Incident light is absorbed by molecules in the fundus, which are excited and in turn emit photons of specific wavelengths that are captured and processed by a sensor to create a metabolic map of the fundus. Studies on the growing number of FAF platforms has shown each may be suited to certain clinical scenarios. Scanning laser ophthalmoscopes, fundus cameras, and modifications of these each have benefits and drawbacks that must be considered before and after imaging to properly interpret the images. Emerging clinical evidence has demonstrated the usefulness of FAF in diagnosis and management of an increasing number of chorioretinal conditions, such as age-related macular degeneration, central serous chorioretinopathy, retinal drug toxicities, and inherited retinal degenerations such as retinitis pigmentosa and Stargardt disease. This article reviews commercial imaging platforms, imaging techniques, and clinical applications of FAF.Fundus autofluorescence (FAF) has allowed  The same imaging concepts from fluorescein angiography (FA) and indocyanine green angiography (ICGA) apply to FAF imaging, except the substances imaged are fluorophores already present in retinal structures instead of a systemically administered dye. The AF signals generally must be amplified due to much lower fluorescence than the dyes and may require additional wavelength filters, light path modifications, or detectors. This review aims to provide a clinically relevant overview of ophthalmic fluorophores, commercial imaging platforms, and updated clinical applications for selected conditions.Fundus autofluorescence (FAF) imaging is based on detecting physiologically and pathologically occurring fluorophores primarily in the photoreceptors and retinal pigment epithelium (RPE) to map the metabolic profile of the fundus. Originally described for ABCA4 dystrophy, retinitis pigmentosa, rod-cone dystrophy, Bietti crystalline dystrophy, CRB1 autofluorescence, pigmented paravenous retinochoroidal atrophy, macular telangiectasia, pseudoxanthoma elasticum, hydroxychloroquine, deferoxamine, pentosan polysulfate, retinal detachment, and retinoschisis. Reviews were referenced if original claims were made, but references to original data in the review were referenced themselves. Data for imaging systems were obtained from either official company websites or user manuals or information from company representatives. The World Health Organization website was used to reference statistics and to confirm published claims.Several literature searches were conducted prior to and during the drafting of this review. Both PubMed and Google Scholar were queried using the following search terms: fundus autofluorescence, short wave autofluorescence, near infrared autofluorescence, lipofuscin, scanning laser ophthalmoscope, fundus camera, wide-field autofluorescence, quantitative fundus autofluorescence, fluorescent lifetime imaging ophthalmoscopy. These were used alone, as well as in combination with the following terms: age-related macular degeneration, geographic atrophy, exudative macular degeneration, retinal pigment epithelial tears, subretinal drusenoid deposits, reticular pseudodrusen, central serous chorioretinopathy, diabetic retinopathy, diabetic macular edema, Stargardt disease, macular dystrophy, fundus flavimaculatus, pattern dystrophy, multifocal pattern dystrophy, adult-onset foveomacular vitelliform dystrophy, reticular pattern dystrophy, fundus pulverulentus, butterfly dystrophy, Best macular dystrophy, autosomal recessive bestrinopathy,  First, an incident photon is absorbed by electrons in the fluorophore, which is elevated to an excited state. Second, the fluorophore rapidly dissipates some of the absorbed energy through transfer primarily either to its surroundings via kinetic energy or to other vibrational states within the same molecule. The electrons in the molecule may relax to several different vibrational states during this period. Last, when this vibrational energy level overlaps with specific molecular energy levels, the excited electrons may transition to a different vibrational level in a lower electronic level or to its ground state. During this transfer, a photon of energy particular to the electronic structure of the molecule may be emitted. Due to the multiple transfers of energy and natural entropy, the emitted photon is always of a lower energy than the absorbed photon. The excitation wavelength of the incident photon is in turn always shorter than that of the emitted photon.Fluorescence is an electronic process occurring at the atomic, molecular, or nanostructural level.When the fluorophore is polyatomic, the electronic transitions encompass excitation and emission spectra of a broad band of wavelengths. However, each molecular spectrum has a wavelength maximum at which the fluorophore results in greatest intensity of fluorescence. Similarly, the emission spectra has a wavelength of maximum intensity emission. The excitation and emission spectra wavelengths may overlap, which leads to a resultant decrease in contrast with imaging. This can be ameliorated with selection of appropriate excitation and emission filters to select desired wavelengths and remove overlapping wavelengths of lower intensity. At the start of phototransduction, 11-cis-retinal chromophore of rhodopsin absorbs a photon of light and isomerizes to all-trans-retinaldehyde [Figure 1]. This form may be released from the activated rhodopsin and react with phosphatidylethanolamine (PE), also in the disc membrane, to form N-retinylidene-PE (NRPE). Another molecule of all-trans-retinal often escapes conversion to the alcohol form and combines with a molecule of NRPE. This intermediate is then taken up by the RPE and converted to N-retinylidene-N-retinylethanolamine (A2E), a stable pyridinium bisretinoid and the most well-known LF component. When subjected to blue light, A2E is photo-oxidized and generates reactive oxygen intermediaries that can destabilize cellular membranes, interfere with cholesterol metabolism, and trigger apoptosis.,7,8Lipofuscin (LF) is a cellular metabolic by-product resulting from incomplete lysosomal degradation of shed photoreceptor outer segments by the RPE and represent the primary substance imaged with short-wavelength autofluorescence (SW-AF).,10 These compounds may then cross-link with themselves and other intracellular molecules to form complex end-products.,12 Photooxidative processes affecting cellular proteins and membrane lipids likely contribute to the LF milieu. Proteomic studies of purified LF granules identified A2E adducts in addition to an abundance of photo-oxidated proteins and lipids, with only 2% by weight of the LF being amino acids.Combinations of at least 20 compounds formed during incomplete visual pathway biosynthesis or subsequent photooxidation may form LF granules.,15 RPE LF extracts have consistently emitted light in the yellow–orange spectrum.,16 Debate continues on the origin of these emissions and whether measuring them clinically is useful.Moreover, although the fluorescent products of lipid oxidation maximally emit light in the blue–green spectrum,,17 They have a peak excitation in the blue range , 439 nm (A2E), 449 nm (A2PE), 426 nm (isoA2E), 490 nm (A2-DHP-PE), and 510 nm .,19,20 Therefore, although individual bisretinoids may fluoresce with blue 488 nm FAF excitation, their different absorbance spectra lead to different emission intensities. Moreover, all-trans-retinal and its ester, physiologic intermediaries during phototransduction, do not contribute significantly to the FAF pattern at 488 nm excitation.The various bisretinoids making up LF are the primary source of the retina AF signal noted with SW-AF.First detected during FA as preinjection fluorescence, normal fundus SW-AF signal demonstrates a dark area centered on the fovea, dark vessels strikingly contrasted against a bright retinal background, and a dark optic disc. The foveal pigments, including lutein and zeaxanthin, reach a maximum concentration at the fovea and decreases exponentially with eccentricity. These pigments are primarily concentrated in the photoreceptor layer axons but can be found in the inner plexiform layer. Normal fundi increased NIR-FAF signal in the fovea corresponding to a higher melanin concentration and the luteal pigment [Figure 2]. The maximum NIR-FAF signal in the fovea is very slightly nasal to the maximum SW-FAF signal, and it is always of lower contrast and exposure to the SW-AF signal, even with significantly higher radiant power and detection sensitivity, and may require dilated pupils for sufficient image quality. The optic disc and retinal vessels have dark signal similar to SW-AF images but with less contrast compared to the surrounding retina. Melanin in the RPE and choroidal layers contribute to NIR-FAF that can be noted with RPE hyperpigmentation, choroidal nevi, and the outline of choroidal vessels, which appear dark. Subacute blood, orange–yellow deposits or chronic subretinal fluid over choroidal melanomas or nevi, and some choroidal neovascular membranes (CNVM) can be bright on NIR-FAF.Near-infrared FAF (NIR-FAF) was first detected while evaluating pre-ICG injection fluorescence with standard ICG angiography filters.Near-infrared reflectance (NIR-R) utilizes an infrared light source . Histopathology confirmed these findings, with diseased RPE noted to be more highly fluorescent than normal RPE on whole flat mounts. However, accumulation of A2E-related compounds and other fluorophores can occur posterior to and in many structures anterior to the RPE.AF was first reported in the 1970s, when autofluorescent signal of optic nerve head drusen was noted prior to the injection of fluorescein dye. disruption of luteal pigment causing unmasking of foveal AF, bare sclera, calcification, optic disc drusen, or any condition that increases LF accumulation and/or migration, such as BD, STGD, and the pattern dystrophies.,30Areas of increased SW-AF, or hyper-AF, result from buildup of AF compounds, an unmasking of these compounds from depleted photoreceptor or luteal pigments, and/or presence of structures that also autofluoresce at the excitation wavelength. Areas of decreased SW-AF, or hypoautofluorescence (hypo-AF), result from loss of AF compounds or RPE, or blockage from overlying substances or structures. Increased SW-AF can be noted with porphyrins from subacute to chronic retinal hemorrhage, Increased FAF signal from the RPE can be achieved by “bleaching” of the photoreceptor pigments with high intensity light and photoisomerization. Bleached rhodopsin can lead to up to 30% increased FAF signal compared with its dark-adapted state, indicating the importance of the photoreceptor layer in interpretation of FAF.Molecules in the pathway of incident light and/or the emitted photons can interfere with the detected signal. Rhodopsin chromophores in the photoreceptor outer segments filter the excitation and emission light, but may also contribute to the FAF signal in certain retinal pathologies. Importantly, it filters lower wavelength light (292–400 nm) to prevent retinal damage from ultraviolet light. However, increased post-translational modifications to lens proteins during aging shifts the absorption maxima more into the visible range. This yellowing of the lens results from covalent modifications to the protein chain, oxidation of aromatic amino acids, and development of advanced glycation end products. In addition, intrinsic lens fluorescence in the UV-A and UV-B ranges increases with age, although likely contributes less to artefacts in FAF.The crystalline lens contributes to absorption of excitation and emitted light.The human cornea has an excitation peak at 365–480 nm and an emission peak maximum at 620 nm, although it can have increased AF in the visible range in diabetic patients.Although abnormal AF signal may be readily apparent, subtle differences from normal or progressive changes on subsequent visits may be qualitatively undetectable. A need to standardize and quantify FAF signal for reproducible and comparable measurements led to the development of qAF. Physiologically higher qAF levels have been demonstrated with increasing age, increasing eccentricity up to 15° from the fovea, the superotemporal fovea, and females. Levels are also higher in Whites compared with Blacks and Asians. To obtain accurate measurements, it is critical to have high-quality images with a skilled ophthalmic photographer, dim and usual lighting, ideal focus, and consideration of significant media opacities that may confound interpretation.,38 Good inter-operator agreeability and between-session repeatability have been shown with trained technicians and standardized imaging procedures.,39Gray pixel values for FAF on imaging systems are normally assigned a value from 0 to 255 but can vary depending on system calibration. Delori et al normalized a FAF system to a standard reference and compensated for factors such as laser power, refractive error, and media opacity variations, in order to calculate the qAF value from the gray level measurements. Every fluorophore harbors a unique excitation and emission spectra as well as a fluorescence lifetime. Quantifying the time from excitation to emission can be measured directly or by modulating flicker light and measuring the phase delay of emission. Problems with the complexity of measuring the decay at each pixel in an image with computational exponentials can be overcome by transforming the histogram of time delays at each pixel into a phasor. The details of this imaging are beyond the scope of this review, but this approach can allow identification of molecular species at each pixel and their proportions. In healthy eyes, increased AF lifetimes have been noted with age and increased distance from the fovea, likely from macular pigment distributions. In general, a significant increase in lifetime is seen in diseased retina and RPE, often due to the buildup of LF with its relatively long lifetime. Disease-specific changes have been noted with AMD, macular telangectasia type 2 (MacTel), Stargardt disease (STGD), hydroxychloroquine retinopathy, and choroideremia. While clinical utility is currently limited, results of these studies are encouraging for monitoring disease course for observation and therapeutic trials.As opposed to qAF which measures the standardized intensity of fluorescence, FLIO measures the time delay from excitation to emission and the decay curve of emission intensity, which are not necessarily related to fluorescent intensity. but has evolved significantly over the years. All SLO systems utilize a laser source to generate a “flying” laser spot that passes through a series of tunable mirrors and lenses to scan horizontally and vertically in a raster pattern over the retina. At each point, the backscattered light from the retina is directed into a detection arm for analysis by software and image creation. Advantages of SLO include lower required light intensity, high frame rate, increased sensitivity, and smaller aperture, that is, smaller pupil, requirements. Disadvantages include the lack of true color imaging, artefacts from eye movements, and spatial distortions.The design of the first scanning laser ophthalmoscope (SLO) was first presented in 1980 Suppression of out-of-focus light increases at further distances from the focal plane; thus, signal from vitreous opacities, lens aberrations, and the cornea are minimized. This provides improved 2D resolution and contrast as well as 3D imaging capabilities.However, some modern systems implement confocal components, which allow imaging of specific planes in the pathway of the laser. By modifying the position of the mirrors and directing the light through a pinhole, the photodiode detector can amplify reflected light from the desired plane of focus while blocking out-of-focus light.Fundus cameras rely on a high-energy white flash that passes through a wide-band excitation filter and series of mirrors and apertures to illuminate the retina. Issues with these systems include low FAF signal, AF of light pathway media, and inherent non-confocality of the system.Inherent limitations in FAF imaging include low AF signal when compared with FA or color fundus imaging, high image noise, low contrast, and AF artifacts from more anterior structures. Modern systems utilize strategies to increase the true posterior FAF while reducing anterior segment interference. Imaging with NIR-AF is also provided with a 787-nm excitation and  The Nidek F-10 and Mirante systems use 490-nm blue light with an emission filter at 510 nm and a 40–60°field of view.,54 They also harbor an infrared imaging mode at 700 nm to detect laterally scattered light, which may be more sensitive for drusen detection. The EIDON device by Centervue is a cSLO with FAF capabilities that uses a larger than standard pinhole.Modern confocal SLO (cSLO) systems include the Heidelberg Retinal Angiograph (HRA) on SpectralisⓇ platforms , the Nidek F-10 and Mirante systems , and the EIDON device  [Table 1]. The Heidelberg SpectralisⓇ HRA2 SW-AF utilizes blue light, with a solid state laser at 488 nm for excitation and a The Rodenstock cSLO and Zeiss prototype SM 30 4024 cSLO are no longer commercially available.,9,55,56 New red-shifted modifications of the Spaide filters can be swapped in with excitation of 535–585 nm and emission filters of 615–715 nm. These modified filters create lower AF signal from the crystalline lens and cornea. The bandpass excitation filter incorporates wavelengths above that absorption spectra of fluorescein, allowing FAF images to be taken after fluorescein injection.The commercially available Zeiss , Canon CX-1/CR-2 , Topcon TRC-50DX/50IX  systems and their specifications are listed in Table 1. These traditional-style fundus cameras all incorporate single flashes of light with bandpass excitation filters in the blue–green range and emission filters in the near- infrared range.Zeiss produces the Visucam family of retinal cameras, the FF 450 Plus IR device, and the Clarus cameras. For FAF imaging, the Visucam and FF450 use excitation filters of 510–580 nm and emission filters with a bandpass of 650–735 nm. The Clarus 500/700 systems are marketed as a true-color, ultra-wide field system using Broad Line Fundus Imaging (BLFI) technology with illumination in two broad wavelengths, using two excitations at 435–500 (blue) and 500–585 (green) nm and emission filters of 532–650 and 630–750 nm, respectively. The Clarus captures a wide range of SW-AF and can image up to 200° with two montaged images.,9,59,60 A scanning laser is reflected off a concave mirror and rasterized to visualize up to 180–200° of the retina in a single capture. As opposed to the HRA2, a green laser at 532 nm is used for excitation, and an emission filter at 540 nm acquires emitted light in the 540–800 nm range [Figure 3]. The laser and pathway are the same for all current models as well as the earlier 200Tx model. The rapid scanning speed with the ability to capture wide angles in a single image can be advantageous in patients with poor vision or poor fixation. An additional advantage of UWF imaging is improved acquisition ability through small pupils  OptomapⓇ ultra-widefield (UWF) imaging platforms are non-mydriatic cSLO with FAF capabilities.The optic nerve SW-AF signal varies depending on both the imaging system and wavelength used. For example, on the Spectralis , F-10/Mirante , and EIDON (450 nm blue), the optic nerve appears very hypo-AF and almost black, but on the Optos , the nerve appears less hypo-AF and almost isoautofluorescent to the fovea, despite all systems being cSLO systems [Figure 2]. Generally, the optic disc appears hyper-AF on fundus cameras, which are mostly green SW-AF. With the Clarus, the optic disc appears very hypo-AF with green SW-AF (500–585 nm) and isoautofluorescent to retina with blue SW-AF (435–500 nm). The reasons for these discrepancies may be due to the varying focal ranges, excitation and emission barriers, and/or light scattering.Comparisons between imaging platforms must take into consideration not only cSLO versus fundus camera but also the technical aspects of the machine, wavelength spectra, and the clinical entity being investigated. As AMD primarily affects the RPE-Bruch's membrane complex, FAF changes, particularly on SW-AF, have been shown to be an extremely useful diagnostic and prognostic tool, even with the advent of high-definition OCT platforms .Advanced forms of AMD such as geographic atrophy (GA) and neovascular AMD (wAMD) are leading causes of vision impairment worldwide, especially in industrialized countries. Atrophic or degenerating RPE cells with less LF content will be hypo-AF, while hyperplastic or dysfunctional RPE cells with LF accumulation will be hyper-AF. In a study by Bindewald et al, certain AF patterns in early and intermediate AMD were delineated, but drusen were not necessarily correlated with a particular FAF change. Speckled and patchy FAF patterns were most frequent, but FAF changes did not correlate topographically with changes on color fundus imaging. Delori et al noted variable FAF patterns over hard and soft drusen, but with hyper-AF surrounding drusen, suggesting changes in RPE metabolism. Focal vitelliform deposition over drusen may increase FAF signal, but otherwise the intrinsic FAF signal from drusen is variable and cannot be used clinically to differentiate drusen types reliably.Early lesions in AMD can be detected more readily with SW-AF than on color imaging as focal hypo-AF and hyper-AF, and a variety of early FAF patterns have been described. Although first described on blue light imaging, they are most easily seen on near-infrared reflectance (NIR-R), NIR-AF, and SW-AF but are readily detectable on HD-OCT and red-free imaging.,68 They are apparent on IR or AF en face imaging as an interlacing network of small dot-like variations. These correlate to small subretinal hyperreflective deposits on HD-OCT. Thinning of the ONL with disruption of the EZ has been described above the SDD, which may lead to the aforementioned FAF changes. Although not a therapeutic target, SDD should be looked for routinely, as evidence suggests increased risk for progression to GA and association with higher genetic risk scores.,72Reticular pseudodrusen, or subretinal drusenoid deposits (SDD), represent an independent risk factor for progression to advanced AMD. The death of RPE and loss of intrinsic fluorophores correlates well with the sharply demarcated borders on FAF and can be correlated with HD-OCT patterns. The pattern of FAF in the junctional zone surrounding the GA has been associated with the rate of GA enlargement, with more diffuse hyper-AF changes generally representing faster rates. The rate of growth on FAF imaging is variable, with large studies ranging from approximately 0.5 to 2.5 mm Multimodal imaging evaluation with VA and tests of visual function noted significant predictors of worse VA with greater areas of confluent hypo-AF and involvement of the fovea with confluent or granular hypo-AF. Larger areas of confluent hypo-AF were also associated with decreased reading speed and contrast sensitivity. Measuring tools on imaging software or vascular landmarks can be used to discern progression of GA.GA has been shown to be readily and accurately detected and monitored with FAF. More foveal-involving patterns were correlated with a worse retinal sensitivity on microperimetry (MP). Patterns on SW-AF and NIR-AF may overlap but are not identical, indicating different involvements of LF and melanin in AMD pathophysiology. In the presence of GA, both SW-AF and NIR-AF are comparable in measuring lesion size. However, because SW-AF can be blocked by foveal luteal pigments, SW-AF has been shown to overestimate foveal GA compared with NIR-AF and MP, while NIR-AF overestimates non-foveal GA. The combination of FAF wavelength modalities may provide more accurate measurements, but the clinical utility of the combination remains to be seen.The utility of NIR-AF is less established than that of SW-AF. Imaging with NIR-AF in early AMD also demonstrates characteristic patterns: normal foveal signal, fovea-sparing hyper/hypo-AF spots, fovea-involving hyper/hypo-AF spots, and a patchy AF pattern. Evaluation of the fovea in fovea-sparing GA is limited by the absorption of blue light by luteal pigments, which makes the fovea appear dark. Although GA area is quantified reproducibly with either green FAF or blue FAF + NIR, slightly better inter-reader agreement with green FAF was noted in one study. Comparison with HD-OCT is important to assess for exudation, CNV formation, and drusen-associated atrophy, in which outer retinal and RPE atrophy has begun overlying drusen and may be variably autofluorescent.The Classification of Atrophy Meeting recommended SW-AF adjunctive to HD-OCT in order to monitor the size of GA and detect peri-atrophy AF changes incipient to progression.,22,84 Classic CNV, or type 2 MNV, shows decreased FAF in the area of leakage on FA, while eyes with occult CNV, or type 1 MNV, shows spots of hypo-AF or normal AF.,86 As the type 2 MNV lesion is subretinal, it may block the AF signal from the RPE. The type 1 MNV may cause focal RPE atrophy and overlying hypo-AF. No FAF pattern over the MNV has been shown to be predictive of outer retinal integrity. However, the presence of an hyper-AF ring surrounding the MNV prior to injections has been associated with increased baseline SRF, increased number of injections, and greater likelihood of EZ disruption on HD-OCT after SRF resolution. This ring suggests more widespread involvement than apparent on OCT or FA. Eyes with FAF alterations have also been associated with worse initial VA and smaller VA gains after anti-VEGF therapy compared with those without FAF changes. Therefore, while the mainstay of modern wAMD monitoring utilizes HD-OCT, ancillary FAF testing may provide useful prognostics for progression and visual acuity.Moreover, wAMD, or macular neovascularization (MNV), has a variable appearance on FAF depending on the integrity of the RPE, the presence of hemorrhage or exudation, and type of MNV. Intraretinal exudates and hemorrhage will block FAF and cause hypo-AF, but with chronicity, hemorrhage may organize and form breakdown products with resultant intense hyper-AF on SW-AF and NIR-AF.,90 occurring in approximately 10–12% of patients with wAMD. They traditionally occur in type 1 MNV, with a large, vascularized, and often peaked PED, and contracture of the MNV is thought to produce tensile forces on RPE attachment junction, eventually resulting in tearing of the RPE. As this is a structural complication of the RPE, SW-AF is particularly suited for imaging this feature. The area of Bruch's membrane/bare choroid shows well-demarcated reduced FAF signal, and the adjacent area of scrolled RPE shows increased AF due to folding of the RPE monolayer. Longitudinal FAF imaging demonstrated centripetal reconstitution of the AF signal on the area of bare choroid and suggests recovery by the RPE cells. A grading system of RPE tears has been suggested based on size on FAF and FA , flavin adenine dinucleotide (FAD), and advanced glycations end-products (AGE), may affect the FAF signal. Impaired mitochondrial function before pericyte cell death from chronic hyperglycemia alters the cellular balance of these molecules. One study found increased quantitative FAF of flavoproteins in NPDR eyes, suggesting modified retinal metabolomics. Other studies have found more notable DR changes on FAF than color imaging, as well as greater FAF pixel intensity and variation in diabetic patients without DR compared with controls. Additionally, studies have found abnormal FLIO parameters in eyes with NPDR as well as without detectable DR. Both blue and green light sources were used in these studies, so any increased sensitivity of blue imaging must be balanced against interruption from the crystalline lens in phakic patients. Lens and vitreous opacities are common in eyes with DR, and cSLO, particularly UWF, can improve signal penetration and decrease imaging time. The prognostic and therapeutic implications of these findings are still not fully explored, however, and the role of FAF in DR remains investigative.DR affects approximately 35% of the more than 422 million adults suffering from diabetes worldwide.,108,109 Increased FAF signal has been significantly correlated with ellipsoid zone (EZ) loss and showed an association with poorer visual acuity, independent on the severity of DME. Another investigation noted increased FAF and diminished MP sensitivity in areas of DME, as well as significant association between abnormal FAF and OCT loci. On the other hand, green FAF has not shown use in identifying DME. A comparative series using UWF green FAF did not show correlation with central retinal thickness (CRT) or the pattern of DME distribution, although macular hemorrhage and microaneurysms could still be visualized.As DR and DME primarily affect inner retinal layers, FAF is less useful than other modalities such as FA or OCT for clinical evaluation. However, FAF can provide useful adjunctive information. Blue macular FAF can increase in the presence of intraretinal fluid, while microaneurysms or blood are more easily visualized with FAF. Foveolar hyper-AF correlated with CME on OCT, and foveal lobules of hyper-AF corresponded to pooling of fluorescein dye on FA. As Xu et al previously demonstrated that perivascular and subretinal microglia are the primary sources of retinal LF, it was hypothesized that the displacement of macular pigments, which have absorption peaks at 460 nm, by cystoid cavities results in the abnormal signal.Bessho et al studied FAF with BRVO and DME-related CME and noted abnormal FAF signal with 488 nm excitation but not 580 nm excitation. Furthermore, a cystoid-appearing NIR-FAF pattern was related to poorer VA and increased central sub-field thickness (CST) but not to ELM status or presence of hyperreflective foci, compared with eyes without cystoid patterns.Yoshitake et al studied DME with cSLO NIR-FAF and found a correlation with a mosaic fluorescent pattern and cystoid spaces on SD-OCT, poorer VA, presence of hyperreflective foci, and disrupted ELM.There is limited role for routine FAF for diagnosis and management of DME given the advent of HD-OCT. However, evidence suggests correlations between FAF findings and cystoid changes, visual acuity, and tests of retinal sensitivity, and routine integration into multimodal DME evaluation may allow longitudinal monitoring of outer retinal and RPE integrity. obstructive sleep apnea, type A personalities, and sleep disturbances have been suggested. Both acute and chronic forms of CSCR have been described, with chronic arbitrarily being defined as longer than three to six months. Acute CSCR with serous retinal detachments and focal leakage on FA usually display normal AF signal, while those with more chronic fluid demonstrate moderately increased AF in the area of detachment. In those with years of symptoms, AF over the affected areas can be irregular with both hyper-AF and hypo-AF areas due to variable degeneration of photoreceptor outer segments and RPE. More homogenous hypo-AF is associated with complete RPE atrophy, while hyper-AF corresponds to retinal thinning and outer retinal atrophy with an intact RPE, and is often seen at the border of the hypo-AF patches but can be isolated [Figure 5].,119 Granular AF represents either variable RPE atrophy and/or hyperreflective foci in the outer retina on HD-OCT.CSCR causes idiopathic serous detachments of the retina and RPE, and associations between corticosteroid use, A recent study provided six retina specialists with anonymous clinical data to correlate with multimodal imaging including OCT, SW-AF, FA, and ICGA for 100 patients. Significant discrepancy between diagnostic descriptions, especially among chronic CSCR cases, was noted, with 36 different terms used by graders. Small amounts of fluid could be easily identified on OCT. However, a “subclinical” term was frequently used when hypo-AF on FAF was present without fluid on OCT, highlighting the importance of FAF for diagnosis when neither FA nor OCT show clinical activity.Cases are often assumed “chronic” in the presence of non-resolving fluid or with degenerative RPE changes without obvious fluid. Duration of symptoms was shortest in the blocked FAF group, followed by mottled FAF, hyper FAF, hyper/hypo FAF, and descending tract. Additionally, blocked FAF had the best mean VA, which is reasonable given likely shorter duration of SRF in this group. The presence of EZ disruption, OS elongation, and RPE proliferation on HD-OCT were more likely with increasing duration of symptoms; all “blocked FAF” eyes had fully intact EZ layers. Increased duration of SRF likely resulted in variable accumulation of shed photoreceptor outer segments and resultant increased FAF.A study by Han et al described five patterns of acute CSCR on FAF: blocked FAF, with no changes or uniform hypo-AF in the region of SRF; mottled FAF, with grainy AF signal when compared with normal surrounding background AF; hyper FAF when compared with surrounding AF; hyper/hypo FAF with mixed areas of AF; and descending tract, with inferiorly directed hypo-AF originating in the macula. These gravitational tracts were noted in 49% of eyes, which was similar to the rate reported in another study. Foveal hypo-AF was seen in 75% of eyes, and foveal AF change was the only FAF feature that correlated with poorer VA at last visit.At the final visit in a large study of chronic CSCR, FAF showed variable morphologies, including mottled hyper-AF and hypo-AF changes, multifocal posterior pole lesions, and inferior gravitational tracts. Another study evaluated macular FAF compared with MP sensitivities and VA in chronic CSCR. The mean retinal sensitivity in regions of iso-FAF  was normal, while it was reduced at points with both hyper-AF and hypo-AF.In a study by Imamura et al, visual acuity decreases were significantly associated with age, confluent hypo-AF in the macula or peripapillary region, and granular hypo-AF in a multivariate regression analysis. Peripheral hyper-AF lesions may indicate activity in the absence of macular involvement and warrant additional imaging.More CSCR abnormalities can be imaged with UWF-FAF than traditional FAF, with 57% of CSCR eyes in one study containing extensive peripheral retinal involvement. Multimodal imaging demonstrated that RPE apertures corresponded to areas of RPE interruption or attenuation mostly at the apex but also at the bases of the PED, and both FA and OCTA showed no evidence of CNV. These apertures should be distinguished from RPE rips, which assume neovascularization, in order to prevent unwarranted treatment.Iovino et al used the term RPE aperture to describe hypo-AF areas within PEDs in CSCR. In addition, regions of hypo-AF on NIR-AF correlated with the site of RPE detachment with or without leakage on FA. However, whether or not NIR-AF provides additional information for clinical decision-making is unclear, and most data support a multimodal approach with HD-OCT, FAF, and FA to guide treatment.Although most studies used SW-AF, there is suggestion that NIR-AF may be more sensitive in detecting retinal abnormalities in CSCR. Best vitelliform macular dystrophy (BD), retinitis pigmentosa/rod-cone dystrophies (RP), central areolar choroidal dystrophy (CACD), and the pattern dystrophies. Indeed, a deep-learning neural network was able to distinguish between STGD, BD, RP, and healthy retinas with an accuracy of 95% based on FAF alone.Many retinal dystrophies are particularly suited for evaluation with FAF due to the degeneration of photoreceptor and RPE with or without the accumulation of LF. AF patterns have been shown to aid diagnosis in a multitude of dystrophies including STGD/fundus flavimaculatus, With improved speed, lower costs, and high accuracy of newer genetic panels, the genotype of the dystrophy has become the paradigm for diagnosis of inherited retinal diseases, especially with the introduction of targeted gene therapies. Regardless, multimodal imaging, especially FAF given its cost-effectiveness and accessibility, has become paramount in monitoring disease progression for prognostication and for clinical trial outcomes.,136However, significant phenotypic overlap can exist among many different genotypes. These so-called “pisciform” flecks are variably hyper-AF due to the buildup of RPE LF. It is classically associated with autosomal recessive (AR) mutations in the ABCA4 gene although autosomal dominant (AD) ELOVL4 and PRPH2/RDS have been reported.,140The most common juvenile retinal dystrophy, STGD is a phenotype characterized by variable macular atrophy with white–yellow deep retinal lesions in the posterior pole and extending to the mid-peripheral retina. The age of this AF ceiling has been shown to depend on both the severity of ABCA4 mutation as well as gene affected. Sparing of the peripapillary retina on SW-AF is characteristic of STGD. Lois et al showed normal, low, high, or mixed FAF patterns in the macula could be found . Lower macular AF values were associated with more atrophy and peripheral rod and cone dysfunction. The subclinical buildup of LF results in increased qAF levels in regions of normal-appearing retina that may precede visual impairment. One case of presumed STGD demonstrated normal qAF and was thereafter found to have X-linked retinitis pigmentosa.Fundus of Stargardt patients display generalized increased AF which reaches a peak value at a certain age and then declines, with the development of dark flecks and/or atrophic changes.Although SW-AF is the predominant modality in existing clinical trials and cross-sectional studies, both SW-AF and NIR-FAF sufficiently image the abnormalities in STD. Cicinelli et al found that while NIR-FAF and SW-AF agreed on baseline assessments, they measured different rates of progression on follow-up and were therefore not interchangeable. on UWF-FAF imaging. Type I consisted of only macular lesions. Type II consisted of macular atrophy with variable peripheral FAF flecks and atrophy. Type 3 contained both macular and peripheral atrophy with three subtypes based on extent of atrophy. Peripheral abnormalities outside of the standard 55° FAF imaging were noted in 72.4% of cases. These included more extensive atrophic or hyper-AF flecks, a posterior polar zone of increased AF in a circular pattern outside of the arcades, and occasional semicircular or zonal abnormalities.Three patterns were described by Klufas et alLonger SW-AF lifetimes for individual flecks on FLIO have been noted after one year of follow-up.Conditions characterized by the accumulation of subfoveal vitelliform material include BD, autosomal recessive bestrophinopathy (ARB), adult onset vitelliform macular dystrophy (AOVMD) (one of the pattern dystrophies), and both idiopathic and paraneoplastic acute exudative polymorphous vitelliform maculopathy (AEPVM). Vitelliform material was shown to be formed from shed photoreceptor debris and LF accumulation in the subretinal space.BEST1, the gene encoding bestrophin-1, whose product localizes to the basolateral plasma membrane of the RPE. Progression of BD is classically described by five stages with variable rates of progression: previtelliform , vitrelliform, pseudohypopyon, vitelliruptive, and atrophic/cicatricial.One of the more common early onset macular dystrophies, BD results from AD mutations in  The hyper-AF pattern on SW-AF was associated with earlier stages and the best BCVA, while the hypo-AF pattern was seen more often in later stages and the poorest BCVA. Areas of hyper-AF on SW-AF generally correspond to subretinal deposits of vitelliform material on HD-OCT. The pseudohypopyon stage exhibits hyper-AF in the region of the vitelliform material, with hypo-AF elsewhere on the SW-AF. The NIR-AF images in this stage show generalized hypo-AF with more hyper-AF foci. In the vitelliruptive phase, both hyper-AF and hypo-AF zones can be noted on SW-AF and NIR-AF. The atrophic stage is marked by mostly absent AF signal on both SW-AF and NIR-AF.,149Parodi et al described six main FAF patterns in BD: normal, hyper-autofluorescent, hypo-autofluorescent, patchy, spoke-like, and multifocal.BEST1 mutations were noted before any changes on SD-OCT or SW-AF evolved.,150 Parodi et al suggest this relates to RPE dysfunction and impaired melanin exocytosis and dispersion, leading to hypo-AF in NIR-AF.,150 Areas outside the central lesion do not display increased qAF levels, as opposed to STGD.Interestingly, preclinical central hypo-AF on NIR-AF in patients with confirmed  These include areas of vitelliform deposition with hyper-AF, as well as zonal swaths of hyper-AF and/or mottled hypo-AF. The hyper-AF regions may correspond to outer retinal thickening and subretinal depositions, or to outer retinal atrophy.AF findings in ARB are similar but often more extensive and variable, especially on UWF-FAF imaging. and paraneoplastic AEPVM have been described, and both present with acute vision loss associated with multiple, diffuse, yellow–white, variably shaped deep chorioretinal lesions, and serous macular detachments. In early stages, the serous fluid is not hyper-AF on SW-AF, but as the yellow–white subretinal material develops, it autofluoresces brightly. These lesions can evolve with gravity dependence and dissolution, forming punctate hyper-AF and hypo-AF patterns with eventual normalization of the FAF pattern.,155Both idiopathic They are genetically heterogeneous but are most frequently caused by PRPH2 gene mutations. The gene product is an integral membrane protein that functions in photoreceptor outer segment morphogenesis.The pattern dystrophies are grouped as such due to patterns of abnormal LF accumulation and RPE damage in the posterior pole. Parodi et al examined AOVMD with SW-AF and NIR-AF. On SW-AF, maculas with AOVMD displayed a normal pattern, focal hyper-AF, or patchy AF. They found BCVA to be decreased compared with controls in all AOVMD, with the lowest BCVA in the patchy group. With NIR-AF, the fovea can display central hypo-AF with surrounding variable hyper-AF, occasionally with a central hyper-AF lesion.Although classified as a pattern dystrophy, AOVMD is phenotypically diverse and shares many features with the bestrophinopathies. It usually presents in middle age with bilateral, subfoveal, vitelliform lesions less than one-half DD in width but may occasionally be multifocal.ABCA4-related STGD, although it usually occurs at an older age. On SW-AF, multiple yellow flecks in the peripheral macula are hyper-AF and may be surrounded by small adjacent hypo-AF zones. Atrophy of the posterior pole is evident usually at ages past 50, much older than STGD in which atrophy usually presents by adolescence.Multifocal pattern dystrophy (MPD) simulating fundus flavimaculatus/STGD shows similar findings to ,159Butterfly pattern dystrophy is characterized by a butterfly wing-shaped pattern of pigment deposition at the macula. A mixed pattern of SW-AF can be noted, with focal hyper-AF corresponding to the yellowish LF deposits or pigmented RPE hypertrophy, with hypo-AF in surrounding atrophic areas.Reticular pattern dystrophy is rare and presents with net-like pigmentation of the macula. This is delineated on SW-AF with hyper-AF with surrounding hypo-AF.Fundus pulverulentus is very rare pattern dystrophy noted by coarse pigment mottling in the macula. Multiple hypo-AF spots with interposed reticular hyper-AF lesions may be present. It is usually AD, with PRPH2 mutations being the most common, although AR cases have been reported.CACD is an inherited retinal dystrophy causing dysfunction of photoreceptors and visual acuity usually between ages 30 and 60, resulting in a well-demarcated macular area of outer retinal, RPE, and choriocapillaris atrophy. Stages 1 and 2 may mimic intermediate AMD. A speckled pattern of hypo- and hyper-AF on SW-AF may present that is more well-demarcated and more regular than in eyes with AMD, with less extramacular extension of lesions. Stages 3 and 4 are marked by central GA. This GA in CACD is more sharply demarcated and round to oval as opposed to patchy and more ill-defined GA in AMD. Additionally, FAF changes in AMD often extend beyond the macula and are less well-demarcated than in CACD, and HD-OCT will demonstrate sub-RPE deposits surrounding the GA.Differentiation between CACD and GA from AMD can be difficult and is often disregarded given the extreme difference in prevalence. Four clinical stages of increasing chorioretinal damage are present in CACD. Full-field ERG is the gold standard diagnostic test, but in more advanced disease this test loses clinical utility. The widespread and often unique outer retinal and RPE manifestations lend themselves well to FAF imaging.RP is a blanket term given to hereditary retinal dystrophies that feature rod and cone photoreceptor degeneration. It affects approximately 1 in 4,000 individuals worldwide and may feature AD, AR, or X-linked inheritance. This constriction is marked by a decrease in EZ band diameter on HD-OCT and suggests progression of structural disorganization of photoreceptor. Moreover, retinal sensitivity on MP and both static and dynamic perimetry has been shown to be relatively preserved within the AF ring and impaired outside the ring. Retinal sensitivity correlated well with outer retinal thickness on HD-OCT but not with total or inner retinal thickness.A characteristic hyper-AF parafoveal ring may be seen in many genotypes of RP on both SW-AF and NIR-AF . It is thought to represent an area of increased photoreceptor phagocytosis and LF accumulation. This ring may often constrict over time as the disease progresses, as opposed to cone–rod dystrophies in which the ring may expand. Robson et al demonstrated that radius of the hyper-AF ring was linearly correlated with pERG amplitude, that is, a larger ring showed greater response on pERG, presumably because the hyper-AF ring demarcates the area between markedly abnormal peripheral retina and more normal central retina. Dowd-Schoeman et al evaluated 31 patients with genotype-confirmed RP with various FAF modalities and described macular patterns of central foveal hyper-AF, perifoveal ring hyper-AF, macular ring hyper-AF, and a bull's eye AF. They further noted extramacular AF patterns of a mid-peripheral hyper-AF ring, extramacular hyper-AF spots, patchy hypo-AF, and diffuse peripheral hypo-AF. Oishi et al imaged 75 patients with various RP genotypes using UWF-FAF. They described three types of macular FAF: type 2 demonstrated ring-shaped perifoveal hyper-AF, type 3 showed abnormal foveal hyper-AF, and type 1 showed neither finding. Regardless of peripheral pathology, type 3 eyes had worse VA than type 1 or 2. The percentage of abnormal peripheral hypo-AF correlated well with visual field area, VA, duration of symptoms, and mean deviation (MD) on static perimetry.Murakami et al categorized three SW-AF ring patterns with RP: abnormal ring AF (most common), abnormal central AF, and neither abnormal ring nor central AF. Another study by Duncker et al noted much lower NIR-AF signal outside of the ring compared to inside the ring, and the inner border of the ring was closer to the fovea on NIR-AF than SW-AF. The inner NIR-AF border corresponded to a location where the EZ band was partially intact on HD-OCT, suggesting NIR-AF can detect RP progression prior to the development of SW-AF-sensitive fluorophores.Schuerch et al demonstrated with non-UWF imaging that SW-AF qAF values internal to the ring were abnormal in only 29% of patients, while qAF values external to the ring were abnormal in just 53% of eyes. Longer lifetimes were found in areas of photoreceptor atrophy and even longer lifetimes in areas of photoreceptor and RPE atrophy. The perifoveal ring of prolonged lifetimes was wider than that seen on qualitative SW-AF. However, although the measured FLIO values just outside the ring were statistically longer than those just inside of the ring, they were not significantly longer than healthy eyes. This is in line with the above findings of abnormal qAF findings external and internal to the ring in only a subset of patients.Dysli et al evaluated FLIO patterns in patients with RP. macular dystrophies, Leber's congenital amaurosis (LCA), Best disease, some cone–rod dystrophies, and X-linked retinoschisis (RS). This shared findings suggests common mechanisms of retinal degeneration in certain retinal dystrophies.An autofluorescent ring is not specific to RP, however, and can be found in pigmented paravenous retinochoroidal atrophy (PPRCA), The cumulative area of hypo-AF on UWF-FAF were significantly correlated with Goldmann perimetry scotomata in another study, but areas of peripheral mottled FAF did not necessarily correlate with VF defects. Retinal sensitivity is better correlated to outer retinal thickness and therefore photoreceptor damage, which is often demarcated by the hyper-AF macula ring, and severe peripheral VF defects can be seen in the absence of RPE loss and marked hypo-AF. Other studies found female carriers of X-linked RP with RPGR mutations to demonstrate variable radial FAF patterns that can extend to the periphery.,185 An inferonasal boundary of abnormal FAF patterns may be noted in patients with RP or STGD and is thought to be related to closure of the optic fissure.As a pan-retinal degeneration, RP may be better imaged with UWF modalities. Peripheral hypo-AF may be punctate/mottled or nummular/patchy, with more extensive patchy changes seen in older patients or those with longer symptom durations.CYP4V2. Along with diffuse retinal and RPE degeneration, it is marked by crystalline deposits in the retina and occasionally the cornea. These deposits are not easily detectable on SW-AF, and they may disappear with progressive chorioretinal degeneration and while they are hallmarks of BCD, they can be seen in other retinal conditions. Crystalline deposition on fundus photographs slightly precedes mottled hypo-AF RPE degeneration on SW-AF that can begin in the posterior pole and peripapillary region. This eventually leads to diffuse hypo-AF with remaining isoAF irregular islands of RPE. However, hypo-AF and hyper-AF lesions are not spatially correlated with the crystalline deposits on color imaging,,190 and NIR-R appears to visualize the crystalline deposits best, although this is also variable. Sclerotic choroidal vessels are typical in BCD and underlie regions of chorioretinal atrophy that can be seen on color imaging. These sclerotic vessels have been shown to be hyper-AF on SW-AF, while non-sclerotic choroidal vessels remain hypo-AF.Bietti's crystalline dystrophy (BCD) is an RP phenotype caused by AR mutations in CRB1 gene can lead to both LCA and RP with distinctive FAF features. Typical findings of peripheral bone-spicule retinal and RPE degeneration with or without hyper-AF macular rings can be found. However, hyper-AF optic disc drusen and a peculiar peri-arteriolar sparing of the RPE with normal AF can be seen. Peripapillary and peripheral hamartomas have been reported with variable SW-AF patterns.,193Mutations in the  Paravenous hypo-AF from RPE atrophy and pigment clumping can be found, with adjacent zones of hyper-AF . These zones of hyper-AF may be found along peripheral veins and may precede frank RPE atrophy and pigment clumping.PPRCA is a degenerative phenotype marked by retinal, RPE, and choriocapillaris atrophy with pigmentation primarily along the retinal veins. Inflammatory, infectious, genetic, and idiopathic etiologies have been described, but the unifying pathogenesis remains unclear.NR2E3-linked RP and attributed to this mutation, but was thereafter also noted in USH2A-linked RP. In a study by Trichonas et al, the pattern of peripheral UWF-FAF was noted to differ among patients with certain genetically confirmed RP mutations. Distinct changes such as the double hyper-AF ring with USH2A or diffuse peripheral hypo-AF and dark appearance with USH2A or PRPH2/RDS mutations were demonstrated. However, a recent study also noted the double hyper-AF ring in a patient with RHO-linked RP and a patient with RPGR-linked RP, as well as significant overlap in other FAF patterns among genotypes. Although many patterns of FAF have been described in RP, and some show predilection for certain genes, it is difficult to determine the RP genotype by FAF pattern alone. However, FAF can be helpful in differentiating RP from other conditions in patients with unexplained visual acuity or visual field loss with or without abnormal findings on fundus exam. FAF findings in RP are generally symmetrical and often include hypo-AF spots or patches in the midperiphery with or without macular AF changes. In addition, FAF is helpful in assessing progression of the disease over time; this may include an increase in hypo-AF spots and a reduction in the size of macular hyper-AF ring.Certain FAF patterns have been thought to be pathognomonic for certain RP genotypes. For example, a double concentric hyper-AF ring was first described in  After baseline evaluation, routine annual screening after five years is recommend by the American Academy of Ophthalmology (AAO), with SW-AF recommended as one of the ancillary screening tests. In non-Asians, HCQ toxicity manifests with parafoveal hyper-AF rings, corresponding to photoreceptor degeneration, that eventually becomes less hyper-AF or even hypo-AF with progression to RPE atrophy. In Asians, there is a higher chance for additional pericentral, peripheral macular damage, or even pericentral changes alone. Patients with pericentral damage also tend to have longer duration of HCQ therapy and larger cumulative doses.HCQ (brand name Plaquenil) is commonly used for treatment of systemic lupus erythematosus, rheumatoid arthritis, and other inflammatory systemic and dermatologic conditions. Patients using  Prolonged FLIO lifetimes have been noted in a parafoveal distribution corresponding to regions of toxicity.The maculopathy may be asymmetric, often starting inferiorly and progressing circumferentially, but may be more symmetric on NIR-FAF. Early changes may be more detectable on qAF as well. and current guidelines still recommend HD-OCT and VF as primary screening modalities.Despite this, HD-OCT has been reported as more sensitive for detecting HCQ-related structural damage than either SW-AF or NIR-AF, Findings included subtle vitelliform deposits and patchy paracentral RPE atrophy that on SW-AF were far more apparent as mottled, irregular patterns of FAF in the posterior pole. Further studies demonstrated a wider phenotypic spectrum, from subtle pattern-like macular changes to confluent pattern of hyper-AF and hypo-AF spots and reticular changes, to widespread nummular chorioretinal atrophy.,209 The hyper-AF lesions localized with yellow deposits and hyperpigmented spots on color imaging. These hyperpigmented spots localized to focal thickening of the RPE on HD-OCT. Most patients show relatively preserved VA in the range of 20/20 to 20/60 but may have subjective visual complaints such as blurring or nyctalopia.A pigmentary maculopathy associated with pentosan polysulfate sodium (PPS), a commonly used treatment of bladder pain and interstitial cystitis, was recently described by Pearce et al.,208 A mathematical analysis of one cohort found the greatest correlation with standardized cumulative PPS dose adjusted to weight, with 75% (n = 3) of patients with accumulated dose of ,211While toxic dose thresholds are still unclear, Wang et al noted no affected patient with cumulative doses  Patterns of SW-AF abnormalities are far more prominent than color imaging and have been categorized into minimal changes, focal hyper-AF with a background of reticular hypo-AF, patchy hyper-AF in a pattern-like distribution, or a diffuse speckled pattern of FAF changes. Studies noted more severe visual abnormalities with the patchy or speckled patterns.,214 Pattern-like changes described include a butterfly-like, fundus flavimaculatus-like, fundus pulverulentus-like, and vitelliform-like distributions.DFO is an iron-chelating compound given for treatment of chronic iron overload in conditions such as β-thalassemia that require frequent blood transfusions. Retinal complications of long-term use include macular and peripheral pigmentary changes accompanied by mildly reduced VA and diminished ERG amplitudes.ABCC6, that affects the skin and connective tissues, including ocular structures. Ophthalmic findings include peau d'orange, optic nerve head drusen, RPE atrophy, angioid streaks, “comet-tail” deposits, and CNV. Macular SW-AF changes include hypo-AF subretinal deposits similar to SDD on HD-OCT, hypo-AF subretinal deposits that appear yellow–brown and cause a pattern dystrophy-like appearance, and hyper-AF subretinal deposits that may be accompanied by SRF. While dark orange–red on color imaging, angioid streaks appear hypo-AF with isoAF specks and are often accompanied by hypo-AF peripapillary atrophy. Progressive RPE atrophy with or without CNV formation is hypo-AF, often with intervening hyper-AF specks, and may extend from the macula and peripapillary area into the periphery. Calcification of Bruch's membrane posteriorly, termed “coquille d'oeuf” (eggshell), is peripherally bordered by the classic peau d'orange. This calcification may be very subtly hypo-AF and more detectable by decreased qAF. Comet lesions and optic disc drusen are hyper-AF. Widespread posterior pole atrophy may obscure many classic findings, and UWF-FAF may allow detection of peripheral lesions to aid diagnosis.PXE is a recessive genetic disorder, marked by mutations in  Retinal atrophy, cavitation, crystal deposition, microaneurysms and vascular abnormalities with leakage, and CNV may result with disease progression. Impaired storage of pigments such as lutein and zeaxanthin, purportedly by Muller cells, results in depletion of macular pigments and increased central macular AF as well as increased FLIO. This pigment loss starts temporally and spreads throughout the fovea, but pigment clumping may cause discrete plaques that are hypo-AF. The presence of increased macular AF or clumps of pigment causing decreased AF were found to be associated with worse VA at baseline and were predictive of slow visual decline at two years, although the natural history of the disease is slowly progressive. Retinal crystals, extent of involvement, and angled dipping vessels (right-angle venules) are better represented on color imaging than FAF. However, SW-AF can detect subtle, usually temporal hyper-AF that can precede HD-OCT, MP, and angiographic changes, but whether this is clinically useful given current lack of effective treatments is to be seen.MacTel is an idiopathic bilateral condition causing characteristic macular changes and affecting the juxtafoveolar capillary network.While HD-OCT imaging can clearly delineate RRD versus RS, technical challenges of capturing peripheral images limit its clinical utility. The advent of UWF imaging provided quick and reliable monitoring of peripheral pathologies but without the depth resolution of OCT. A number of studies have examined the FAF signal in RRD, RS, and RS detachment (RSD) given previous work noting FAF changes related to retinal layer separations and metabolic changes. This hyper-AF PM corresponded to shallow SRF leading up to the frank RRD. After PPV, disappearance of the hyper-AF PM with demonstration of a demarcation line often occurred. Another study examined FAF changes after primary SB and noted FAF changes in the area of cryopexy depending on the amount of RPE destruction, and buckling elements produced hypo-AF shadows at their posterior edge, with radial hyper-AF streaks approximately 50% of the time.Witmer et al demonstrated hypo-AF over the detachment in all eyes, with a hyper-AF leading edge or posterior margin (PM) in all macula-involving RRDs and 75% of macula-on RRDs. All eyes with RS showed no or minimal AF change over the main RS cavity, while the PM was variable. Pooled analysis found that hypo-AF in RRD had a positive predictive value (PPV) of 95% and negative predictive value (NPV) of 76%, while hyper-AF in RSD indicating SRF had a PPV of 100%.Navaratnam et al expanded on these findings and included eyes with RS and RSD. Although finding similar changes with acute RRD, they noted isoAF or hypo-AF in areas of chronic RRD (Francone et al correlated HD-OCT with UWF-FAF in patients with RS complicated by RRD and also found mostly isoAF change over the RS cavity with a hypo-AF posterior border in bullous RS. However, hyper-AF at or extending past the PM of the RS corresponded to SRF or full-thickness RRD on HD-OCT in all eyes.Hypo-AF over the RRD in these studies was purported to result from blockage of RPE signal from edematous retina and SRF, while the hyper-AF PM was suggested to result from buildup of fluorophores in either photoreceptor outer segments or RPE from impaired metabolism. Careful examination of RS with serial UWF-FAF imaging or peri-operative UWF-FAF imaging of RRDs can be useful to detect progression of SRF or conversion of schisis cavities into full-thickness detachments.in vivo topographical imaging of retinal metabolic changes and general health of the photoreceptor and RPE layers. Advances in imaging technologies have demonstrated more peripheral FAF changes as well as earlier detection of macular metabolic changes that can be invaluable for clinical decision-making. Several imaging platforms are available, each with their advantages and disadvantages, and careful consideration should be made both in system utilization and imaging interpretation based on published findings with each modality. While SW-AF has historically been most studied, further improvements and accessibility for other techniques such as NIR-FAF, qAF, and FLIO may increase diagnostic and monitoring capabilities.FAF allows noninvasive and This review has been supported, in part, by an unrestricted grant to the Department of Ophthalmology of the University of Southern California from Research to Prevent Blindness, New York, NY.There is no conflict of interest.


Thyroid storm is a rare but life-threatening endocrinological emergency with significant mortality ranging from 10-30% with multi-organ involvement and failure. In view of the rarity of this condition and efficacy of established first line medical treatment, use of extra-corporeal treatments are uncommon, not well-studied, and its available evidence exists only from case reports and case series. We describe a 28-year-old man who presented with an out-of-hospital cardiac arrest secondary to thyroid storm. Despite conventional first-line pharmacotherapy, he developed cardiogenic shock and circulatory collapse with intravenous esmolol infusion, as well as multi-organ failure. He required therapeutic plasma exchange, concurrent renal replacement therapy, and veno-arterial extra-corporeal membrane oxygenation, one of the few reported cases in the literature. While there was clinical stabilization and improvement in tri-iodothyronine levels on three extra-corporeal systems, he suffered irreversible hypoxic-ischemic brain injury. We reviewed the use of early therapeutic plasma exchange and extra-corporeal membrane oxygenation, as well as the development of other novel extra-corporeal modalities when conventional pharmacotherapy is unsuccessful or contraindicated. This case also highlights the complexities in the management of thyroid storm, calling for caution with beta-blockade use in thyrocardiac disease, with close monitoring and prompt organ support. Thyroid storm (TS) is a life-threatening exacerbation of the hyperthyroid state characterized by multi-organ dysfunction of the cardiovascular, thermoregulatory, gastrointestinal-hepatic and central nervous systems. While the incidence among hospitalized patients is estimated to be low at 1-2% . The diaWe present a case of TS in a young patient with undiagnosed Graves’ disease, presenting with an out-of-hospital cardiac arrest. Initially hemodynamically stable following return of spontaneous circulation, he developed circulatory collapse after intravenous esmolol infusion, initiated for control of tachycardia. There was consequent multi-organ failure which contraindicated the use of standard anti-thyroid drug therapy. He required three extra-corporeal systems of continuous renal replacement therapy (CRRT), veno-arterial extra-corporeal membrane oxygenation (VA-ECMO) and therapeutic plasma exchange (TPE) for stabilization, one of the few reported cases in the literature.A 28-year-old male presented with an out-of-hospital ventricular fibrillation (VF) arrest, preceded by an acute respiratory illness. There was return of spontaneous circulation after 60 minutes of resuscitation with bystander cardiopulmonary resuscitation and external defibrillation by paramedics. In the Emergency Department, he was febrile at 40.5 degrees Celsius, hypertensive with a blood pressure of 146/83mmHg and tachycardic with a heart rate of 155 beats per minute. Physical examination was unremarkable, except for a Glasgow Coma Scale of 3. No goiter was seen on examination. Corroborative history from his family confirmed symptoms of heat intolerance, loss of weight, hand tremors and palpitations in the preceding two months, as well as a maternal history of Graves’ thyrotoxicosis.Initial investigations showed elevated inflammatory markers, mixed respiratory and metabolic acidosis, raised troponin I, but with normal electrolyte levels. Electrocardiogram confirmed sinus tachycardia. Chest radiograph showed prominent pulmonary vasculature without evidence of pneumonia. Point-of-care echocardiogram showed impaired left ventricular systolic function without other obvious abnormalities; the marked sinus tachycardia precluded accurate estimation of the left ventricular ejection fraction (LVEF). Computed tomographic (CT) scan of the brain was normal, and urine drug screen was negative. A coronary angiogram performed was normal, and a provisional diagnosis of acute myocarditis was made. Thyroid function test, sent as part of investigations for myocarditis, showed thyrotoxicosis with an elevated serum free thyroxine (FT4) level of 42.1pmol/L (reference range: 8.0-16.0pmol/L) and a suppressed serum thyroid stimulating hormone (TSH) at <0.01mIU/L (reference range: 0.45-4.50mIU/L)  in TS management. On review of the medical literature of articles in English, there has only been four case reports describing the concurrent use of TPE and ECMO in patients with thyrotoxicosis with circulatory collapse –7, with Characterized by extreme multi-systemic manifestations of thyrotoxicosis, TS is uncommon but potentially fatal, with a mortality rate of 10-30% . StandarTPE is an extra-corporeal blood purification technique used for eliminating large molecular substances. Currently, TPE has a Class II indication for TS in the 2019 American Society for Apheresis (ASFA) guidelines, either as a standalone or adjunct therapy, although the grade 2C level of recommendation suggests the evidence arises mostly from case reports and case series, with an absence of prospective randomized controlled trials . TPE is ECMO is an established life-saving treatment option for patients who develop acute cardiopulmonary failure , althougNovel therapies are also increasingly considered for TS, using principles similar to TPE by removing protein bound thyroid hormones. Case reports with the use of dialysis has been proposed in management of thyroid storm, most notably with CRRT, which is preferred due to its better tolerability in hemodynamically unstable patients due to its slower rate of exchange of fluids and solutes. Parikh et al.  and KobaThese cases provide some evidence of the use and benefits of extra-corporeal systems in the management of TS, after conventional pharmacotherapy is unsuccessful or contraindicated. Owing to the efficacy of pharmacotherapy and risks of extra-corporeal systems, conventional pharmacotherapy should be always be instituted as initial therapy. Comparison trials between pharmacotherapy and extra-corporeal systems or randomized controlled trials are unavailable due to the rarity of TS, and are unlikely to be performed now given the established efficacy of first line pharmacological agents. Retrospective analysis from the National Inpatient Database in Japan has shown that use of extra-corporeal systems is associated with higher mortality. It reported increased mortality in patients requiring hemodialysis and TPE with adjusted odds ratio for mortality at 4.81. The mortality was 61.9% in 13 out of 21 patients, compared to a mortality of 43.3% requiring either hemodialysis or TPE, and 7.8% requiring neither support. The use of ECMO had a trend towards increased mortality , with a mortality of 72.2% among 13 of 18 patients, as compared to 9.3% in patients not requiring ECMO . These nWhile our patient received guideline-directed TS pharmacological therapy in a timely fashion, the development of circulatory collapse with consequent multi-organ failure following intravenous esmolol infusion, an ultra-short acting beta-blocker, deserves further discussion.The cardiovascular effects in TS are driven largely by T3, leading to increased chronotropy and inotropy, improved diastolic relaxation and decreased peripheral resistance, eventually resulting in high cardiac output (CO) heart failure (HF), estimated to be seen in 6% of patients with thyrotoxicosis. This is thought to be reversible with treatment with thyrotoxicosis, with a small study showing improving in LVEF from 28% to 55% . Cardiomversus 2.3 hours respectively) and duration of action, as well as its relatively higher beta 1-selectivity (Tachycardia is almost always present in TS, and patients with tachycardia exceeding 150 beats per minute are associated with a higher mortality rate in a retrospective Japanese cohort . Accordiectivity . The comectivity . Regardlectivity . The aboectivity  reviewedWe highlight a case of TS presenting with out-of-hospital cardiac arrest, with further hemodynamic decompensation following beta-blockade and multi-organ failure which limited therapeutic options. Despite prompt initiation of CRRT, VA-ECMO and TPE, he sustained hypoxic-ischemic brain injury. Underscoring the complexities in TS, this case calls for caution with beta-blockade in thyrocardiac disease, close monitoring and prompt organ support, and consideration of early TPE when conventional options fail. A review of the use of TPE and other extra-corporeal systems shows that TPE may be an underutilized rescue treatment for severe thyroid storm not amenable to conventional pharmacotherapy or contraindicated due to side effects or multi-organ involvement. Further study of novel extra-corporeal therapies for TS is needed to uncover its therapeutic potential, especially in the Intensive Care setting.All authors were involved in the management of the patient. SL, KW, and SY wrote the first draft of the manuscript. PL and KR reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Maternal pre-pregnancy overweight/obesity and uncontrolled third-trimester glycemia among GDM-negative women both have adverse effects on offspring growth within 24 months. With the combination of increasing pre-pregnancy BMI and maternal third-trimester FPG, the possibility of offspring catch-up growth increases.Intrauterine modifiable maternal metabolic factors are essential to the early growth of offspring. The study sought to evaluate the associations of pre-pregnancy BMI and third-trimester fasting plasma glucose (FPG) with offspring growth outcomes within 24 months among GDM-negative pregnant women. Four hundred eighty-three mother –offspring dyads were included from the Shanghai Maternal-Child Pairs Cohort. The pregnant women were categorized into four mutually exclusive groups according to pre-pregnancy BMI as normal or overweight/obesity and third-trimester FPG as controlled or not controlled. Offspring growth in early life was indicated by the BAZ (BMI Z-score), catch-up growth, and overweight/obesity. Among those with controlled third-trimester FPG, pre-pregnancy overweight/obesity significantly increased offspring birth weight, BAZ, and risks of overweight/obesity  within 24 months. Those who had uncontrolled third-trimester FPG had a reduced risk of offspring overweight/obesity within 24 months by 47%. The combination of pre-pregnancy overweight/obesity and maternal uncontrolled third-trimester FPG increased 5.24-fold risk of offspring catch-up growth within 24 months ( Early life is a sensitive period characterized by adipocyte proliferation; once the number of adipocytes is set, it does not decrease . GloballThe incidence of GDM in China is 17.5% , and appAmong women with or without GDM, in contrast to a normal pregnancy BMI, pre-pregnancy obesity is a risk factor for overweight/obesity in children ,17. Offs1c levels, which has clinical value [Currently, glycemic monitoring is performed mainly in the second trimester, and assessing glycemic control in the third trimester is not yet part of routine medical care for women in most countries ; howeveral value ,28. Moreal value . Higher al value ,28. The We aim to evaluate the associations of pre-pregnancy overweight/obesity and third-trimester glycemia, based on the categorized groups of the two-factor cross combination, in relation to the growth of offspring in early life. In addition, we wanted to assess whether pregnancy management recommendations for healthy overweight/obesity women need to be optimized.n = 483) who met the criteria to detect significant statistical difference. No significant difference was found between characteristics of the study participants compared to the subset used for complete analysis in Participants were selected from the ongoing Shanghai Maternal-Child Pairs Cohort (Shanghai MCPC), which began in April 2016 . Birth-weight-standard-deviation scores, standardized as the Z score for sex and gestational age, were calculated. The catch-up growth category in early life was divided into two groups based on the BAZ (BMI Z-score) increment: catch-up growth (≥0.67). A gain in BAZ greater than 0.67 SD scores between the zero and different months indicated clinically significant catch-up growth, and evidence suggested that children with catch-up growth in early life had a higher risk of central obesity at 5 years of age [Infant anthropometric data were body length and body weight of the offspring at birth and at eight subsequent time points . The Z score of growth was calculated with the WHO Anthro software (s of age . Overweis of age .2), with the results determined with one decimal place. Maternal pre-pregnancy BMI was divided into two groups  by the Chinese BMI cutoffs [Data for height and pre-pregnancy weight were obtained from the pregnant women’s baseline interview at 12–16 weeks of gestation, which were highly correlated with the weight and height that the researchers subsequently measured around the 21 weeks of gestation (correlation coefficient = 0.94 for weight and 0.97 for height). Height was measured in m  and weight in kg , was recalled by the pregnant women and surveyed by a nurse. Body mass index (BMI) was calculated by dividing body weight (kg) by height-squared , age at delivery, last menstrual period, blood pressure taken within 1 month before delivery, prenatal weight) from medical records. In addition, data on educational level, maternal smoke or alcohol during pregnancy, family income, delivery mode, mode of infant feeding within the first 6 months, breast feeding duration, physical activity level, and anxiety and depression in the third trimester were collected by means of professional questionnaires ,38,39. Ep-value < 0.05 was considered statistically significant. All of the analyses were performed with the statistical software packages R 3.4.3 and SPSS (v22.0).We categorized women into four mutually exclusive groups according to categories of pre-pregnancy BMI and FPG in the third trimester: Group 1 , Group 2 (overweight/obesity with pre-BMI and third-trimester FPG being controlled), Group 3 , and Group 4 (overweight/obesity with pre-BMI and third-trimester FPG not controlled). Differences among mean continuous variables by BMI/GDM group were evaluated using ANOVA with Dunnett’s or Bonferroni’s significant difference adjustment for multiple comparisons. Pre-pregnancy BMI and third-trimester glycemia were transformed into continuous variables, and the interaction term of both variates was added to the fully adjusted model to investigate the potential effect by multivariate regression. For all months, overweight/obesity rates of offspring were over 10%. To avoid using odds ratios (ORs) and overstating the risk, we calculated relative risks (RRs) instead by log-binomial regression or Poisson regression with robust variance estimation and linear regression models to evaluate the differences in offspring anthropometric outcomes  among different monthly time intervals between offspring in different groups. Generalized estimating equations were used to study the longitudinal development of offspring. Confounders were chosen based on previous studies and backward selections in primary analyses. Collinearity among the exposure variables was examined, with a maximum variance inflation factor of 2, suggesting no evidence of collinearity. Missing covariate data were imputed using multiple imputation. Significance tests were two-tailed, and a Of all studied GDM-negative pregnant women, the mean pre-pregnancy maternal BMI was 21.3, with 18.6% of women being overweight/obesity, and the proportion of women in the third-trimester FPG not-controlled group was 8.1%. p = 0.003).To identify the independent and combined effects of pre-pregnancy BMI and third-trimester FPG on offspring anthropometric outcomes within 24 months, p < 0.05) and had an increased BAZ in the first 9 and 12 months of life, as well as an increased risk of overweight/obesity in offspring at 9 months of age.Forest plot of the effect of maternal pre-pregnancy BMI and third-trimester FPG on offspring growth and development from 1–24 months according to groups is shown in We further compared the independent and combined effects of the four groups of pre-pregnancy BMI and third-trimester FPG on offspring growth . If mateAmong a group of pregnant women with normal pre-pregnancy BMI, third-trimester third-trimester FPG not controlled was a protective factor against overweight/obesity offspring within 2 years of age, reducing the risk by 47% . However, it seemed to increase the risk of offspring catch-up growth by 1.17 times, though this was not significant. In Model 2, an additional adjustment was made for gestational weight gain, but the results were almost the same as those in Model 1. The combination of pre-pregnancy overweight/obesity and maternal third-trimester FPG not controlled increased the risk of offspring catch-up growth within 24 months after birth .The present study focused on pregnant women without GDM. When women had a controlled FPG status, pre-pregnancy overweight/obesity might still significantly increase birth weight, the BAZ and the risk of the offspring being overweight/obesity during the first two years of life. Uncontrolled third-trimester glycemia can reduce the risk of overweight/obesity among offspring within two years after birth but can increase the likelihood of catch-up growth, even among the offspring of women who had a normal pre-pregnancy BMI. The combination of these two factors has a greater impact on the likelihood of offspring catch-up growth than each factor alone.The birth weight of newborns increased significantly with increasing pre-pregnancy BMI after controlling the third-trimester FPG, similar to another study . One of Systematic reviews  have invMonitoring glycemic control in the third trimester plays a relevant role in children’s health . SurveysPrevious research  establisSummary of previous research indicate that intrauterine hyperglycemia, which includes GDM and controlled third-trimester FPG, is related to childhood overweight/obesity in offspring ,24 and iGlobally, the highest prevalence of GDM was reported in Europe (31.0%) , which mThe longitudinal study allows for more latitude in discerning causality vs. association. By combining pre-BMI and FPG in the third trimester into groups, we explored the effects of the two factors on the growth of offspring within 24 months. We adjusted for several potential confounders, such as family history of diabetes, gestational weight gain, gestational physical level, gestational hypertension, anxiety and depression in the third trimester, and socioeconomic parameters, to improve the reliability of the results. Second, the subjects of our study were Chinese mother–child pairs, which minimized the confounding effects by ethnic background. There were still some limitations. Some subjects were lost to follow-up at multiple time points in early life, and the relatively small number of pre-pregnancy overweight/obesity women in the third-trimester FPG uncontrolled group may limit our power to detect significant differences among the groups for growth outcome comparisons and lead to unstable estimates. However, the use of longitudinal models would subsequently have resulted in increased power. Due to the limitation of the number of overweight and obese women before pregnancy in this study, they are combined for analysis. Being overweight or having obesity may have a differential impact on offspring, but it still needs to be verified by future studies. If pregnant women received treatment  for elevated glucose, associations with outcomes could be impacted by the treatment. Women would not get special treatment  if they were not diagnosed as GDM; meanwhile, we adjusted the energy intake and physical activity level in late-pregnancy to maintain the credibility of the results. We regret not having collected information of children’s own energy intake, as it has been recognized as an important factor for children growth and development. Additionally, the effect of blood glucose levels during pregnancy on the development of offspring is also reflected in indicators such as fat percentage and waist circumference, but we did not have data for these indicators. The use of FPG as a study indicator also has some clinical significance; whether FPG or PPG is more sensitive needs to be further studied in the future. Children in the Shanghai MCPC will be followed up until five years of age, and these observations will be used to evaluate the long-term repercussions of our findings.Both maternal pre-pregnancy overweight/obesity and uncontrolled third-trimester glycemia have negative effects on offspring growth within 24 months, which were mainly manifested in the increased risk of offspring overweight/obesity and increasing the probability of catch-up growth, respectively. Future primary prevention of childhood overweight/obesity needs to focus on screening for overweight/obesity women planning for pregnancy. At the same time, a negative GDM test during pregnancy is not the only signal of normal blood glucose levels; additional attention may be paid to the third-trimester glycemia.


Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen–surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin–ligand interaction, supported by present high-throughput “omics” technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19. Consequently, adhesins can be used to specifically enrich pathogens. Below, we explore this concept from a variety of viewpoints.The severity of an infectious disease depends on the level of invasiveness and the extent of host cell and tissue damage caused by the pathogen involved . A varyiThe core technologies used by the routine microbiology laboratory are still mostly microscopy- and culture-based. Immunological tests detecting pathogen-specific antigens and antibodies are also routinely used in diagnostics. Furthermore, over the past years, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and molecular (nucleic acid-targeting) testing have been introduced successfully in the routine clinical microbiology laboratory . The main technologies in routine high-throughput laboratories are relatively slow, usually taking at least one overnight incubation. They are limited in terms of sensitivity and specificity, suggesting that there is room for improvement ,20. CultDevelopment of better diagnostic tools requires an integrated and interdisciplinary research environment drawing on engineering, biomedical sciences, and product development activities in both academia and the IVD industry . SharingAn important recent example of the benefits of such an integrated approach was provided during the COVID-19 pandemic. All parties involved generated a huge portfolio of diagnostic tests, immunological and molecular, many of which were rapidly authorized for emergency use  by the US Food and Drug Administration (FDA) ,24. The From a purely technological perspective there are a number of competing approaches that will significantly influence the use of adhesion in clinical microbiological diagnostics. Three of these technologies need a brief assessment. First, PCR and related nucleic acid amplification technologies are sensitive, increasingly cheap, can be deployed widely and essentially detect all microbial species and resistance genes. This technology can be used directly on clinical specimens and it strongly depends on the number of pathogen cells available whether adhesion-based enrichment of such pathogens is required or not. Second, next generation (genome) sequencing (NGS) has become faster and more cost-effective over recent decades, and this is likely to continue. Its performance will soon equal or better that of amplification testing. This suggests that NGS will be routinely used in the microbiology lab. Finally, there is an increase in the rapid availability of high-affinity specific binding reagents other than functional adhesins, such as  antibodies, adhirons and aptamers ,27. ThesIn the following sections, we describe the major interactions between microbial adhesins and host ligands or receptors. We try to define what further structural biology studies are needed, and how these might be useful in the development of novel diagnostic tests.The initial interaction between pathogens and their hosts is defined at the molecular level by the selective interaction of pathogen adhesins with their host receptors. This specific interplay can be exploited in various stages of the classical microbiological diagnostic workflow. To do so, we must extend our understanding of the basic principles of pathogen adhesion so we can apply adhesion assays in the initial capture and enrichment of (complete or parts of) pathogens . All micThe emergence of SARS-CoV-2 has made it clear that not all adhesins have been identified yet. New ones can be detected using nucleic acid sequencing strategies and searches for new structures that are homologous to known adhesins. Otherwise, proteomic research can be applied to detect cellular surface proteins that provide adhesive characteristics. Random knock-out mutagenesis can also generate cells deficient in adhesion and reverse genetics then allows the functional analysis of the genes and proteins involved. Biophysical technologies can be used to define adhesin structures (see sections below).Viruses, with relatively small genomes, have a limited repertoire of adhesin structures per individual viral lineage, although an individual adhesin structure is repeated frequently on a single virion. It has to be noted that viruses that are becoming endemic or pandemic exist, even in single hosts, as a species swarm with differing receptor affinities. Recent examples include the SARS-CoV-2 variants such as the B.1.1.7 , B.1.351 , P.1  and B.617.2  variants of concern . The virBox 1The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). The precise mechanisms of disease are still incompletely understood  and replThe nature of the bacterial adhesion molecule varies from distinct organelles such as flagellae or fimbriae to surface exposed, cell wall- or cell membrane-attached proteins, lipids, and sugar (poly- or oligo-saccharide) moieties . AdhesinIn certain bacterial adhesins, such as the trimeric autotransporter adhesins (TAAs) , the indSurface exposed adhesion domains are external moieties and exposed parts of the proteins may be subject to strong environmental selection and possible natural adaptation ,66. HencThere are few structures of bacterial adhesins complexed with their ligands, even though there are many of virus-receptor complexes ,36. ThisNMR spectroscopy, X-ray crystallography, cryo-electron microscopy  showed how they are involved in entero–hemorrhagic pathogenicity [Nano-biosensors for miniaturized detection of adhesion, (cryo)EM for visualization of adhesion and identification of molecular partners, X-ray crystallography for the definition of global adhesin structure and more generic tools like NMR for local information on binding partners, and advanced bioinformatics all play essential roles in the further optimization of structure determination and translational applications ,90,91. Ial cells . The strgenicity ,95. Anotgenicity ). Adhirogenicity . Well-chExploiting the adhesion capacity of pathogens for in vitro diagnostics is a relatively new concept ,99, but E. coli O157:H7 has been successfully enriched from contaminated water samples [Listeria monocytogenes cells [Bacterial or viral detection and identification is a complicated multi-step process starting with the collection of clinical samples of diverse origin  and consistency . Several IVD development projects aim to optimize existing diagnostic tests or to develop novel, specific, and preferably ‘point-of-care’ (PoC) diagnostic tools . We envi samples ; and it es cells . PathogeIn many cases the sample needs to be pre-treated in order to prepare it for the actual diagnostic process. Unfortunately, uniform processing methods for samples of diverse origin are rare , a surface protein expressed by ylococci . SpA hasylococci . Bindingylococci ,130. SpAylococci . Howeverylococci . With a mixtures .S. non-aureus strain or a new Coronavirus spike protein are significant examples. The question in novel sample enrichment approaches is whether there is a need for test devices that can capture most if not all clinically relevant pathogens or whether sequential testing would be a better option. In any case, new technologies should preferably allow the direct enrichment of pathogens from low-titer samples.The development of diagnostic tools requires detailed testing in artificially spiked and “real-life” samples from clinical, environmental, and industrial origins. Institutional or commercially available pathogen strain collections should be used to define the natural variation in the adhesins and the effect of such variation on adhesion efficiency and, hence, test quality . This coHigh content proteomics , electroCurrently used physical test platforms range from microfluidic biosensors and nanowires to more classical Raman spectroscopy-, electrochemical- or PCR-based equipment. Furthermore, enrichment platforms frequently utilize nanorods or -wires when straightforward analytical signal detection is required . When, after the initial adhesion test, follow-up testing is required in a more preparative manner, magnetic beads are by far the most common approach. The entities to be detected can vary from intact cells, through simple enzymes, to the products of nucleic acid amplification reactions. Of note, nucleic acids rarely play a role in microbial adhesion though biofilms contain relatively large amounts of these molecules and are thought to function as adhesins under biofilm conditions . Still, E. coli and S. aureus, representatives of Gram-negative and Gram-positive pathogens, respectively. All methods described generate results in 7 min to 3 h, show high sensitivity (to about ten colony-forming units at their most sensitive) and are of great quality; still, their implementation into routine use is sparse. The most commonly used procedures at the level of detection are coloration, fluorescence measurement, and detection of amplified nucleic acid. The most frequent model organisms used are Biosensors, analytical devices that detect and quantify biomolecules or cells, are composed of three elements: the bioreceptor , a transducer , and the display set-up . Their aE. coli strain ORN178 can be detected through the binding of type-1 fimbriae to α-D-mannose by attaching the sugar to a nanomechanical cantilever in the biosensor [E. coli and ECM proteins in the presence of polysaccharides [E. coli binding using poly-sulfated polysaccharides. In this work, the binding of pathogenic E. coli O157:H7 was also evaluated. SPR and electrochemical impedance spectroscopy (EIS) have been used for rapid detection of E. coli through lectin binding using concanavalin A immobilized as a self-assembled monolayer onto a gold electrode surface. These biosensors could be used for screening bacterial load in water samples [There is promising biosensor-mediated research for different microbial pathogens. iosensor . Upon bicharides  in a mod samples .Legionella collagen-like (Lcl) adhesin binds ECM components and mediates bacterial binding to host cells. Lcl has been used to detect glycosaminoglycans (GAGs) via SPR and EIS onto gold electrodes and gold screen-printed electrodes [L. pneumophila lung infection. In addition, haemagglutinin (HA), a homo-trimeric glycoprotein expressed on the surface of the influenza virus, shows high affinity towards sialic acid-terminated trisaccharides of epidermal cell membranes. Researchers developed both a mechanical and an electrochemical biosensor for the detection of the human influenza virus based on this interaction [ectrodes . The Lcleraction . BindingThere is still an urgent need both for improved insight into how to capture and enrich pathogens from complex clinical samples for downstream analytical diagnostics, and for the design and study of anti-adhesive compounds (to help prevent non-specific binding) . For insCurrent diagnostics for bacterial and viral pathogens are typically unspecific and include cultivations of uncertain sensitivity, as pathogen concentrations as low as one viable count per mL can be indicative of infection . EnrichmBox 2E. coli, constitute a special category of such anti-ligands [The development of anti-adhesive materials with minimal fouling, based on ‘grafting-from’ approaches, may be useful to inhibit adhesion. The development of ‘anti-ligands’ to inhibit adhesion provides additional therapeutic approaches. Pilicides, which inhibit the first steps in biofilm formation for -ligands ,167, and-ligands . The tar-ligands . Their d-ligands ,171,172.The IVD workflows show the overall state of readiness for innovation in clinical microbiology . ExpansiCOVID-19 has, at last, made the general public aware of the global impact of infectious diseases and the need for and relevance of rapid diagnostics . We must