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.

ICTER scientists re-engineer the study of the cornea

A press release related to the paper “Multimode fiber enables control of spatial coherence in Fourier-domain full-field optical coherence tomography for in vivo corneal imaging” by Egidijus Auksorius, Dawid Borycki, and Maciej Wojtkowski, has been published on AlphaGalileo on July 29th, 2021.

Here is the AlphaGalileo press release:

A pioneer eye imaging discovery: ICTER scientists re-engineer the study of the cornea.


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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Dr. Andrzej Foik – leader of the Ophthalmic Biology Group got granting under SONATA 16 funded by NSC

Source: www.ncn.gov.pl

On May 20, 2021, the Polish National Science Center announced the results of the SONATA 16 competition. One of the winners was our colleague, OBi group leader, Dr. Andrzej Foik. The topic of the winning competition is the role of the basal part of the crescentic brain in visual information processing. Find more information about the winning Project.


NSC announced the results of the competition for Polish-Lithuanian research projects – DAINA 2. Prof. Wojtkowski and Dr. Auksorius are among the winners

Source: www.ncn.gov.pl

On May 4, 2021 Polish National Science Center announced the results of the competition for Polish-Lithuanian research projects – DAINA 2. Prof. Wojtkowski and Dr. Auksorius are among the winners The topic of their winning project is Volumetric image reconstruction with filtering of redundant phase information. Find more information about the winning Project.


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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