light being from about 1 to about 4 times the area of the portion of the retinal 15 exposed to the target stimulus/In a specific example, the portion of the retina exposed to the photohleaching light is about 3 times the area of the portion of the retinal exposed to the target stimulus.

In still a further example, the portion of the retina exposed to the photobleaehing light is: located on the inferior vertical meridian or the superior vertical meridian. Such 20 localization allows for symmetry1 between the right; andleft eye.

In yet anotherexample, thp:portion of thetretiha exposed to tite photobleaehing light has a distinctive shape. In certain eases, the photobleacMng process: piav produce an after image:. When a target stimulus is used, sudt as in: conjunction with a psychophysical test, the subject may confuse the after image with the target stimulus. By providing a distinctive 25:· shape to the photobleaehing light such confusion is minimized. The shape may be a circle, a square, a triangle, a diamond, a polygon, a star or other shape as desired. In a specific: example, the photobleaehing light and the target stihiulus have different shapes, if desired:, color may bo substituted lor shape, or both color and;shape may be used. in yet another altsniate; embodiment of theiphotobleaeltingvmeihod described herein, 30 the photobleaehing method utilizes a bleaching light with an intensity that ts at or below the 20 intensity of ambient' daylight levels. For the purpose of this disclosure, the intensity of ambient daylight is in the range of SO ίο 4ΐϊθ ed/rn" or 3,15 to 4.05 log soot TO see5. Prior photobleaching methods add devices utilizing such methods, especially those used for measuring dark adapiahom utilized a photobleaching light having an intensity that was well : 5 above the intensity of ambient daylight. This brute force approach was used to ensure a 2014208236 31 Μ 2014 uniform state of phofobfeaehing, or adaptation starting point, for ail pa ti ents , However,,ills also possible to ensure a unilbnn state of phombieaching with a photobleaching light having an intensity that is at or below the intensity of ambient daylight. For example, the patient eairbe taken from anrhient daylight trtto a dark room, allowed to dark: adapt brielly to a level Ip below ambient daylight, and then pheipbleached using a Hash of light having an intensity at or below the intensity of ambient daylight. Alternatively, the patient can he taken from ambient daylight into a dark room, exposed to a steady photobleaching light having an intensity below the intensity of ambient daylight until such time as the steady photobleaching light is clearly visible to the patient, thus effectively arresting dark 15 adaptation for all pahents at a epfomon starting level bdfow ambient daylight conditions. In the fatter altematiy^ the steady photobleaching light can be a randomly selected shape that the patient must identify to the test operafpr before dark adaptation testing can proceed, thereby verifyingihaf the: patient is appropriately pre-conditioned, Use of a photobleaching light with an intensity that, is at or below the intensity: of ambient daylight levels: offers: 20: several advantages, fn-paridetiiar, th§-patfobfc:burdeit is^gducecl. In addition, as illustrated in Example 4 below, die overall dark adaptation test timecao: be. shortened,,

The described photobleaching methods may utilize pnef two or all three of the above described improvements, in any combination. For example, a photobleaching method may be provided using a photobleaching light emitting a light consisting essentially of a speeifre 25 w aveiengfo pf light or a tailpred speetrum of wavelengths centered on a specific wavelength of light- In another example, a photobleaching method may be. provided using a photobleaching light eniitiingia light consisting essentially of a specific wavelength ofjighi or a tailored specifum of wavelengths: centered op a specificf wavelength of light in combination with only a particular area of the retina phoiobleaehed. In yet another example, 30 a photohicaching method may he provided using a photobleaching light having an intensity that is at or below the intensity of ambient daylight; 21

Furthermore, the described pbotobieachipg methodsmay also be incorproated into an apparatus, machine· or devises used to administer a psychophysical test that requires pboiobleaehing, such as, but not limited to a dark adaptomeier. Such apparatus, machines or devices are «11 known in the art and may be modified to meorporaie the photpbleaehmg 5 methods described herein. Such a modified apparatus, machine or device is also within the scope of the present disclosure. For example, the dark adaptomeier disclosed in US Patent No. 10/5^1,230 ebnld he modified to incorporate the phptobleaeMng methods described herein. Likewise, a photohleaebing light source capable of emitting a tailored range of wavelengths or a particular wavelength suitable to photobleach a desired population of rod, 10 cone or ganglion cell photoreceptors or a photobleaching light emitting a light having an intensity at or below the intensity of ambient daylight are also within the scope of the disclosure, as well as the use of such phbtbbleaching light sources in an apparatus, machine or deri ce used to administer a psychophysical test that req uires photobleaching. 2014208236 31 Jul2014

The present disclosure also provides a combination of a photobleaching light as 15 described herein and $*n apparatus to administer a psychophysical test to monitor a response to: the photobleaching light. The photobleaching light may he a part of the apparatus. As discussed above, the nature of the apparatus may he determined by the psychophysical test: administered. For example,dark adaptometem fbr/hipphotqnteiers) are used to measure dark adaptation and diagnose age-related macular degeneration, preferential hvperacuity 20 perimeters are used to measure: Vernier aenily and assess; the severity of age-related: macular degeneration* ETDRS charts are: used to measure: Spatial: resolution acuity, Pell-Rohson contrast sensitivity charts ^:'Ρ$#Γίρ· rnmbrm sensitivity, the Farasworth-Muhsell 100: Hue Test is used to measure: color vision, frequency doubling perimeters are used to measure: frequency doubling;visual illusion, and field analyzers are used to measure; visual 25 field:and diagnose glaucoma,

Psychophysical tests using visual stimuli include, lor example, dark adaptometry, visual sensitivity tests*, spatial resolution acuity tests, contrast sensitivity tests,:, flicker photometry, photostress tests,. Veriiier acuity tests, colorimetry, motion detection tests,; object recognition, and perimetry. The: combination can. be used: to assess the status of visual 30 .functions including,, tor example, dark adaptation, photopic sensitivity, scotopie sensitivity, visual acuity, color sensitivity, contrast sensitivity, color discrimination, and visual field. 22 2014208236 31Jul2014

Furthermore,. the combianUon can be used ίο diagnosis the risk, presence or severity of eye diseases including, for example, age-related macular degeneration, vitaminA deficiency». Sorsby’s fondus: dystrophy, autosomal dpmiifont late-onset degenefotidn, rod-cone dystrophies, color Mindness, oculartumors, cataract, diabetic retinopathy, and glaucoma,

5 EXAMPLES

Example 1 - Effect of Bieachinu Liaht Spectrum on the Shape and Kinetics of Dark

Ih this example, a comparison was made between dark adaptation curves generated using a phofohleaching light emitting an achromatic white light comprising a broad spectrum ID of wavelengths' and foafk: adaptation curves generated using: a photobl^lfogdi'^htiei^i^ihg a tailored spectmm of wavelengths centered on only the bine, green and red portions of the achromatic White photobldachiuglight.

Dark adaptation was measured using an AdaptDx dark adaptomeier (Apeliotus Technologies, Info) accofoing to the nianuiaeturer’is instructions, using methods known in 15 the art. Tie intensity of the xenon are photobleaehing light (administered as a flash) incorporated in the dark, adapiometer was set at 7:.:03 Iog:: scoi: Td sec’5 and masked to phoiobleach an area of the retina, covering , about 4* of visual angle centeredvat 6" on the inferior vertical meridian. The spectrum: of the photobleaehing: light was varied for each of .four dark adaptation «reasufomehtSfe: In one case, the photobleadhing light emitted the #0.: essentially white, 5:500 Kelvin color temperature broad, spectrum light (consisting of wavelengths from about: 400 nm to: about 700 nni)::generated by the: xenon arc source: (FIG. 4A), Ip the other'three cafes, the photobleaehing: light was tailored to emit a spectrum Of light: consisting essentially of wavelengths in the narrow blue (about 405 nm fo: about 425 nm), green (about :400 nm to about 510 uni) and narrow red (about 040 nm. to about: 060 rtm|> 25 spectrufos (FIGS. 4B~D, respectively). As Used invilie pfosenf;' the disclosure, the tern) -‘abotif* when, used in reference to; a wavelength or range of wavelengths it is meant: to encompass a rang©: of wavelengths on either side of the designated wavelength equal to the error in genentiion or 'measurenwnt of the: designated wavelength; all recitatidhs of wavelength in die prescnti speci tleation may be considered to be modified by the:term: about 30 if desired The: spectrums detailed above: were: generafocl by placing narrow bandpass intertemoce filters (Edmund Optics 19X43-158, NT43-169 and !fT43-T-89, respectively} d9er die face offfeifc&amp;jpmare Tiash window. The test eye was phofobleaeheb while the subject was focused on a fixation light to ensure dint the proper retinal location was bleached. Seofopic threshold measurements for the target iimulus began immediately after 5 pfwfobieaehoffset The target stimulus was a circular spot covering about 2°- of visual angle presented at S0 on the; inferior vertical meridian with a wavelength spectrum centered on 500 nm. Buring threshold measurement the sul^eet focused on die fixarion light and responded when the stimulns was judged to be present by pushing a button. Threshold was estimated using a 3fooWn/l~up modified staircase procedure. Starting at a relatively high intensity 10 (5;O0 ed/nfifi the target was presented every 2 or 3 seconds for a 200-ms duration. If the 2014208236 31 Μ 2014 subject did not respond the target stimulus was of the target stimulus remained unchanged until the subject responded the target stimulus was visible. IF the subject indicated foe target stimulus was visible, the intensity of the target Stimulus was decreased for each successive presentation in steps of 03 log «nits ('3foownn) until the 15 participant stopped responding that foe target stimulus was present. After the subject indicated that foe target stimulus was invisible by not pushing the button, the. intensity of the target stimulus was increased for each successive presentation in steps of 0.1 log units £Ί~ up”) until the suhjeei responded foal the target stimulus was once again visible. This intensify was defined as the threshold estimate. Successive threshold measurements were 20 obtained starting with a target stimulus intensity 0.3 log units brighter than the previous threshold estimate. The snfoeet had a 30-second rest period between threshold measurements. Threshold estimates were made about once a mimite for the duration of the measurement protocol. About twenty threshold measurements Were made during each dark adaptation test. 25 TIGS. 4AT> show four dark adaptation curves from the same test subject generated in response ip the four different photobleaehing light spectrums described above. The subject shows a stereotypical dark adaptation curve in response to the white photobleaehing light as expected (FIG, 4A). Bse of a photobleaehing light tailored to emit a spectrum of light consisting essentially of wavelengths in fhwfofiS© ofabout 490 nm to about 510 M« 30 (green spectrum), presemes foe stereotypicaf shape of the dark adaptation function because the rods are still Strongly bleached, in ednfiast, use of a phofobleaching light tailored to emit 24 a spectrum of light consisting essentially of wavelengths in the range of about 405 mil to about 425 : nib (blue speGtsiifri!) (EIG. 4C) on a photobleaehmg light tailored to emit a spectrum of light consisting essentially of wavelengths in the range of aboutOAO nm to: about 600 nm. (red spectrum) (ItiGi 419) felled to produce a stereotypical: dark adaptation response $i curve beeaase the rods Were only weakly photobleached, In addition, the:dark adaptation response obtained1 ttsingn.: pbotobieachiog light tailored to emit; a speetrwm of light consisting, essentially of wavelengths in die range of about 490 am to about 510: nm (green spectrum;) gave results more quickly than using a phoiobleaching light enhtting a broad spectrum Of light (compare FIGS 4A and AS). Recovery occurs fester because the additional 10 phbtohieaehihgcontribution from the blue and red components of the white spectrum, which is largely outside the rod response speeUung has been eliminated. 2014208236 31 Jul2014

Therefore, the rise of a phmbhleaehing light emitting a tailored spectrum of light consisting essentially of wavelengths in the range of about 49Q nm tp:^bpdf:5;f'0tapv(jgreen spectrum) was shown to give essentially the same dark adaptation response as a 15 photohleaching light emitting a broad achromatic white bleach and to provide the results more quickly. Ibis example shows that, for this particular objective (measuring dark adaptation), a photohleaching light emitting a tailored speetrUm of light consisting esseniialiy of wavelengths in the range of about 490 nm to about 5!0 nm (green spectrum) is an improvement overa photoMeaehihg light emitting an achromatie broad spectrum of white 20 light. However* it should be noted that lot other olhbciiveSj: the use a photohleaching tight: emitting a tailoted speetnnn of wavelengths other than that shown in this example may also: be useful. M Pfomary, essentially the same dark adaptation response: is obtained with:less patient burden·* both because only a fraction of the total energy impinges on the retina and because the most irritating short wavelength portion of the spectrum, is eliminated (he,, the 25: bMe .speetrttm}>: Moreover, the result is obtained more quickly.

Example- Iheferenee Test tor White Flash vs. Green Flash. in this example*: a preference test Was conducted comparing a photobleaching light comprising a broad wavelength spectrum of about 400 nm to about yOO mn generated by a xenon are light and a photobleaching light that was tailored to emit a spectrum 01 light 30 consisting of about 490 run to about 510 nm (green spectrum). 25

These, photobleaching light spectra were analyzed for theii to generate classical dark adaptation curves in Example i above and shown ίο produce generally similar dark adaptation curves. The phpfobleaehmg light;-m eaefoci^iya$ generated using a commercial camera flash system (SunTak 621 Super Pro). This system uses a xenon arc light source that 5 generates a broad,relativelyfiatspectrum of light (5501) Kelvin color terapemtnre) spanning the entire range of cone and rod sensitivity (about 400 opr to about 700 nm). The flash whs set at its maximum intensity of 7,48 log scot fd seof The ‘'green^ flash was created by placing a narrow (about 490 nm to about 51(1 pro) haa^|^^:^d#:re«ee filter (Edmund Optics; MT43-169) over the face of the xenon arc flash window. The broad wavelength 10 “white"’ spectrum photobleactung light was Creafed by placing a clear glass blank (essentially 1,0(1% transmittance at ail wavelengths) over the face of the xenon are flash window, so that the test subjects weiP confronted with similar ednfigufaiions in both eases. 2014208236 31 M2014

For each participant, one eye was exposed to the “white” phofobleachiog light comprising a broad wave!engtlrspectrum of aboth 41)0 nm to about 700 nm and the opposite 15 eye was expo^d td the ‘‘gfecn” photobleachi eg light that was tailored to emit a spectrum of light consisting esseniiafly of waveiengfbs of about 490 nm to about 510 nm. The flash: unit was held apprbxituately 20 ern in from of the test eyepwith the nonTest eye covered. The right eye was always exposed to the photobleaching: light first; however, the tests were eounterbalaheed with regard to sequence, alternating between the first: flash being -"white” 20 photobSeaching light with the properties described above and the first flash beihS '‘Stu^n” photobleaching light with the properties described above. There was an interval of approximately 1 minute between the two flashes. Immediately after exposure to each of the “white” and “green” photobleaching lights, the participants were askpd to rate disepmfori on a scale of 1 to 10, with I being “no discomfort, 1 would look at it all day” and 10 being 25 “highly uneomfortabie, I would not want to look at it again”. At the conclusion of the entire sequence, the participants were asked if they had to be exposed to one of the “white” or “green” photobleaching lights again which of the two they would prefer. A total of eleven naive participants were tested. There were six females and five males, all Caucasian, with a mean age of 30.6 years (range 22 ip 47). The age distributions (mean and range) for the two sexes Were eomparable. The results are shown in Figure 5:, There was a clear preference for the “green” photobleaching light, with an average rating of 26 2,8 for the .“greenT photahleaching light (rang© 1 to 51 vs, 5,1. forthe ‘'white” photbbleaehing light (range 2 to 8), and 91%2of the rsubjects indicated a preference for the ''green’ photoblcaching light if they were to be tested agam. While the subpopiiiahoh humbers are small» there was a consistent preference for the “green” phoiobleaching light regardless of sexj age (under 30 vs. over 30) or the order of the “white”·- “green” photobleaehing light sequence. There was a tendency for young females lo be mors sensitive :;to the “white” ifoptohIt aching light and to show a sfoonger preference for the “green” photohfeacbing light. 2014208236 31 Μ 2014 3 - Effect of Plmiobleaching light Spectrum on Variability in Dark Adaptation Measurements due lo Lens Op ac i tv. ΙΟ; In tiiis example» a comparison was made between dark adaptation curves generated using a photobleaehing: light that was tailored to emit: a speefoum of light consisting essentially of wavelengths of about 490 pm to about 510 mb (green spectrum) and a photobleaehing light that was Mimed to emit; a spectrum of light consisting essentially of wavelengths Of abo ut 440 nm to about 460 nm (blue· spectrum), both with and without a blue 1.5 absorption TjIter in front of the lest subject's eve. The blue absorption filter simulates the preferential absorption of shorter wavelengths due: to fens opacity,

Dark adaptation functions were measured using an AdaptDx dark adapiometer (Apebotus Technologies, Inc») as described In Example 1 above; as: modified below. To generate the photobleaehing:: light with The grObh: spectrum, the intensity of tlie xenoh arc; 2G: light source Incorporated in the dark: adaptometer was set at 7,()3 log scof Tdsecfo and a narrow green: (about 49() nm to about 510 nm) bandpass interference filter :(Edmund Optics 1S1T43-169) was placed over the lace of the xenon arc flash window,: To generate the photobleaehing light with the blue spectrum, the intensity of the xenon arc light source incorporated in the dark adaptometer was set at 7.60 log scot Td sec’1, and a narrow blue :25 (about 440 nm to about 460 nm) bandpass imerferenee filter (Edmtmd Optics NT43-163) was placed over the lace of the xenon arc flash window. BIOS. 6A-D show the resulting ditrk; adaptation fuhctiobS; Placing; the blue absorption .filter in front of the test subfect’s eye to lower the. transmission of slforhwaveiength lig|i in a fashion similar to that encountered with lens opacity, such as caused by cataracts: and age-related increases in lens opacity, had 30 mihimal impact On the dark adaptationcurves generated using the green spectrum 2? photobleaching light (compare FIG. 6A, designated control, vs, FIG. 6B, designated simulated lens opacity). lens opacity had a major impact pn dte 2014208236 31 Μ 2014 dark adaptation curves 'bliaevspectrum photobleaching light (compare FIG. 6C\ designated control, vs, FIG, 6Pj designated simulated lens opacity). 5 These results show that the use of a photohleachingTight tailored to emit a spectrum of light consisting essentially of wavelengths of about 490 urn to about 510 am (green spectrum}minimizes the variability in dark adaptation responses, and associated diagnostic measurements, due to the filtering effects of lens opacity, such as caused by cataracts and age-related increases in lens opacity. 10 Example 4 Photobleaching Below Ambient Daylight Levels.

In this example, a comparison was made between dark adaptation curves generated using a photobleaching light having an intensity above The intensity of ambient daylight and a photobleaching light having an intensity below the intensity of ambient daylights Dark adaptation speed was determined using a sensitive and reliable benchmark known as the rod 15 intercept. 'The;f#.interceptisthe time for seotopic sehsitfvity^to^recoverto 5x10'4 edrinf

Dark adaptation functions were measured using an AdaptOx dark adaptometer (Apeliblns Technologies, Inc.) as described in Example 1 above as rnodified below. Dark adaptation curves were generated using methods known in the: art, FIG. 1 compares two dark adaptation curves from the same test subject, lit the first case (FI<3: 7A), To phoioMeaehmg was accomplished using a brighh; achromatic white flash photobleach generated by a xenon arc light source producing a broad, relatively fiat spectrum of light (5500 Kelvin color temperature) spanning the entire range; of cone and rod sensitivity and having an intensity of A 38 log sept Td sec'5, which is well above the intensity of ambient daylight. In the; second case {FIG, 7B) photobleaching. was aeepmphshed using with a dim 25 (TO cd/m~) uniform bleaching: field for 1-minute, which is well below the intensity of ambient daylight levels. The rod intercept in response to the: bright, achromatic white flash photobleach was 6.97 nurmtes: compared with 1:54 minutes; for the dim 'background photon leaching. The cone-mediated portion of the dark adaptation function (the; first four thresholds in FIG. 7A) Is effectively eliminated by using the dim1 phbiehleaehipg procedure.: 28

Thus, use-«F a photbbleaching li^khaytogless limn the intensity bf ambient daylight can dramatically shorten the duration of a dark adaptation test. 2014208236 31 Jul2014

IS e 5 -- Effect of Eccentricity on Dark Adaptation: In Age-related Mueulonathv:; to this example, a comparison washmade between dark adaptation ..curves generated 5 by measuring dark adaptation ;at positions 5° and 12 s' on tbs inferior vertical, meridian, using both normal, test subjects and test: subjects: with age-relatediniaculdpaiby^ARM'},

Dark adaptation iunetions were .pleasured using: an. AdaptDX dark aduptometef (Apehotus Technologies, Inc.) as described in Example: 1 above as: modified below. Dark adaptation .function was measured in response to a 4C diameter photofelsaching light 10 (provided as a dash) with an intensity of 6.38 log scot I'd sec"’, fe this example, the photobieachiiig light was the essentially white, MOO Kelvin color temperature broad specirnm light (having wavelengths: from about 400 nm fe about ?f)Dmm) generated by the xenon arc source incorporated in the dark adapfometer, The target stimulus light was a 2W diameter, SOO-mn eireolar spot centered within the area subjected to photobleachingv 15 Scotopic threshold ineasurcmentsbegan immediately after photobleach effect. During threshold measurement the subject focused on the fixation light and msponded when the stimuhis was; judged to be present by pushing a button, Threshold was estimated using a 3-tiown/1-up modified staircase procedure, Approximately one threshold was measured each minute for 2fi minutes, Dark adaptation speed was determined using a sensitive and reliable 20 bench.mark known, as the rod Metcept The rod Intercept is the time for scotopic sensitivity io recover to 5x1 O’* edduo The dark adaptation. Impairment of the ARM patients was· calculated relative to a control group of age-matched adults. 25

Anatomical studies have shown that file area of greatest rod dysfunction associated with ARM is within-the parafoveal region (3-40:5° eccentricity) of the retina. The pattern of Scotopic sensitivity impatriilent:exhibited by ARM patients is consistent with, the anatomical findings; that is, scotopic sensitivity impairment is greatest in the parafoveal region and decreases as a function of eccentricity towards the retinal periphery, in this example; we examined whether darkadaptation impairment hasa Similar pattern of dysfimctiOn. A total of 5 normal old adults and 8 ARM patients were tested. Group assignment 30 was based on grading of iimdus photographs using the ARBDS AMD Severity Classification 29 2014208236 31 Jul2014

System. Besiwotrebted visual acuity (ETDRS chart) and contrast sensitivity (Pelti-Robson. chart) were measured «η thm-'dayfof testing. Dark .adaptation function was measured as described above. Each. participant had: their dark adaptation function measured 'ph the interior vertical meridian at S*:!and 12° on separate testing days. Soil! groups were:similar in 5:· age. test eye acuity, and test: eye contrast sensitivity. Dark adaptation inipairmCUtdbr the AM id group relative to the normal old adults was almost 5 at. 12°. Furthermore, for the AMD group dark adaptation was;, ahnost 3 mtntUes slower at 5° than at 12° tor the AMD grpitp, whereas for the normal old adults:: dark: adaptation was almost 3 minutes foster at 5° than at 12*. 10 Patients in the ARM -group, exhibit greater dark adaptation impairment in the parafoveal region compared to an area adjacent to the macula, In. general, the AMD: group’s dark adaptation was slower in the parafoveal region compared with the more peripheral point; whereas, the normal did adults exhibited the opposite pattern. These results shpfolhat tailoring the region of the retina that Is subject to photobleaciiiug and sfoiseqnent testing -to; 15 the pattern of dysfunction for a particular disease. Ibr example, by choosing a region of the retina with maximum disease susceptibility, or by comparison of one or more areas haying different; disease susceptibilities in a single test, may be useful in the design of a diagnostic aimed at detecting the earliest stages of a disease. While this principle wasTIluAfated in the current example using ARM, it is equally applicable to other disease states. 20 fkmnnle„.6..-. Effect of Photobleaching Lb

MasajgBS&amp;fc

In this example, a comparison was made between dark adaptation curves generated by measuring dark adaptation with achromatic and green photobleaohing lights, upng both normal test subjects and test subjects with age-related maeulopathy (ARM). 25 Dark adaptation fonettons were measured «sing an AdaptDx dark adaptometer (Apeifotus 1'echnologies, Inc.) as described in Example 1 abovC asripOdiSed below. Id One case* an achromatic bleacbing/llghi (essentially whiter liSOO Kelvin color temperature broad· spectrum;.consisting of wavelcnglhktffom: about 400 nm to about ?00 tan) was generated by the xenon are light source incorporated: in the dark adaptometer, with the intensity of the 3D: flash set at 6,38 log scot Td see'-|EI(j, 8A). In (he other case, a green bleaching light (about: >490 sim to about Si 0 urn) was generated by placing a namow green bandpass interference filter fbdniund Opiici.l^43-l:b^)>over>tb^vfeeio of the xenon arc flash incorporated in the dark adaptomcter, with the intensity of the fiash set at f .03 log scot Td sec:?(TlG, SB). 2014208236 31 M2014

These two conditions produce pearly equi¥aient photohleaching of the photoieeeptor visual 5 pigments.

In both cases a normal: adult and an ARM patientweretested. The response patterns for the achromatic and green phoiobleaehing lights are the: same* with the ARM patient /exhibiting markedly slowed dark adaptation relative to the normal adult; in. both eases, iaekson and Edwards (A Short-Puratton Dark Adaptation Protocol for Assessment of Age-10 Related Maeufopathy, Journal of Ocular Biology, Diseases, and Infophatics;: in press 2008, ineotporated herein in its entirety by reference) haye shown. that measurement of dark adaptation using an achromatic photobleaching light is a sensitive and specific diagnostic fbr ARM,, The results of this example: show that the; ability to discriminate ARM: is preserved; when using a green bleaching light, allowing the added benefit of lower patient burden and, IS lower confound from lens, opacity without loss of diagnostic utility.

The foregoing/description illustrates and describes the methods and other teachings of the present disclosure. Addition ally, the disclosure shows: and describes only certain embodiments of the methods and other teachings disclosed,but, as mentioned above, it is to be understood that the teachings of the present disclosure are capable of use in vari ous other 20 combinations, mod ifications, andenvironments and is capable of changes or modifications within the scope of the teachings as expressed herein, commensurate with the skill andfor knowledge of a person having ordinary skill in the relevant art,The embodiments described hereinabove are further intended to explain best modes known of practicing the methods arid other teachings of the present disclosure and to enable others skilled in the art to utilize the 25 teachings of the present disclpsure in such, or other, embodiments and with the various modifications required by the particular applications or uses; Accordingly, the methods and other teachings of the present disclosure are not intended idlimit the exact embodiments and examples disclosed herein. All references cited herein are incorporated by reference as if fully set forth in this disclosure. 3.1 A reference herein to a patent document or other matter which is given as prior art is not taken as an admission that that document or prior art was part of common general knowledge at the priority date of any of the claims. 2014208236 31 Μ 2014

With reference to the use of the worths) “comprise” or “comprises” or “comprising” 5 in the foregoing description and/or in the following claims, unless the context requires otherwise, (hose words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that each of those words is to he so interpreted in construing the foregoing description and/or the following claims. 32

Claims (36)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. A method for photobleaching a subject's eye, the method comprising the steps of: a. exposing a portion of the retina of the subject’s eye to a target stimulus light; b. spatially tailoring a region of the retina of the subject's eye that is subject to photobleaching by exposing only a selected region of the retina to a photobleaching light to photobleach at least a portion of at least one visual pigment in the selected region, wherein the area of the selected region is less than 200 times the area of the portion of the retina exposed to the target stimulus light; and c. using dark adaptometry to monitor a response to the photobleaching light to determine a status of dark adaptation.
2. 2. A combination for photobleaching a subject's eye, the combination comprising: a. a photobleaching light and a target stimulus light; b. an apparatus for spatially tailoring a region of the retina of the subject's eye that is subject to photobleaching so that only a selected region of the retina is exposed to the photobleaching light and an apparatus for exposing the target stimulus light to a portion of the retina, wherein the area of the selected region is less than 200 times the area of the portion of the retina exposed to the target stimulus light; and c. an apparatus to administer a dark adaptometry psychophysical test to monitor a response to the photobleaching light to determine a status of dark adaptation.
3. 3. A combination when used for photobleaching a subject's eye, the combination comprising: a. a photobleaching light and a target stimulus light; b. an apparatus for spatially tailoring a region of the retina of the subject's eye that is subject to photobleaching so that only a selected region of the retina is exposed to the photobleaching light and an apparatus for exposing the target stimulus light to a portion of the retina, wherein the area of the selected region is less than 200 times the area of the portion of the retina exposed to the target stimulus light; and c. an apparatus to administer a dark adaptometry psychophysical test to monitor a response to the photobleaching light to determine a status of dark adaptation.
4. 4. The combination of claim 2 or 3, wherein the apparatus for spatially tailoring a region of the retina of the subject's eye restricts the photobleaching light to expose the selected region of the retina to the photobleaching light.
5. 5. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the photobleaching light has a tailored wavelength spectra within the visible spectrum to accentuate or minimize a response of a subset of photoreceptors to the photobleaching light.
6. 6. The method or combination of claim 5, wherein the tailored wavelength spectra consists essentially of a wavelength selected from the group consisting of: a wavelength of about 505 nm, a range of wavelengths centered on 505 nm, a wavelength of about 419 nm, a range of wavelengths centered on 419 nm, a wavelength of about 531 nm, a range of wavelengths centered on 531 nm, a wavelength of about 558 run, a range of wavelengths centered on 558 nm, a wavelength of about 460 nm, a range of wavelengths centered on 460 nm, a wavelength of about 650 nm, a range of wavelengths centered on 650 nm, a wavelength of about 410 nm, a range of wavelengths centered on 410 nm, a wavelength of about 570 nm, or a range of wavelengths centered on 570 nm, and a wavelength or range of wavelengths over about 480 nm.
7. 7. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the photobleaching light is restricted to expose the selected region of the retina to a photobleaching light.
8. 8. The method or combination of claim 7, wherein the photobleaching light is restricted using a mask, projection onto only the selected region, orientation of the subject's retina or a combination of the foregoing.
9. 9. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the target stimulus light has a spectra.
10. 10. The method or combination of claim 9, wherein the spectra of the photobleaching light and the spectra of the target stimulus light are different from one another.
11. 11. The method or combination of claim 9, wherein the spectra of the photobleaching light and the spectra of the target stimulus light are the same as one another.
12. 12. The method or combination of claim 9, wherein the target stimulus light source has a tailored wavelength spectra.
13. 13. The method or combination of claim 9, wherein the selected region is exposed to the photobleaching light and the target stimulus light.
14. 14. The method or combination of claim 9, wherein the selected region is exposed only to the photobleaching light.
15. 15. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the selected region is selected as a function of eccentricity from the fovea to the peripheral retina.
16. 16. The method or combination of claim 15, wherein the method or combination is used to determine if the subject is suffering from or at risk for age-related macular degeneration.
17. 17. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the selected region is outside the fovea, entirely inside the macula, is in the peripheral retina, is located on the inferior vertical meridian, is located on the superior vertical meridian, is located at about 0 degree to about 0.5 degree eccentricity, is located at about 2 degree to about 10 degree eccentricity, is located at 3 degree to 10 degree eccentricity, or is located at about 10 degree to about 30 degree eccentricity.
18. 18. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the selected region is entirely inside the macula, is in the peripheral retina, is located on the inferior vertical meridian or is located on the superior vertical meridian.
19. 19. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the selected region is located at about 0 degree to about 0.5 degree eccentricity, is located at about 2 degree to about 10 degree eccentricity, is located at 3 degree to 10 degree eccentricity, or is located at about 10 degree to about 30 degree eccentricity.
20. 20. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the selected region is an annular region.
21. 21. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the selected region is an annular region completely excluding the fovea.
22. 22. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the selected region is an annular region completely excluding the fovea, the annular region having an inner edge located at or outside about 2 degree and an outer edge located at or inside about 10 degree eccentricity.
23. 23. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the selected region covers an area of about 4 degree of visual angle to about 6 degree of visual angle.
24. 24. The method or combination of any preceding claim, further comprising exposing more than one selected region of the retina of the subject's eye to a photobleaching light to photobleach at least a portion of at least one visual pigment in each of the more than one selected regions.
25. 25. The method or combination of claim 24, wherein each of the more than one selected regions have a different degree of eccentricity.
26. 26. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the photobleaching light has a set intensity.
27. 27. The method or combination of claim 26, wherein the set intensity is at or below 4.05 log scot Td/sec or at or below 3.15 log scot Td/sec.
28. 28. The method or combination of any one of the preceding claims, wherein the method or combination is used to determine the health of the subject's eye.
29. 29. The method or combination of any one of the preceding claims, wherein the method or combination is used to determine a parameter selected from the group consisting of: the health of the subject's eye, if the subject is suffering from an eye disease, if the subject is at risk for developing an eye disease and the severity of an eye disease.
30. 30. The method or combination of anyone of the preceding claims, wherein the subject burden is reduced as compared to prior art bleaching methods, the subject discomfort is reduced as compared to prior art bleaching methods or a combination of the foregoing are reduced.
31. 31. The method of any one of the preceding claims, wherein measurement variability is reduced as compared to prior art bleaching methods, measurement bias is reduced as compared to prior art bleaching methods or a combination of the foregoing are reduced.
32. 32. The method of any one of the preceding claims, wherein a time course for administering the psychophysical test using the photobleaching light is reduced as compared to prior art bleaching methods.
33. 33. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the selected region is located at 2 degree to 10 degree eccentricity.
34. 34. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the area of the selected region is less than 100 times the area of the portion of the retina exposed to the target stimulus light.
35. 35. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the area of the selected region is less than 9 times the area of the portion of the retina exposed to the target stimulus light.
36. 36. The method of claim 1, or the combination of any one of claims 2 to 4, wherein the area of the selected region is less than 4 times the area of the portion of the retina exposed to the target stimulus light.