Er-doped fiber laser

Femtosecond lasers are used in many techniques in biophotonics, including multiphoton microscopy, two-photon fluorescence ophthalmoscopy, and two-photon microperimetry. These methods require precisely selected parameters of ultrashort pulses to ensure noninvasive and efficient imaging of the sample under study or examination of the patient. Titanium-sapphire lasers or parametric oscillators are most commonly used for this purpose. However, these laser sources are very expensive, complicated to use, require water cooling, and are not mobile. A solution to these problems may be using femtosecond fiber lasers, which offer equally short pulse durations but are much more compact and easier to use and transport, enabling clinical translation. The lasers are being developed at the Wrocław University of Science and Technology, and researchers at ICTER are working on their applications.

The first application is multiphoton microscopy, particularly fluorescence microscopy with two-photon excitation. In this application, it is crucial to minimize the average excitation power of the laser, thus reducing the thermal interaction with the sample under study. For this purpose, a femtosecond fiber laser with an adjustable repetition rate and very short pulse duration (less than 60 fs) was developed. The laser operates in the near-infrared spectral range (central wavelength around 780 nm) by doubling the frequency of a laser doped with erbium ions (operating at 1560 nm). The laser was developed as a compact, easy-to-use prototype [1].

The second application, from the field of ophthalmology, is two-photon fluorescence ophthalmoscopy. This method allows noninvasive imaging of autofluorescence induced in the retina and retinal pigment epithelial layer. In this application, the key is to reduce the power of the average laser beam used to excite the fluorescence. This has been achieved by using a femtosecond fiber laser with an adjustable repetition rate and a very short pulse duration [2].

A recent application, also from the field of ophthalmology, is two-photon microperimetry and the study of the phenomenon of two-photon vision. Another femtosecond fiber laser with a tunable wavelength, ranging from 872 to 1075 nm, was developed for this purpose. Such a wide tuning range allowed a better study of humans’ scotopic spectral sensitivity of two-photon vision [3].

Autor: Jakub Bogusławski, PhD


Jakub Bogusławski, PhD jboguslawski@ichf.edu.pl

Marcin Marzejon, PhD mmarzejon@ichf.edu.pl

Katarzyna Komar, PhD kkomar@ichf.edu.pl

Prof. Maciej Wojtkowski mwojtkowski@ichf.edu.pl


  1. D. Stachowiak, J. Bogusławski, A. Głuszek, Z. Łaszczych, M. Wojtkowski, G. Soboń, “Frequency-doubled femtosecond Er-doped fiber laser for two-photon excited fluorescence imaging,” Biomedical Optics Express 11(8), 4431 (2020).
  2. Jakub Boguslawski, Grazyna Palczewska, Slawomir Tomczewski, Jadwiga Milkiewicz, Piotr Kasprzycki, Dorota Stachowiak, Katarzyna Komar, Marcin J Marzejon, Bartosz L Sikorski, Arkadiusz Hudzikowski, Aleksander Głuszek, Zbigniew Łaszczych, Karol Karnowski, Grzegorz Soboń, Krzysztof Palczewski, Maciej Wojtkowski, “In vivo imaging of the human eye using a two-photon excited fluorescence scanning laser ophthalmoscope,” The Journal of Clinical Investigation 2022;132(2):e154218.
  3. Dorota Stachowiak, Marcin Marzejon, Jakub Bogusławski, Zbigniew Łaszczych, Katarzyna Komar, Maciej Wojtkowski, Grzegorz Soboń, “Femtosecond Er-doped fiber laser source tunable from 872 to 1075 nm for two-photon vision studies in humans,” Biomedical Optics Express 131(4), 1899-1911 (2022).


Multi-spot measurements of air-induced corneal deformations (IMCUSTOMEYE project)

The IMCUSTOMEYE project involves the cooperation of 10 partners, both academic and industrial, began in 2018. From day one, as a consortium, we have focused on developing new, non-invasive, imaging-based methods to change the paradigm in the diagnosis and treatment of various eye diseases.

POB group’s researchers, were tasked with constructing a compact, low-cost device to measure 3D dynamic corneal deformation of the human eye. As it is in life, and especially in physics, we had to make some compromises with respect to the prototype being constructed. Even if full three-dimensional imaging of a corneal deformation process lasting only 20 ms is possible, it would require considerable complication of the measurement system and generate unacceptable costs. We proposed an intermediate solution of simultaneous measurements at multiple points on the cornea, including the center of the cornea and 4 pairs of points placed opposite along 4 directions (horizontal, vertical and corresponding directions rotated by 45 degrees). This approach made it possible to prepare a prototype compact system to be placed in an eye clinic. In addition, we preliminarily verified the possibility of both further miniaturization of the system and the potential for a significant reduction in manufacturing costs.

Clinical prototype

Our clinical prototype has not only survived the 300+ kilometer trip to the clinic in Bydgoszcz, Poland, but has also measured more than 100 eyes to date. It is worth noting that the prototype has been prepared from the hardware and software side in such a way that it could be successfully operated by eye clinic staff.

To analyze the data, we extract temporal corneal deformation for each spot. The biomechanical asymmetry can be assessed by comparison of opposite spots. To provide more intuitive presentation of the results, we introduced “asymmetry vector” that can be plotted for any deformation parameter (e.g., displacements amplitudes, deformation area, deformation slopes). For each pair of opposite spots, we create a vector pointing towards spot with higher value of selected parameter with a magnitude given by the differences of values for both spots in pair.

Data analysis pipeline

Having vectors for all 4 pairs of spots we can calculate overall vector to show global effect. This approach was applied already to some of our early clinical data to show differences in biomechanical asymmetry between healthy and keratoconus corneas (presented here for displacement amplitude and area).

Early clinical results

Text: Dr. Karol Karnowski


Karol Karnowski, PhD

Jadwiga Milkiewicz, MSc

Angela Pachacz, Eng

Onur Cetinkaya, BEng

Rafał Pietruch, Eng

Andrea Curatolo, PhD

Prof. Maciej Wojtkowski


  1. D. Alonso-Caneiro, K. Karnowski, B. Kaluzny, A. Kowalczyk, and M. Wojtkowski, “Assessment of corneal dynamics with high-speed swept source Optical Coherence Tomography combined with an air puff system”, Optics Express, Vol. 19, Issue 15, pp. 14188-14199 (2011)
  2. S. Marcos, C. Dorronsoro, K. Karnowski, M. Wojtkowski, „Corneal biomechanics From Theory to Practice: OCT with air puff stimulus”, Kugler Publications 2016, edited by C.J. Roberts, J. Liu
  3. K. Karnowski, E. Maczynska, M. Nowakowski, B. Kaluzny, I. Grulkowski, M. Wojtkowski, “Impact of diurnal IOP variations on the dynamic corneal hysteresis measured with air-puff swept-source OCT”, Photonics Letters of Poland, (2018)
  4. E. Maczynska, K. Karnowski, K. Szulzycki, M. Malinowska, H. Dolezyczek, A. Cichanski, M. Wojtkowski, B. Kaluzny and I. Grulkowski, “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 (2019)
  5. A. Curatolo, J. S. Birkenfeld, E. Martinez-Enriquez, J. A. Germann, G. Muralidharan, J. Palací, D. Pascual, A. Eliasy, A. Abass, J. Solarski, K. 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)

Spatio-Temporal Optical Coherence Tomography (STOC-T) 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 and, as shown below, precludes visualization of deep retina layers.

To solve this problem, we developed a 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 obtaining in vivo high-resolution, volumetric images of human skin, retina, and cornea at unprecedented speeds.

In STOC imaging, we have extended FD-FF-OCT with a spatial phase modulator (SPM). The SPM dynamically modulates the phase of incident light by generating time-varying transverse mode (TEM) patterns. This is accomplished through the use of active modulators or long multimode optical fiber. The resulting signals are processed and averaged to produce noise-free volume images of the sample. Phase modulation acts here as an additional optical gating mechanism that isolates the signal used to create images. The result is improved images of the sample.

However, the en face images (XY projections) are distorted by eye or sample-induced aberrations. We overcome them in post-processing using the computational aberration correction (CAC). The CAC algorithm proceeds as sketched in the figure. Specifically, we iteratively (in the computer) correct the phase of the spatial spectrum until we optimize the image sharpness metric:

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 (indicated earlier). Specifically, we render the choroid, which was impossible with conventional Fourier-domain FF-OCT (without phase modulation).

Text: Dawid Borycki, PhD


Egidijus Auksorius

Dawid Borycki

Piotr Węgrzyn

Kamil Liżewski

Sławomir Tomczewski

Maciej Wojtkowski


  1. M. Wojtkowski, P. Stremplewski, E. Auksorius, and D. Borycki, “Spatio-Temporal Optical Coherence Imaging – a new tool for in vivo microscopy,” Photonics Letters of Poland 11, 45-50 (2019). https://photonics.pl/PLP/index.php/letters/article/view/11-15
  2. Borycki, D. et al., Control of the optical field coherence by spatiotemporal light modulation, Opt. Lett., 2013 38(22): p. 4817-4820.
  3. 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.
  4. Stremplewski, P., et al., In vivo volumetric imaging by crosstalk-free full-field OCT. Optica, 2019. 6(5): p. 608-617.
  5. Auksorius, E., et al., 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.
  6. 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.
  7. Borycki, D., et al., Computational aberration correction in spatiotemporal optical coherence (STOC) imaging. Opt Lett, 2020. 45(6): p. 1293-1296.
  8. Egidijus Auksorius, Dawid Borycki, Maciej Wojtkowski, Multimode fiber enables control of spatial coherence in Fourier-domain full-field optical coherence tomography for in vivo corneal imaging, Opt Lett, 2021. 46(6): p. 1413-1416.
  9. Auksorius E., et al., Spatio-Temporal Optical Coherence Tomography provides advanced imaging of the human retina and choroid, arXiv preprint arXiv:2107.10672 (2021).
  10. Auksorius E., Fourier-domain full-field optical coherence tomography with real-time axial imaging, Opt Lett, 2021., Vol. 46(18): p. 4478-4481.
  11. Auksorius E., et al., Multimode fiber as a tool to reduce cross talk in Fourier-domain full-field optical coherence tomography. Opt Lett, 2022. 47(4): p. 838-841.
  12. Tomczewski S., et al., Light-adapted flicker optoretinograms captured with a spatio-temporal optical coherence-tomography (STOC-T) system. Biomed Opt Express, 2022 13(4): p. 2186-2201.
  13. Auksorius, E., et al., Spatio-temporal optical coherence tomography provides full thickness imaging of the chorioretinal complex. iScience, 2022 25(12) 105513.


“Two photon vision and two photon eye imaging (2×2-PhotonVis)” project 

The “Two photon vision and two photon eye imaging (2×2-PhotonVis)” project was carried out within IChF PAN (Instytut Chemii Fizycznej PAN) and later ICTER from December 2017 to September 2022. The main goal of this project was to develop novel and original optical methods and instrumentation for functional testing of human and animal vision, using two-photon absorption and two-photon excited fluorescence processes.

The project broadened our knowledge about the optical properties of human and rodent retina and its susceptibility to non-linear optical processes of two-photon isomerization of rhodopsin chromophores and two-photon excitation fluorescence in RPE cells. The project resulted in nine papers published in JCR-indexed journals.

Figure 8, paper: JCI Insight – Two-photon imaging of the mammalian retina with ultrafast pulsing laser

The POIR.04.04.00-00-3D47/16 project is carried out within the TEAM TECH programme of the FNP Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund.

Author: Slawomir Tomczewski, PhD
Grant awardee and project leader: Prof. Maciej Wojtkowski
Website of the #TeamTech project: https://2photon.icter.pl/

Related paper: JCI Insight – Two-photon imaging of the mammalian retina with ultrafast pulsing laser

Two-photon imaging of the mammalian retina with ultrafast pulsing laser


Noninvasive imaging of visual system components in vivo is critical for understanding the causal mechanisms of retinal diseases and for developing therapies for their treatment. However, ultraviolet light needed to excite endogenous fluorophores that participate in metabolic processes of the retina is highly attenuated by the anterior segment of the human eye. In contrast, 2-photon excitation fluorescence imaging with pulsed infrared light overcomes this obstacle. Reducing retinal exposure to laser radiation remains a major barrier in advancing this technology to studies in humans. To increase fluorescence intensity and reduce the requisite laser power, we modulated ultrashort laser pulses with high-order dispersion compensation and applied sensorless adaptive optics and custom image recovery software and observed an over 300% increase in fluorescence of endogenous retinal fluorophores when laser pulses were shortened from 75 fs to 20 fs. No functional or structural changes to the retina were detected after exposure to 2-photon excitation imaging light with 20-fs pulses. Moreover, wide bandwidth associated with short pulses enables excitation of multiple fluorophores with different absorption spectra and thus can provide information about their relative changes and intracellular distribution. These data constitute a substantial advancement for safe 2-photon fluorescence imaging of the human eye.


Grazyna Palczewska, Patrycjusz Stremplewski, Susie Suh, Nathan Alexander, David Salom, Zhiqian Dong, Daniel Ruminski, Elliot H. Choi, Avery E. Sears, Timothy S. Kern, Maciej Wojtkowski, Krzysztof Palczewski


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).



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.

Link to publication



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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