Non-invasive Optoretinography (ORG)

For many years visual inspection of fundus photography and examination of images acquired with optical coherence tomography (OCT) have been used by ophthalmologists for eye disease diagnosis and monitoring therapy progress thanks to their ability to detect morphological biomarkers of pathophysiology. However, early retinal degeneration might affect photoreceptor physiology and their functional response to light stimuli long before disrupting retinal morphology at a scale visible by clinical instruments. The anomalies in physiological response can be measured with Electroretinography (ERG), by recording the electrical currents generated directly by retinal neurons in combination with contributions from retinal glia. The drawback of ERG is that it measures an average response from large portions of the retina, and it might miss physiological changes occurring only in small areas. This issue can be partially solved by multifocal ERG, which measures the response from specific retinal regions. However, discriminating photoreceptor degeneration from that of the neural retina remains a problem.

More recently, a new technique called optoretinography (ORG) has been developed. In this technique, the physiological response to a single pulse light stimulus is measured with the use of OCT. In our work, we focus on the development of ORG that can measure response to a flicker stimulus. Similar measurements have been performed with ERG multiple times, and they have proven instrumental in the analysis of retinal light adaptation and critical flicker frequency (CFF) variations between the macula and periphery.

Our results have already demonstrated that we could detect the photoreceptor response to different flicker frequencies in a repeatable fashion. We also demonstrated the ability to spatially detect the response to a patterned stimulus with light stripes flickering at different frequencies. These results highlight the prospect for a more objective study of CFF variations across the retina or complete characterization of the spatially resolved temporal frequency response of the retina with flicker ORG perimetry and other novel accurate retinal functional studies for early detection of retinal degeneration and therapy monitoring.

Text: Sławomir Tomczewski, PhD, e-mail: stomczewski@ichf.edu.pl


Novel, combined macro- and microscopic methodology for detecting the Keratoconus disease

The information below is based on the paper: “Detection of Subclinical Keratoconus With a Validated Alternative Method to Corneal Densitometry” by Alejandra Consejo; Marta Jiménez-García; Ikram Issarti & Jos J. Rozema.

Keratoconus is an eye disease that affects the cornea, the clear transparent lens outside our eyes. Keratoconus is a progressive disease that affects 1 in 1000 people and, if untreated, might lead to blindness. However, the early detection of keratoconus is still a clinical challenge.

This work was developed in the frame of MAiCRO project, financed by National Science Center, at ICTER  in collaboration with colleagues from Volantis, from the Antwerp University Hospital in Belgium. 

In this paper a reinterpretation of already available clinical data to enhance the early detection of keratoconus is proposed. During a standard ophthalmologist examination, doctors focus mainly on analyzing macroscopic data: corneal shape, thickness, radius, and other geometrical parameters. The analysis of this information allows the ophthalmologists to diagnose different eye diseases, but it has repeatedly proven to be not enough for a proper diagnosis, specially in the early cases.  

What innovation brings the approach proposed by Consejo, Jiménez-García, Issarti & Rozema? The authors define a diagnostic tool based not only on traditional macroscopic parameters, but also on microscopic data of the cornea’s tissue, combining both approaches (macro- and microscopic information) in a methodology denominated MAiCRO. In particular, in this article, corneal tomographies of sixty right eyes were obtained from the Department of Ophthalmology at the Antwerp University Hospital. The patients were divided in three study groups: controls (20 eyes), clinical keratoconus (20 eyes), and subclinical keratoconus (20 eyes) – subclinical keratoconus are eyes that have not developed the disease yet. The study defined biomarkers that account for tissue transparency and compared these biomarkers between study groups. To define those biomarkers different techniques of image processing and statistical modelling of light intensity distribution (in other words, using information of the journey of the light through the cornea) were applied.

The results of the study validated with a ROC analysis confirmed a discrimination success of 97% when differentiating between subclinical keratoconus and control eyes, which is a much higher clinical diagnosis rate success than clinical standards.

One of the benefits of this methodology is the reinterpretation of commonly available hospital data obtained through non-invasive ophthalmological tomography based on Scheimpflug technology. Using MAiCRO methodology, doctors do not need to take more measurements, or perform additional tests in order to be able to diagnose Keratoconus more effectively than before.

Dr Alejandra Consejo is a Postdoctoral fellow collaborating with ICTER.

Supported by funding from the National Science Centre (Poland) under the OPUS 19 funding scheme (project no. 2020/37/B/ST7/00559).


Two-photon microperimetry

Microperimetry is a subjective visual field testing method that enables the assessment of retinal function at various specific and focal locations. Two-photon microperimetry is an extension of this technique. In contrast to traditional microperimetry, which uses a visible stimulus, two-photon microperimetry utilizes pulsed infrared lasers as a source of stimulating radiation. The subject perceives such a stimulus as a color one due to the two-photon vision phenomenon [1].

The applicability of two-photon microperimetry depends largely on the parameters of the laser used for experiments. Therefore, in ICTER, we conduct extensive research on the influence of parameters of pulsed infrared laser, like pulse duration, pulse repetition rate, wavelength, on the perception by humans [2, 3]. Moreover, we perform a clinical assessment of two-photon microperimetry usefulness for earlier and more effective eye visual function abnormalities [4]. We hope that a deeper understanding of the phenomenon of two-photon vision, optimization of visual field test procedures, and clinical tests enable us to provide a useful tool for ophthalmologists worldwide. 

[1] Ruminski et al., BOE 10(9), pp. 4551-4567 (2019). DOI: 10.1364/BOE.10.004551

[2] Marzejon et al., BOE 12(2), pp. 462-479 (2021). DOI: 10.1364/BOE.411168

[3] Marzejon et al., Proc. SPIE 11623, 116231N (2021). DOI: 10.1117/12.2582735

[4] Komar et al., AOVS 62(8), 2009 (2021).

Text: Marcin Marzejon, MSc


Two-photon microperimetry: sensitivity of human photoreceptors to infrared light

Daniel Ruminski, Grazyna Palczewska, Maciej Nowakowski, Agnieszka Zielińska, Vladimir J. Kefalov, Katarzyna Komar, Krzysztof Palczewski, and Maciej Wojtkowski


Microperimetry is a subjective ophthalmologic test used to assess retinal function at various specific and focal locations of the visual field. Historically, visible light has been described as ranging from 400 to 720 nm. However, we previously demonstrated that infra-red light can initiate visual transduction in rod photoreceptors by a mechanism of two-photon absorption by visual pigments. Here we introduce a newly designed and constructed two-photon microperimeter. We provide for the first time evidence of the presence of a nonlinear process occurring in the human retina based on psychophysical tests using newly developed instrumentation. Since infra-red light penetrates the aged front of the eye better than visible light, it has the potential for improved functional diagnostics in patients with age-related visual disorders.

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STOC imaging

Conventional scanning OCT combines time- with confocal gating enabling high-speed, high-resolution cross-sectional imaging of the human retina. Classic OCT, however, does not provide high-resolution en face images of the outer retinal layers due to eye aberrations and the fundamental tradeoff between imaging depth and transverse resolution.

This tradeoff is reduced by a full-field OCT (FF-OCT) method that uses a two-dimensional camera instead of a single-element photodiode. However, an attempt to boost the FF-OCT imaging speed by Fourier-domain (FD) detection resulted in another severe limitation – spatial coherence of the laser generates coherent artifacts, which reduces the spatial resolution.

To solve this problem, we developed the new way of controlling the optical phase called STOC (Spatio-Temporal Optical Coherence). Application of STOC to Fourier-domain full-field optical coherence tomography (FD-FF-OCT) is called STOC tomography (STOC-T) or STOC imaging, and enabled obtainining in vivo high-resolution, volumetric images of skin [1], the retina [2], and cornea [3].

Fig. 1. STOC imaging enables high-resolution imaging of the retina by spatial phase modulation (a). Computational aberration correction enables to correct the data in post-processing to remove aberrations (b). By repeating measurements at different locations, and stitching together the resulting images we obtain high fidelity wide area retinal images (c).

FF-OCT is significantly different from the classic OCT. FF-OCT is closer to multi-color (or multi-wavelength) digital holography than scanning microscopy, especially with the Fourier detection (FD) with the tunable laser. FD-FF-OCT uses a tunable laser and an ultra-fast area scan camera. The tunable laser encodes depth information about the sample. We extended FD-FF-OCT by the spatial phase modulator (SPM) [Fig. 1(a)]. The SPM dynamically modulates the phase of incident light. The resulting signals are processed and averaged to produce noise-free volumetric images of the sample. Phase modulation works here as an additional gating mechanism that rejects the multiply scattered light effectively [last column in Fig. 1(b)]. However, as shown in Fig. 1(b), the en face images are distorted by eye aberrations. We overcome them computationally using the computational aberration correction (CAC) [4].

CAC proceeds, as sketched in Fig. 1(b). The complex data (amplitude and phase) representing the layer at a depth z_l, U(x,y,z=z_l) of the sample is 2D Fourier transformed to achieve the spatial spectrum U ̃(k_x,k_y,z=z_l). The latter is multiplied by the variable phase mask, M(k_x,k_y) and the resulting modified spatial spectrum U ̃(k_x,k_y,z)M(k_x,k_y) is inverse Fourier-transformed to achieve the corrected field U_c (x,y,z=z_l). We then evaluate the image quality metric on |U_c (x,y,z=z_l )|^2. Here, for the metric, we use the kurtosis. The phase mask is M(k_x,k_y,z_l )=exp[i∑_(n=1)^N▒〖α_n (z_l ) 〗 Z_n (k_x,k_y)], where α_n (z_l) are the depth-dependent adjustable parameters, and Z_n (k_x,k_y) are the Zernike polynomials. The representative estimated real part of the resulting phase mask is shown in the second column of Fig. 2(b). The last column of Fig. 2(b) demonstrates that CAC enables to depict otherwise invisible photoreceptor cone mosaic, the primary sensing element of the human eye.

To achieve wide-field retina images we perform measurements at different locations, and then stitch resulting volumes together to render high resolution, high fidelity retinal images at different depths [Fig. 1(c)]. Specifically, we render the choroid, which was not possible with conventional Fourier-domain FF-OCT (without phase modulation).

Text: Dawid Borycki, PhD, e-mail: dborycki@ichf.edu.pl


  1. Borycki, D., et al., Spatiotemporal optical coherence (STOC) manipulation suppresses coherent cross-talk in full-field swept-source optical coherence tomography. Biomed Opt Express, 2019. 10(4): p. 2032-2054.
  2. Stremplewski, P., et al., In vivo volumetric imaging by crosstalk-free full-field OCT. Optica, 2019. 6(5): p. 608-617.
  3. Auksorius, E., D. Borycki, and M. Wojtkowski, Crosstalk-free volumetric in vivo imaging of a human retina with Fourier-domain full-field optical coherence tomography. Biomed Opt Express, 2019. 10(12): p. 6390-6407.
  4. Auksorius, E., et al., In vivo imaging of the human cornea with high-speed and high-resolution Fourier-domain full-field optical coherence tomography. Biomed Opt Express, 2020. 11(5): p. 2849-2865.
  5. Borycki, D., et al., Computational aberration correction in spatiotemporal optical coherence (STOC) imaging. Opt Lett, 2020. 45(6): p. 1293-1296.


A decade ago, two scientists from our Institute – prof. Wojtkowski and dr Karnowski – published the world’s first air-puff Optical Coherence Tomography research [1]. The proposed method for direct measurements of apex corneal deformation was explored in several follow-up studies [2-4].

Over the last 4 years, prof. Wojtkowski and dr Karnowski lead locally (at the Institute of Physical Chemistry Polish Academy of Sciences) a group of researchers within the IMCUSTOMEYE – a 4-year project funded by the European Commission’s Horizon 2020 Programme under the Photonics 2017 KET topic. The IMCUSTOMEYE project focuses on the progress of the space of the air-puff OCT method towards three-dimensional measurements [5]. The ultimate goal is to enable the characterization of the ocular mechanical behavior in vivo using a cost-effective imaging technology that provides results in almost real-time. The techniques will enable the construction of patient-specific models that can predict with high accuracy the mechanical response of eyes to disease and treatment.

Our role, as experts in biomedical optics and photonics, is to develop compact, affordable OCT device to image dynamic corneal deformation in a three-dimensional manner.


[1] David Alonso-Caneiro, Karol Karnowski, Bartlomiej J. Kaluzny, Andrzej Kowalczyk, and Maciej Wojtkowski, “Assessment of corneal dynamics with high-speed swept source Optical Coherence Tomography combined with an air puff system,” Opt. Express 19, 14188-14199 (2011)

[2] Carlos Dorronsoro, Daniel Pascual, Pablo Pérez-Merino, Sabine Kling, and Susana Marcos, “Dynamic OCT measurement of corneal deformation by an air puff in normal and cross-linked corneas,” Biomed. Opt. Express 3, 473-487 (2012)

[3] Maczynska, E, Karnowski, K, Szulzycki, K, et al. Assessment of the influence of viscoelasticity of cornea in animal ex vivo model using air-puff optical coherence tomography and corneal hysteresis. J. Biophotonics. 2019; 12:e201800154

[4] Karol Marian Karnowski, Ewa Mączyńska, Maciej Nowakowski, Bartłomiej Kałużny, Ireneusz Grulkowski, Maciej Wojtkowski, “Impact of diurnal IOP variations on the dynamic corneal hysteresis measured with air-puff swept-source OCT”, Phot. Lett. Pol., vol. 10, no. 3, pp. 64-66, (2018)

[5] Andrea Curatolo, Judith S. Birkenfeld, Eduardo Martinez-Enriquez, James A. Germann, Geethika Muralidharan, Jesús Palací, Daniel Pascual, Ashkan Eliasy, Ahmed Abass, Jędrzej Solarski, Karol Karnowski, Maciej Wojtkowski, Ahmed Elsheikh, and Susana Marcos, “Multi-meridian corneal imaging of air-puff induced deformation for improved detection of biomechanical abnormalities,” Biomed. Opt. Express 11, 6337-6355 (2020).

Author: Karol Karnowski, PhD


Estimation of scleral mechanical properties from air-puff optical coherence tomography

David Bronte-Ciriza, Judith S. Birkenfeld, Andrés de la Hoz, Andrea Curatolo, James A. Germann, Lupe Villegas, Alejandra Varea, Eduardo Martínez-Enríquez, and Susana Marcos


We introduce a method to estimate the biomechanical properties of the porcine sclera in intact eye globes ex vivo, using optical coherence tomography that is coupled with an air-puff excitation source, and inverse optimization techniques based on finite element modeling. Air-puff induced tissue deformation was determined at seven different locations on the ocular globe, and the maximum apex deformation, the deformation velocity, and the arc-length during deformation were quantified. In the sclera, the experimental maximum deformation amplitude and the corresponding arc length were dependent on the location of air-puff excitation. The normalized temporal deformation profile of the sclera was distinct from that in the cornea, but similar in all tested scleral locations, suggesting that this profile is independent of variations in scleral thickness. Inverse optimization techniques showed that the estimated scleral elastic modulus ranged from 1.84 ± 0.30 MPa (equatorial inferior) to 6.04 ± 2.11 MPa (equatorial temporal). The use of scleral air-puff imaging holds promise for non-invasively investigating the structural changes in the sclera associated with myopia and glaucoma, and for monitoring potential modulation of scleral stiffness in disease or treatment.

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Telehealth applications

Smartphone-based optical palpation: towards elastography of skin for telehealth applications

Rowan W. Sanderson, Qi Fang, Andrea Curatolo, Aiden Taba, Helen M. DeJong, Fiona M. Wood, and Brendan F. Kennedy


Smartphones are now integral to many telehealth services that provide remote patients with an improved diagnostic standard of care. The ongoing management of burn wounds and scars is one area in which telehealth has been adopted, using video and photography to assess the repair process over time. However, a current limitation is the inability to evaluate scar stiffness objectively and repeatedly: an essential measurement for classifying the degree of inflammation and fibrosis. Optical elastography detects mechanical contrast on a micrometer- to millimeter-scale, however, typically requires expensive optics and bulky imaging systems, making it prohibitive for wide-spread adoption in telehealth. More recently, a new variant of optical elastography, camera-based optical palpation, has demonstrated the capability to perform elastography at low cost using a standard digital camera. In this paper, we propose smartphone-based optical palpation, adapting camera-based optical palpation by utilizing a commercially available smartphone camera to provide sub-millimeter resolution imaging of mechanical contrast in scar tissue in a form factor that is amenable to telehealth. We first validate this technique on a silicone phantom containing a 5 × 5 × 1 mm3 embedded inclusion, demonstrating comparative image quality between mounted and handheld implementations. We then demonstrate preliminary in vivo smartphone-based optical palpation by imaging a region of healthy skin and two scars on a burns patient, showing clear mechanical contrast between regions of scar tissue and healthy tissue. This study represents the first implementation of elastography on a smartphone device, extending the potential application of elastography to telehealth.

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In vivo corneal imaging

Multimode fiber enables control of spatial coherence in Fourier-domain full-field optical coherence tomography for in vivo corneal imaging

Egidijus Auksorius, Dawid Borycki, and Maciej Wojtkowski


Fourier-domain full-field optical coherence tomography (FD-FF-OCT) has recently emerged as a fast alternative to point-scanning confocal OCT in eye imaging. However, when imaging the cornea with FD-FF-OCT, a spatially coherent laser can focus down on the retina to a spot that exceeds the maximum permissible exposure level. Here we demonstrate that a long multimode fiber with a small core can be used to reduce the spatial coherence of the laser and, thus, enable ultrafast in vivo volumetric imaging of the human cornea without causing risk to the retina.

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Two-photon vision phenomenon

Effects of laser pulse duration in two-photon vision threshold measurements

Marcin Marzejon, Łukasz Kornaszewski, Maciej Wojtkowski, Katarzyna Komar


Pulsed near-infrared (NIR) light sources can be successfully applied for both imaging and functional testing of the human eye, as published recently. These two groups of applications have different requirements. For imaging applications, the most preferable is invisible scanning beam while efficiently visible stimulating beam is preferable for functional testing applications. The functional testing of human eye using NIR laser beams is possible due to two-photon vision (2PV) phenomenon. 2PV enables perception of pulsed near-infrared laser light as color corresponding to approximately half of the laser wavelength. This study aims to characterize two-photon vision thresholds for various pulse lengths from a solidstate sub-picosecond laser (λc = 1043.3 nm, Frep = 62.65 MHz), either of 253 fs duration or elongated by Martinez- type stretcher to 2 ps, and fiber-optic picosecond laser (λc = 1028.4 nm, Frep = 19.19 MHz, τp = 12.2 ps).

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