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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Optics in droplet microfluidics

Droplet-based microfluidics facilitates the manipulation of small volumes of liquids of two immiscible phases; e.g., water and oil. Effectively, the technique involves a small reactor in which a chemical reaction or biological process can be performed and observed over time. Microdroplets can be mixed, sorted, incubated, and analyzed. Those operations can be performed in specially designed microfluidic chips, creating small lab-on-a-chip devices. Besides miniaturization, an added advantage is that many replications of one experiment can be performed very quickly.

In our research, we aim to observe the behavior of various clinically relevant bacteria strains. More specifically, we are interested to see how they react to antibiotics. Antimicrobial resistance (AMR) is one of the most urgent threats to global health. It occurs when bacteria, viruses, fungi, or parasites transform over time and are no longer sensitive to medicines. As a result, antibiotics or other antimicrobial drugs become unsuccessful and can no longer treat diseases effectively. The World Health Organization (WHO) has recognized AMR as one of the top 10 threats to global public health.

Monitoring bacterial behavior; i.e., growth, is a challenging and time-consuming task, particularly when we need to monitor thousands or millions of replicate experiments. Optical methods combined with microfluidics enable us to address this problem. Using specially designed chips, we can apply droplets in the path of a laser beam and analyze the light scattered from bacteria cells. The intensity of the scattered light is proportional to bacterial concentration in the droplets, and we can follow it over time. We can monitor more than 1000 droplets per second and analyze them with dedicated software. Additionally, we can also make the system more compact and easier to use by using fiber optics.


Natalia Pacocha, Jakub Bogusławski, Michał Horka, Karol Makuch, Kamil Liżewski, Maciej Wojtkowski, and Piotr Garstecki, “High-Throughput Monitoring of Bacterial Cell Density in Nanoliter Droplets: Label-Free Detection of Unmodified Gram-Positive and Gram-Negative Bacteria,” Analytical Chemistry 93(2), 843-850 (2021).


Text: Jakub Bogusławski, PhD



Adenine base editing

Retinal pigment epithelium (RPE) located at the back of the eye is essential for vision. It supports the photoreceptors, providing molecules required for their function. One of the main proteins produced by the RPE and indispensable for vision is the RPE65 enzyme, which is responsible for chemical signaling at the initial step of visual processing. De novo nonsense mutations in the Rpe65 gene underlie inherited genetic disorders of the eyes, resulting in blindness. To address this problem, we have harnessed the power of adenine base editors (ABEs) with Cas9 – single-guide RNA machinery to target  the mutations in the Rpe65 gene for their repair. We delivered genes coding for ABEs and the Cas9 system subretinally via a lentiviral vector. Our therapeutic manipulation corrected the pathogenic mutation in a mouse model with up to 29% efficiency and with minimal formation of indel and off-target mutations. The ABE-treated mice displayed restored RPE65 expression and its activity in the visual cycle. Moreover, we have observed near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.


dr Andrzej Foik, e-mail: afoik@ichf.edu.pl & dr Anna Posłuszny, e-mail: aposluszny@ichf.edu.pl

Pertinent published article:

Restoration of visual function in adult mice with an inherited retinal disease via adenine base editing

Susie Suh, Elliot H. Choi, Henri Leinonen, Andrzej T. Foik, Gregory A. Newby, Wei-Hsi Yeh, Zhiqian Dong, Philip D. Kiser, David C. Lyon, David R. Liu & Krzysztof Palczewski, Nat Biomed Eng. 2021 Feb;5(2):169-178.



Two-photon optical biopsy of the retina

The retina is an essential part of the eye, as it is responsible for transforming light into electrical signals, which are processed in the brain later on. It effectively works as a photodetector of the eye. Imaging the structure and function of the living retina is crucial for the effective diagnosis and treatment of eye diseases and drug developments. Nowadays, structural information about the retina can be obtained from, e.g., OCT studies. However, functional alterations are the first signs of early pathological processes and often precede structural changes; this information is currently very challenging to obtain.

The retina has a layered structure, which is packed with various fluorophores. For example, retinal pigments epithelium (RPE) contains lipofuscin, a byproduct of the visual cycle. Lipofuscin is accumulated overage, but also with a progression of the disease. Other examples are retinol and retinyl esters (vitamin A derivatives active in a visual cycle), melanin, FAD, NADH, collagen, and elastin. Those substances can provide valuable information about the retina’s health and can be a valuable tool for discovering functional alterations during age-related macular degeneration, diabetic retinopathy, or glaucoma.

Standard ophthalmic autofluorescence imaging visualizes the distribution of retinal fluorophores, but only intensity information is available. As a result, signals from various fluorophores cannot be distinguished. Lipofuscin is a dominant fluorophore, and its strong signal needs to be discriminated from other, usually much weaker, sources. Luckily, various fluorophores differ in fluorescence properties, i.e., fluorescence lifetime and fluorescence spectrum. This adds as an additional discrimination parameter.

The eye is the window to the body, but it also has a specific transmission range. Consequently, many fluorophores with excitation spectra in the UV/blue spectral range (<420 nm) cannot be excited, and the information contained in them is unavailable. Our solution to this problem is to use a two-photon excitation. This scheme uses short (femtosecond) pulses in the near-infrared (twice the wavelength), bypassing those limitations. For example, using femtosecond pulses at 730 nm is equivalent to single-photon excitation at 365 nm, which would not be possible in the living eye. Additional advantages of this method are improved resolution, lower phototoxicity, and lower scattering.

In our research, we aim at visualizing the structure and molecular composition of ocular tissues using both spectral and temporal discrimination. For example, the image shows fluorescence lifetime (FLIM) distribution of retinal pigment epithelium cells of Abca4PV/PV mouse (a model of Stargardt disease in humans). The image reflects differences in the subcellular distribution of endogenous fluorophores in the RPE. Shorter lifetimes (blue-green color) are associated with A2E, a component of lipofuscin. Red granules (longer lifetime) can be associated with retinyl esters.

Similar discrimination can be done by looking at the fluorescence spectra. Pixels displaying spectral features of retinosomes were color-coded in red, while those with A2E-like properties were in blue. The analysis shows that retinosomes are predominantly located near RPE cell borders, and some are near cell nuclei. Such studies require specialized ultrafast light sources, scanning ophthalmoscopes, sensitive light detectors, and dedicated software for analysis.

Grazyna Palczewska, Jakub Boguslawski, Patrycjusz Stremplewski, Lukasz Kornaszewski, Jianye Zhang, Zhiqian Dong, Xiao-Xuan Liang, Enrico Gratton, Alfred Vogel, Maciej Wojtkowski, Krzysztof Palczewski, “Noninvasive two-photon optical biopsy of retinal fluorophores”, Proceedings of the National Academy of Sciences 117(36), 22532-22543 (2020).

DOI: doi.org/10.1073/pnas.2007527117

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, “Two-photon imaging of the mammalian retina with ultrafast pulsing laser”, Journal of Clinical Investigation Insight 3(17), e121555 (2018).

DOI: doi.org/10.1172/jci.insight.121555

Text: Jakub Bogusławski, PhD



Visually evoked potential plasticity

One of the methods for evoking plasticity in the visual system is repeated stimulation with appropriate visual stimuli. Repeated exposure to sensory stimuli can induce neuronal network changes in the cortical circuits and improve the perception of these stimuli in the primary visual cortex (V1). The aim of our studies was to investigate the effect of repetitive visual training on the magnitude of visual responses in the primary visual cortex and in the superior colliculus (SC), the subcortical structure of the extrageniculate visual pathway in rats. Our study showed that a three-hour, passive visual training with light flashes enhanced visual responses both at the cortical level and in the superior colliculus. The next part of our study focused on distinguishing which input projection is responsible for the observed training effect in the SC, especially whether the increase of collicular response depends on the enhancement in the V1. The SC receives information both from the retina and from layer 5 of the V1. The experiment with pharmacological blocking of V1 did not suppress training-related plasticity in the SC. These results for the first time identified the superior colliculus as a possible target for training strategies to improve the efficiency of the visual process; e.g., in the case of primary visual cortex injuries.


dr Katarzyna Kordecka, e-mail: kkordecka@ichf.edu.pl


Cortical Inactivation Does Not Block Response Enhancement in the Superior Colliculus

Katarzyna Kordecka, Andrzej T. Foik, Agnieszka Wierzbicka and Wioletta J. Waleszczyk