System for photoporation of cells, method for photoporation of cells, and use of system for photoporation of cells

Abstract

The invention relates to a photoporation system, a method for photoporation of cells, and a use of such a system, with the goal of increasing the permeability of cellular membranes or disrupting cellular membranes, for further applications.

Description

[0001] SYSTEM FOR PHOTOPORATION OF CELLS, METHOD FOR PHOTOPORATION OF CELLS, AND USE OF SYSTEM FOR PHOTOPORATION OF CELLS

[0002] TECHNICAL FIELD

[0003] The invention relates to a photoporation system, a method for photoporation of cells, and a use of such a system, with the goal of increasing the permeability of cellular membranes or disrupting cellular membranes, for further applications.

[0004] PRIOR ART

[0005] The delivery of various macromolecules into the cytosol of different cell types is required for a variety of applications such as cancer immunotherapy, stem cell therapy and other biomedical research areas. The majority of the macromolecules cannot spontaneously cross the cell membrane as it is non-permeable for many compounds. Therefore, different techniques are needed to allow the cytosolic delivery of such cell-impermeable compounds. For several years, electroporation has been used as a standard delivery tool. Electroporation has the ability to form pores into the cell membrane following the application of electrical pulses. The cell-impermeable compounds can then migrate through these pores from the surrounding medium into the cell's interior. Despite that electroporation can reach high delivery efficiencies for a variety of cell types, several limitations have been reported, including high cytotoxicity, the induction of genomic alterations, the induction of phenotypic changes and a low delivery efficiency in several primary (immune) cells. Hence, novel techniques, which do not affect the genotype or phenotype of the cells and which can achieve a high delivery efficiency with a high viability, are required.

[0006] One of these newer delivery methods makes use of photoporation to create the desired pores in the cell membrane, allowing a much wider range of cells to be experimented on and used. It is clear that, while this is a highly interesting application, the development is still in its infancy, in terms of methodology but especially in terms of supporting hardware. Such methods are amongst others described in WO2022 / 189627, W02021 / 058430, W02020127076, and in applicant's presently unpublished patent applications PCT / EP2023 / 058509, PCT / EP2022 / 064003.

[0007] Currently used systems for photoporation are primarily found in lab settings and are usually not dedicated systems but instead built (largely from scratch) by the researchers, and are not optimized in any way in terms of user friendliness, efficient use of components, increased accuracy, etc.

[0008] The invention aims to solve the above problems, and to provide a comfortable pair of glasses that is easily adjustable.

[0009] SUMMARY OF THE INVENTION

[0010] In a first aspect, the invention pertains to a system for photoporation of cells according to claim 1.

[0011] In a second aspect, the invention relates to the use of a system according to the first aspect, for photoporation of one or more cells, and in particular for such further applications as delivering cargo in to said cell(s).

[0012] In a third aspect, the invention relates to a method of performing a photoporation step of one or more cells, preferably for delivery of cargo into said cell(s), with a system according to the first aspect.

[0013] Further improvements are considered for one or more of the aforementioned aspects of the invention, as described in the dependent claims, as well as in the rest of the description.

[0014] The described invention provide for a clear improvement on the state of the art, which is lacking in well-developed systems, and associated methods and uses, allowing a more versatile, integrated system for photoporation as well as subsequent visualization.

[0015] DESCRIPTION OF THE FIGURES

[0016] Fig. 1 shows a representation of an embodiment of the system according to the invention.

[0017] Fig. 2A and 2B show the system of Fig. 1, with a photoporation laser beam either being obstructed by a shutter or being passed.

[0018] Fig. 3 show a possible realization of a tunable power modulation means, and its operation. Fig. 4 show a lateral view of a further embodiment of the system of Fig. 1 in operation with a photoporation beam propagating.

[0019] Fig. 5 show the system of Fig. 1, with an excitation beam irradiating a sample.

[0020] Fig. 6 show the system of Fig. 1, with a photoporation beam irradiating a sample and the resulting emitted light therefrom being directed to a visualization means.

[0021] Fig. 7 show the system of Fig. 1, with an excitation beam irradiating a sample and the resulting emitted light therefrom being directed to a detection means.

[0022] Fig. 8 show the system of Fig. 1, with an excitation beam irradiating a sample and the resulting emitted light therefrom being directed to a visualization means.

[0023] Fig. 9A and 9B shows an embodiment of the sensor means, with a visualization means and a detection means, and a switching means for defining the trajectory of a returning light beam.

[0024] DETAILED DESCRIPTION

[0025] Unless otherwise defined, all terms used in the description of the invention, including technical and scientific terms, have the meaning as commonly understood by a person skilled in the art to which the invention pertains. For a better understanding of the description of the invention, the following terms are explained explicitly.

[0026] In this document, "a" and "the" refer to both the singular and the plural, unless the context presupposes otherwise. For example, "a segment" means one or more segments.

[0027] When the term "around" or "about" is used in this document with a measurable quantity, a parameter, a duration or moment, and the like, then variations are meant of approx. 20% or less, preferably approx. 10% or less, more preferably approx. 5% or less, even more preferably approx. 1% or less, and even more preferably approx. 0.1% or less than and of the quoted value, insofar as such variations are applicable in the described invention. However, it must be understood that the value of a quantity used where the term "about" or "around" is used, is itself specifically disclosed. The terms "comprise," "comprising," "consist of," "consisting of," "provided with," "have," "having," "include," "including," "contain," "containing" are synonyms and are inclusive or open terms that indicate the presence of what follows, and which do not exclude or prevent the presence of other components, characteristics, elements, members, steps, as known from or disclosed in the prior art.

[0028] The term "cell" refers to all types of biological cells, including eukaryotic cells and prokaryotic cells. As used herein, the terms "cells" and "biological cells" are interchangeably used.

[0029] The terms "alter", "altering" or "alteration" refer to any way to change one or more properties of a cell, for example at least locally, such as a barrier of a cell. Altering includes but is not limited to inducing a local change in a cell's composition, for example in the composition of a barrier of the cell by adding, removing, destroying or reorganizing constituents. Altering comprises for example changing one or more physicochemical properties, such as its viscosity, porosity, density, rigidity, elasticity etc. Altering also includes local destruction or rearrangement of cellular barrier constituents, resulting in a change of the composition and / or physicochemical properties of the cellular barrier. Altering includes amongst other deforming, permeabilizing and perforating.

[0030] The terms "deform", "deforming" and "deformation" refer to any way to alter the spatial organization or structure of a cell, in particular of the cellular barrier, at least partially, for example at least locally. Examples of deforming comprise providing a cellular barrier with indentations or invaginations.

[0031] The terms "permeabilize", "permeabilizing" and "permeabilization" refer to any way to alter the permeability of a cell, in particular of the permeability of the cellular barrier, at least partially, for example at least locally. Examples of permeabilizing comprise altering the barrier composition or structure so that it becomes more permeable to a cargo.

[0032] The terms "perforate", "perforating" or "perforation" refer to any way to provide a cell, in particular the cellular barrier, at least partially, for example at least locally, with one or more openings, holes or pores. By perforating a cellular barrier, openings are created into the barrier allowing the transport of a cargo across or into that barrier.

[0033] The terms "perforate", "perforating", "perforation" and the terms "increase the permeability of", "permeabilize", "permeabilizing", "permeabilization" are interchangeably used. Similarly, the terms "opening", "hole" and "pore" may be used interchangeably herein.

[0034] As used herein, the terms "cargo" or "agent" broadly refer to any chemical (e.g., inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g., biological macromolecule), particle (e.g. a nanoparticle), a combination or mixture thereof, a sample of undetermined composition, or an extract made from biological materials such as bacteria, plants, fungi, or animal cells or tissues. Preferred "cargos" or "agents" include nucleic acids, oligonucleotides, ribozymes, proteins, polypeptides, peptides, peptidomimetics, peptide nucleic acids, antibodies, antibody fragments, antibody-like protein scaffolds, aptamers, photoaptamers, spiegelmers, chemical substances, lipids, carbohydrates, polysaccharides, etc., and any combinations thereof such as gene editing system, e.g. CRISPR / Cas. Depending on the context, the term "agent" may denote a "therapeutic agent" or "drug", useful for or used in the treatment, cure, prevention, or diagnosis of a disease. The cargo as taught herein includes but is not limited to a cargo in solution, and a dried or lyophilized cargo, such as a powder of the cargo.

[0035] Most preferably, "cargo" refers to nucleic acids (specifically DNA (deoxyribonucleic acid) or RNA (ribonucleic acid)) and proteins.

[0036] In embodiments, the cargo may comprise or consist of two or more agents combined with or chemically bonded to each other. In embodiments, the cargo may comprise or consist of two or more agents conjugated (e.g. covalently bound) to each other. For instance, the cargo may be an agent comprising or consisting of two, three, four, five, six or more agents conjugated (e.g. covalently bound) to each other.

[0037] The term "photoresponsive", "photosensitive", "light sensitising" may be used interchangeably and refer to the capacity to respond to electromagnetic radiation, such as e.g. visible light. Particularly, the term refers to the capacity to respond to electromagnetic radiation as produced by the photoporation laser device of the present invention, although not limited thereto necessarily. "Responding" to electromagnetic radiation is to be considered in the context of photoporation, namely in that the response is suitable for creating pores in a cell barrier (cell membrane).

[0038] Preferably, the photoresponsive particles are organic or non-organic nanoparticles. Preferred embodiments of the nanoparticles comprise metal and / or metal-oxides, such as gold, titanium and titanium oxides, such TiO? (titanium dioxide).

[0039] Quoting numeric intervals by the endpoints includes all integers, fractions, and / or real numbers between the endpoints, including those endpoints. The invention relates to a system for photoporation of cells, said system comprising: a. a laser device, preferably a pulsed laser device, for generation of photoporation laser beams, preferably photoporation laser pulses, with a predefined first wavelength; b. an excitation light emitting device, preferably an excitation laser device, more preferably a continuous wave laser device, for generation of excitation light beams with a predefined second wavelength, the second wavelength different from the first wavelength; c. a sample holder configured for holding one or more receptacles with cell material; d. a sensor means, configured for detection and / or visualization of cell material in the sample holder; e. an optical processing assembly, comprising: i. a first optical processing subsystem configured for directing the photoporation laser beam from the pulsed laser device along a first optical path to the sample holder, and configured for processing said photoporation laser beam, the first optical processing subsystem comprising along the first optical path a tunable power modulation means for modulating power of the photoporation laser beam, a first dichroic deflection means configured for reflecting light with a wavelength substantially equal to the first wavelength further along the first optical path towards the sample holder, the first dichroic deflection means being positioned downstream along the first optical path more proximal to the sample holder than the power modulation means, and a focusing lens for focusing the photoporation laser beam, ii. a second optical processing subsystem configured for directing the excitation light beam from the excitation light emitting device along a second optical path to the sample holder; and iii. a third optical processing subsystem configured for directing resultant light from the sample holder along a third optical path to the sensor means.

[0040] The photoporation process has its use in a multitude of further applications. Photoporation itself serves to increase permeability or entirely disrupt (or even destroy) the cellular membrane of one or more cells. This permeabilization step is in itself a precursor to further steps, such as the intracellular delivery of external payloads, which may or may not lead to transfection (in the case the payload is a nucleic acid); the release of intracellular compounds from the cell; killing of cells by severe disruption / destruction of their cell membrane; destabilization / permeabilization of intracellular organelles, such as the nucleus, endosomes, etc.

[0041] The system described in the invention provides for a fully-integrated, optimized assembly for photoporation of cells, including a first laser device for generating photoporation beams, as well as a second laser device that generates laser beams of a different wavelength, for validating the correct functioning of the system.

[0042] The purpose of the photoporation laser device is clear, namely the creation of (transient) pores in the cell membrane via laser irradiation. This can be directly (targeting the cell membrane directly, causing optical breakdown to create the pores) or indirectly, for instance via light-sensitive nanoparticles around the cell, which react to the laser irradiation, absorbing the light and heating up, which then creates the pores in the cell membrane. This requires an accurate laser device, capable of reliably generating high power output.

[0043] The purpose of the excitation light emitting device is in part as a validation tool. In many applications of the photoporation system, the ultimate goal is transfection of payloads into the cell, usually in experimental settings, such as research and development. These payloads are often extremely difficult to produce, and therefore very valuable. The particles are often made up of different components, requiring a separate synthesis process (as well a subsequent processing steps, such as purification), and ultimately assembly. Furthermore, they are often not (yet) produced on large scales, meaning that the production cost itself is far from optimized, typically resulting in a slow and costly process at that point. Additionally, the processes in which such payloads are used, are limited and fairly novel, again leading to a lack of development in production process and facilities. Finally, the payloads often undergo substantial validations (regulatory as well as quality control).

[0044] Therefore, in most experiments, the system is tested with 'fake' payloads, which are cheap, easy to produce, and importantly, have well-known characteristics that can be exploited. In photoporation, the payloads are chosen for their fluorescent characteristics, such that they respond well to irradiation with light beams with the second wavelength, of the excitation light emitting device. This way, the system can check the results with the fake payloads, validate that the photoporation went well and the transfection took place as desired, before using the real, costly payloads.

[0045] In a preferred embodiment, the photoporation laser device is a pulsed laser device. The advantages of using a pulsed laser device are that they can produce laser beams with a much higher peak power. This allows the system to effectively have a much higher range of power which an operator can then define, as reducing power is a very straightforward operation, which cannot be said of the inverse. Accordingly, as opposed to continuous wave laser devices, pulsed lasers can be used to deliver energy to a target material without causing as much total heat generation at the target material, as the laser is only on for a short period of time. This is important for applications where heat input could damage the material or surrounding tissue. Additionally, it can also assist in reducing overall heat generation in the system, as in most cases, the heat generated by the laser devices will affect nearby components. In some instances, this may affect their performance, for instance requiring real-time finetuning, adjustments, calibrations, and in other instances even rendering them inoperational by the extent of the effects of the heat buildup. This disadvantage can be countered by forced cooling of the laser device, but this is another cost, takes up valuable space, and may adversely affect other parts of the system. A pulsed laser device effectively only produces heat part-time, while the CW laser device does so continuously. Finally, pulsed laser devices are capable of greater precision than CW lasers devices, as they have a very finely controlled pulse length and interval between pulses, allowing more control, and discrete targeting. For CW laser devices, discrete targeting for instance requires a shutter and very accurate control thereof.

[0046] In a particularly preferred embodiment, the pulsed laser device is a nanosecond pulsed laser device, meaning that the pulse lengths are in the range of a few nanoseconds.

[0047] In a preferred embodiment, the pulses of the pulsed laser device have a pulse length between 10.0 femtoseconds and 10 ps, preferably between 50 fs and 5.0 ps, more preferably between 250 fs and 2.5 ps, even more preferably between 1.0 ps and 1.0 ps, even more preferably between 5.0 ps and 500 ns, even more preferably between 10.0 ps and 100 ns, even more preferably between 25 ps and 50 ns, even more preferably between 50 ps and 20 ns, even more preferably between 100 ps and 10 ns.

[0048] Building further on the above preferably, the pulse length is at least 10.0 ps, more preferably at least 12.5 ps, more preferably at least 15.0 ps, more preferably at least 17.5 ps, more preferably at least 20.0 ps, more preferably at least 25.0 ps, more preferably at least 30.0 ps, more preferably at least 35.0 ps, more preferably at least 40.0 ps, more preferably at least 45.0 ps, more preferably at least 50.0 ps, more preferably at least 55.0 ps, more preferably at least 60.0 ps, more preferably at least 65.0 ps, more preferably at least 70.0 ps, more preferably at least 75.0 ps, more preferably at least 80.0 ps, more preferably at least 85.0 ps, more preferably at least 90.0 ps, more preferably at least 95.0 ps, more preferably at least 100.0 ps. Building further on the above, preferably, the pulse length is at most 100 ns, more preferably at most 90 ns, more preferably at most 80 ns, more preferably at most 70 ns, more preferably at most 60 ns, more preferably at most 50 ns, more preferably at most 45 ns, more preferably at most 40 ns, more preferably at most 35 ns, more preferably at most 30 ns, more preferably at most 25 ns, more preferably at most 20 ns, more preferably at most 15 ns, more preferably at most 10 ns.

[0049] In a preferred embodiment, the pulsed laser device may be one of the following types: mode-locked solid-state laser device, Q-switched solid-state laser device, or gain switched semiconductor laser device.

[0050] The laser pulses may each have a fluence in the focal plane of the laser (electromagnetic energy delivered per unit area) in the range of 0.001 J / cm2to 100 J / cm2, 0.005 J / cm2to 50 J / cm2, 0.01 J / cm2to 10 J / cm2, or 0.05 J / cm2to 5 J / cm2, e.g. in the range of 0.1 J / cm2to 2 J / cm2.

[0051] The total number of laser pulses used for the photoporation may consist of 1 to 1000 laser pulses, such as 1 to 500 laser pulses, 1 to 100 laser pulses, 1 to 20 laser pulses, and most preferably 1 to 10 laser pulses (per cell). The number of laser pulses may be depending on the photoresponsive particle, the type of cargo, and the type of cells.

[0052] In a preferred embodiment, the laser pulse frequencies may range from less than 1 Hz to greater than 1 GHz. More preferably, the laser pulse frequency is between 10 Hz and 200 MHz.

[0053] In a preferred embodiment, the first wavelength is 532 nm, which is in the visible (green) light range. However, alternative choices are of course possible.

[0054] In an alternatively preferred embodiment, the photoporation laser device is a continuous wave laser device. CW laser devices are typically cheaper and simpler in their build. Additionally, their energy conversion is more efficient as there are no losses associated to the pulse generation of pulsed laser devices. Finally, the power output of a CW laser device is typically more stable, producing a more constant power, which makes the subsequent power control easier.

[0055] In a preferred embodiment, the photoporation laser device comprises or is provided with a cooling system, for forced cooling of the laser device. In a particularly preferred embodiment, the excitation light emitting device is an excitation laser device for generation of excitation laser beams of the second wavelength.

[0056] In an alternative embodiment, the excitation light emitting device comprises one or more of the following list: light-emitting diode (LED), metal-halide lamps, mercury vapor lamp, tungsten-halogen (incandescent) lamp, xenon arc lamp.

[0057] In a preferred embodiment, the excitation light emitting device is a continuous wave laser device. For the excitation light emitting device, increasing the peak power is less important, as it isn't used for photoporation, but instead for light / laser-induced excitation of particles. In that sense, a more stable power output is preferable.

[0058] In an alternatively preferred embodiment, the excitation light emitting device is a pulsed laser device.

[0059] The laser pulses of the excitation laser device preferably have a pulse duration of between 1 fs and 10 ns, preferably between 10 fs and 1 ns, more preferably between 50 fs and 500 ps. Specific preferred subranges can be chosen, for instance 1 to 10 ps, or 50 to 500 ps, or between 60 fs and 200 fs, such as 70 fs or 150 fs.

[0060] The laser pulse frequency for the excitation laser device is preferably between 10 kHz and 1 GHz, more preferably between 1 MHz and 500 MHz. Preferred frequencies are multiples of 10 MHz, such as 10 MHz, 20 MHz, 30 MHz, 40 MHz, 50 MHz, 60 MHz, 70 MHz, 80 MHz, 90 MHz, 100 MHz, etc.

[0061] The power of the pulsed excitation laser device is preferably at most 10 W, and ultimately dependent on the pulse duration. Femtosecond lasers typically have a power around 1 to 10 W, for instance 2 to 3 W, while picosecond and nanosecond lasers have a lower power due to the increased pulse duration, and typically have a power in the range of 100 mW to 1 W, for instance around 500 mW.

[0062] In a particularly preferred embodiment, the second wavelength is 488 nm, which is in the visible (blue) light range. However, alternative choices are of course possible

[0063] The components in the optical processing assembly allow for adjustments to the settings of the system, which can be dependent on the sample, the desired next steps (as discussed above, destruction, transfection, destabilization, ...), and further characteristics (such as type of payload in case of transfection). As a first part, the optical processing assembly comprises a first optical processing subsystem defining a first optical path via which the laser beams of the photoporation laser device are guided to the sample holder.

[0064] The first optical processing subsystem firstly comprises a power modulation means, allowing the subsystem to reduce the power of the laser beam of photoporation laser device to a desired level that is tailored to a particular application. This power modulation means can for instance be an acousto-optic modulator, a device that uses sound waves to control the properties (relating to its interaction with light) of the medium through which the light passes. AOMs typically comprise a transparent crystal, such as quartz or lithium niobate. When a sound wave is applied to the crystal, it creates a periodic variation in the refractive index of the crystal. This variation in the refractive index can be used to diffract the laser beam. By varying the intensity of the applied sound wave, a stronger diffraction of the laser beam occurs, resulting in a decreased power of the (part of the) laser that is transmitted further along the first optical path.

[0065] Another possibility for the power modulation means is an electro-optic modulator (EOM), where instead of using sound waves to control the properties of the medium through which light passes, a controllable electric field is used to affect the medium and its properties. EOMs are typically faster than AOMs, but result in a more complex design and consume more power.

[0066] Preferably however, a configuration is used with a halfwave plate followed by a polarizing beam splitter. The laser beam is passed through the halfwave plate, which rotates the polarization of the laser beam by 180 degrees. The laser beam is then passed through the polarizing beam splitter, and only transmits the laser beam if its polarization is aligned with the transmission axis of the polarizing beam splitter, and reflects the laser beam if its polarization is aligned with the reflection axis of the polarizing beam splitter.

[0067] By rotating the halfwave plate, the polarization of the laser beam can be rotated, and allows control of the amount of light that is transmitted and reflected by the polarizing beam splitter. As a result, the power of the transmitted laser beam can be modulated. The advantage lies in its simplicity of design, low cost and reliability.

[0068] The first optical processing subsystem further comprises a first dichroic deflection means, which is configured for reflecting light with a wavelength substantially equal to the first wavelength (that of the photoporation laser device) further along the first optical path towards the sample holder. Light with other wavelengths is transmitted (or differently deflected) such that it is steered away from the first optical path. A dichroic mirror is a preferred choice for the dichroic deflection means, as it absorbs very little of incident light, and can be very finely processed such that it only reflects a narrow range (stopband) of wavelengths, and transmits the others. A dichroic mirror is produced by building alternating layers of optical coatings with different refractive indices upon a glass substrate. The interfaces between the layers of different refractive index produce phased reflections, selectively reinforcing certain wavelengths of light and interfering with other wavelengths. By controlling the thickness and number of the layers, the frequency of the stopband of the mirror can be tuned and made as wide or narrow as desired. Because unwanted wavelengths are transmitted rather than absorbed, dichroic mirrors do not absorb this unwanted energy during operation and so do not become nearly as hot as the equivalent conventional mirror.

[0069] One advantage thus is that it further filters the incident light (from the photoporation laser device) to the desired wavelength, and removes unwanted wavelengths. Further advantages are the simplicity of such a mirror compared to performance, low cost, compact build and high resistance to heating.

[0070] The first optical processing subsystem further comprises a focusing lens for focusing the photoporation laser beam towards the sample holder. Most preferably, the focusing lens is positioned directly before the sample holder along the first (and second) optical path, and after the other components, to ensure the desired focus point is correctly positioned.

[0071] In this setup, the optical processing assembly allows for a sufficient control of the laser beams generated by the photoporation laser device. It is capable of modulating power towards the needs of the specific application, of filtering the laser beams further towards the needed wavelength, and of focusing the laser beams shortly before incidence at the sample holder.

[0072] The second optical processing subsystem is configured for directing the excitation light beams from the excitation light emitting device to the sample holder along a second optical path.

[0073] The third optical processing subsystem is configured for directing resultant light from the sample holder along a third optical path to the sensor means.

[0074] In a preferred embodiment, the first and second optical paths partially coincide or align, wherein the first and second optical processing subsystem partially overlap in terms of components comprised therein. This allows for the first and second optical processing subsystem to (re)use some of each other's components. It furthermore allows the system to be made more compact (no longer requiring space for fully separate optical paths), and sharing components reduces cost, reduces power consumption and simplifies its operation.

[0075] In particular, the focusing lens is preferably such a shared component, thus focusing both photoporation laser beams as well as excitation light beams. Since the purpose is the same in both cases, having and controlling two focusing lenses would be highly redundant, and would further require two separate optical paths, taking valuable space in the system, as well as adding more heat (and obstructing opportunities to remove said heat).

[0076] More preferably, another shared component is the first dichroic deflection means. While its use is limited for the excitation light beam (as they are simply transmitted, having a different wavelength than the first dichroic deflection means is configured to reflect), aligning the path from the sample holder to the first dichroic deflection means optimizes the use of free space, especially where this is limited such as at the sample holder. In essence, this means that the second optical path will pass through the first dichroic deflection means, and before that point, will diverge from first optical path. The part of the first and second optical paths between the first dichroic deflection means and the sample holder will be shared.

[0077] The first dichroic deflection means thus serves to join (or separate) the first and second optical paths, depending on the point of view.

[0078] In a preferred embodiment, the first and third optical paths partially coincide or align, wherein the first and third optical processing subsystem partially overlap in terms of components comprised therein. As before, this allows the subsystems to share components, to reduce volume of the system, as well as costs, power consumption and complexity.

[0079] Sharing the focusing lens in particular is again preferable, as in both directions (towards and away from the sample holder), focusing the beam will be advantageous.

[0080] The same advantages mentioned before apply for embodiments wherein the first dichroic deflection means is also on the first and third optical path. Additionally, the first dichroic deflection means largely allows full transmission of incident light that arrives from the sample holder, along the third optical path, since this light is the result of fluorescence of materials at the sample holder that are irradiated, and subsequently emit light with a (usually) different wavelength. The first dichroic deflection means only reflects light of the first wavelength, and allows the other wavelengths to pass through without substantial absorption or reflection, further along the third optical path towards the sensor means. In a preferred embodiment, the second and third optical paths partially coincide or align, wherein the second and third optical processing subsystem partially overlap in terms of components comprised therein. As before, this allows the subsystems to share components, to reduce volume of the system, as well as costs, power consumption and complexity.

[0081] In a most preferred embodiment, all three optical paths partially coincide or align, wherein the first, second and third optical processing subsystem partially overlap in terms of components used in each of them (meaning there are one or more components used in both the first, second and third optical processing subsystem). The advantages follow from the aforementioned combinations.

[0082] Ideally, the space around the sample holder is minimally used, by a single focusing lens, and the three optical paths align between the sample holder and the first dichroic deflection means, with the dichroic deflection means allowing passage from the excitation light beams and the light emitted from the sample holder as a result of its irradiation.

[0083] The sensor means can vary in its configuration, and can comprise multiple different sensors at once, such as an imager or a detector.

[0084] In a preferred embodiment, the third optical processing subsystem further comprises a beam collimator for collimating light returning from the sample holder (fluorescent light amongst others), onto the third optical path. Preferably, the beam collimator is positioned such that the collimator is positioned between the movable mirror assembly and the sample holder.

[0085] In a preferred embodiment, the beam collimator is positioned close to the sample holder along the optical paths, preferably between the first dichroic deflection means and the sample holder, but more preferably between the first dichroic deflection means and the focusing lens. However, in some embodiments, the collimator is the focusing lens, with the focusing lens focusing photoporation and excitation light beams along the first and second paths, and collimating (fluorescent) light from the irradiated (cells in the) samples at the sample holder.

[0086] In a preferred embodiment, the second optical processing subsystem comprises a second dichroic deflection means positioned on the second and third optical path, wherein the second dichroic deflection means is configured for reflecting light with a wavelength substantially equal to the second wavelength further down the second optical path towards the sample holder, and for transmitting resultant light returning form the sample holder with a wavelength substantially different from the (first and) second wavelength further down the third optical path towards the sensor means.

[0087] In some variations, the second dichroic deflection means functions as a band pass filter, which deflects / reflects all wavelengths lower than the second wavelength. This allows protection of the components in the third path from high energy wavelengths. It is not required to transmit lower wavelength light towards the detectors since there should not be any as well.

[0088] As with the first dichroic deflection means, this is preferably in the form of a dichroic mirror. By positioning it on the second and third optical path, it serves as a simple separator for light beams from the excitation light emitting device, ensuring that only those with the 'right' wavelength get reflected further along the second optical path towards the sample holder, while the others are transmitted (into a beam dump or the likes thereof). Meanwhile, light returning from the sample holder is not (or barely) filtered, as there is little to none light present with a wavelength equal to the second wavelength that is reflected out of the third optical path. As such, the returning light largely passes through the second dichroic deflection means, further along the third optical path, to the sensor means.

[0089] In embodiments wherein the first, second and third optical paths all align, it should furthermore be noted that the light returning from the sample holder passes through both the first and the second dichroic deflection means but stays largely unchanged, and continues along the third optical path to the sensor means. The other way around, the excitation light beams get reflected by the second dichroic deflection means, further along the second optical path, then pass through the first dichroic deflection means (further along the second optical path), and proceed to the sample holder.

[0090] The first optical path thus originates from the photoporation laser device, and subsequently passes to the power modulation means, to the first dichroic deflection means, to the focusing lens and to the sample holder. The second optical path thus originates from the excitation light emitting device, and subsequently passes to the second dichroic deflection means, to the first dichroic deflection means, to the focusing lens and to the sample holder. The third optical path originates from the sample holder, and subsequently passes to the first dichroic deflection means, to the second dichroic deflection means and ultimately to the sensor means.

[0091] Note that this does not preclude the presence of other components on any one of said paths.

[0092] In a preferred embodiment, the first dichroic deflection means is positioned on both the first, the second and the third optical path, while the second dichroic deflection means is not on the first optical path (but preferably on the third optical path as well as on the second optical path).

[0093] By doing so, part of the optical paths can be combined as mentioned before, from the first dichroic deflection means up to the sample holder. Inversely, positioning the second dichroic deflection means as a shared component in each path (with the first dichroic deflection means not being on the second optical path, and preferably on the third optical path) is possible, but leads to a suboptimal setup since the photoporation laser beams of the more frequently used, and much higher-power photoporation laser device, pass through the second dichroic deflection means each time, which is undesirable. In the first setup, the excitation light beams will pass through the first dichroic deflection means, but the excitation light emitting device is used less often, is lower-power and is of lesser importance.

[0094] In a preferred embodiment, the power modulation means is a tunable power attenuator, the tunable power attenuator comprising a half-wave plate for the first wavelength, and a polarizing beam splitter further along the first optical path than the half-wave plate, wherein the relative orientation of the half-wave plate and the polarizing beam splitter is tunable, thereby allowing the system to modulate the effectively transmitted power. The above embodiment is simple to use, compact, low-cost, easy to calibrate and reliable, as opposed to alternatives such as AOMs and EOMs. The function of the tunable power attenuator is for regulating the power of the photoporation laser beams that arrive at the sample holder.

[0095] In a preferred embodiment, the optical processing assembly comprises a movable mirror assembly with at least one movable mirror for reflecting light following the first or second optical path towards the sample holder, wherein the movable mirror assembly is configured to reflect said light onto the sample holder in a (two-dimensional) scanning pattern, the movable mirror assembly being positioned on the first, the second and the third optical path, between the first dichroic deflection means and the sample holder.

[0096] In a particularly preferred embodiment, the at least one movable mirror is configured for changing the position of incidence of the reflected light on the sample holder two- dimensionally in the plane of the sample holder, by tilting the at least one movable mirror over at least two non-parallel axes. Preferably this is achieved by tilting each at least two movable mirrors over at least one axis in concert with each other, the at least one axis of the at least movable mirrors being mutually non-parallel.

[0097] Preferably, the optical processing assembly comprises multiple movable mirrors, most preferably only two, each rotatable / tiltable over a single axis, wherein each mirror is rotatable / tiltable over a different and not parallel, preferably perpendicular, axis, thereby allowing the mirror set to cover a two-dimensional surface with the scanning pattern. Most preferably, it comprises said multiple, and preferably only two, movable mirrors shortly before the sample holder.

[0098] Alternatively, the optical processing assembly comprises a movable mirror assembly with a single movable mirror for reflecting the light following the first or second optical path towards the sample holder in a scanning pattern. In such a situation, the single movable mirror will be tiltable / rotatable over multiple (different) axes, in order to be able to cover a two-dimensional area with the scanning pattern. This is however used less often, as the control of such a single multi-axially movable mirror is more complex than using (at least) two, while the gain (in space and / or cost) is limited.

[0099] In a particularly preferred embodiment, the movable mirror(s) is (are) a mirror galvanometer or a galvo mirror. Galvo mirrors operate by rotating a mirror part relative to a static part with a galvanometer motor, which generates torque to rotate the mirror by applying and varying a magnetic field. Galvo mirrors allow rotation at very high speeds and with extreme accuracy (both crucial for light and / or laser beam manipulation), which is ideal for reflecting the light and / or laser beams in a scanning pattern onto the sample holder. Additionally, they are compact and lightweight, are relatively cheap and very durable.

[0100] Alternatives for a galvo mirror are amongst others:

[0101] - acousto-optic deflectors (AODs): excellent characteristics in terms of speed and precision; spatial light modulators (SLMs): high precision, and additional functionalities such as modulation of characteristics of the light and / or laser beam;

[0102] - fast scanning mirrors (FSMs): very high speed, relatively cheap, very durable; deformable mirrors (DMs): high speed, high versatility and precision.

[0103] It should be understood that where limitative statements are made, for instance, "only two" movable mirrors, "a single movable mirror", this is to be considered in its context of mirrors that actively take part in generating the scanning pattern. Other movable mirrors may of course still be present in other parts of the optical paths for deflecting / reflecting the light and / or laser beams.

[0104] In a preferred embodiment, the movable mirror assembly is positioned before the focusing lens along the first and second optical path (such that the focusing lens is between the movable mirror assembly and the sample holder). However, in some variations, the order is reversed, with the movable mirror assembly being positioned between the focusing lens and the sample holder.

[0105] In a further preferred embodiment, the power modulation means is configured for modulating the power of the photoporation laser beam to maintain a substantially constant flux as the photoporation beam are reflected in the scanning pattern, taking into account the angle of incidence of the photoporation laser beam on the plane of the sample holder and an associated change in beam area of the photoporation beam on the plane of the sample holder.

[0106] In moving the point of incidence of the laser beam on the sample holder during the scanning pattern, an inevitable result is that the angle of incidence is also changed, resulting in optical aberrations at the sample holder, typically increasing in aberrance the further they are off-axis. One of these unwanted effects is comatic aberration, which is the result of a non-paraxial incidence of light beams on a (focusing) lens, leading to a distorted image (with a comet-like tail). Another effect is that the area of incidence of an obliquely incident laser beam increases with respect to a perpendicular incidence, resulting in a smaller radiant exposure or fluence.

[0107] In order to address such aberrations (especially a reduced fluence), the power of the laser beams can be modulated to compensate for such effects, increasing the transmitted power output in accordance with the increase of area of incidence at the sample holder (and reducing the transmitted power output as the area of incidence reduces). In many cases, the effect of the scanning pattern on the fluence at each point can be modeled / predicted, and / or can be experimentally determined, allowing for a reliable modulation that counters these effects.

[0108] Alternatively, instead of modulating the power based on the angle of incidence to obtain a constant flux across the scanning pattern, the sample holder can be discretely illuminated in a plurality of (overlapping) smaller scan areas over which the flux varies less in order to cover the total scan area that is formed by the overlapping scan areas with a constant flux. The plurality of overlapping scan areas generates a mosaic that represents the total scan area.

[0109] In a preferred embodiment, the first optical processing subsystem further comprises a beam detection means and an optical element which partially deflects the photoporation laser beam to the beam detection means.

[0110] In a further preferred embodiment, the optical element which partially deflects the photoporation laser beam to the beam detection means, comprises a polarizing beam splitter along the first optical path, wherein the polarizing beam splitter removes waste light from the photoporation laser beam and directs said waste light to the beam detection means. The beam detection means is configured for detecting beam information relating to the photoporation laser beam from the waste light. In particular in the case of a photoporation pulsed laser device this is convenient, as it allows monitoring of the pulse regime and its characteristics, in order to correctly control and time operation of the other components of the optical processing assembly, such as the movable mirror assembly, but also the power modulation means. However, for continuous wave laser devices, it is also advantageous.

[0111] Most importantly however, the polarizing beam splitter serves to clean up the photoporation laser beam at that point, removing the waste light, such that the light that passes is linearly polarized.

[0112] As such, the beam detection means and the polarizing beam splitter are preferably positioned before the power modulation means along the first optical path (between photoporation laser device and the power modulation means), such that the laser beams arriving at the power modulation means are already linearized, and ideal for power modulation purposes.

[0113] Alternatively, the optical element which partially deflects the photoporation laser beam to the beam detection means, comprises a non-polarizing beam splitter, such as cube beam splitters, plate beam splitters and pellicle beam splitters.

[0114] In a further preferred embodiment, the scanning pattern comprises a plurality of discrete positions, wherein the at least one movable mirror is controllably moved based on the beam detection information from the beam detection means for incidence of the reflected light from the movable mirror on the discrete positions of the scanning pattern. It should be noted that the movable mirror can be used to perform the scanning pattern for both the excitation light beams and the photoporation laser beams. Preferably, it can also be used to direct light from the (content of the) sample holder back along the third optical path, towards the sensor means, ensuring that the desired area of the (content of the) sample holder can be visualized / analyzed.

[0115] As mentioned before, there are multiple configurations possible for the movable mirror assembly, with a multi-mirror (two preferably) being the preferred option, with the mirror being tiltable / rotatable around two different axes.

[0116] In a particularly preferred embodiment, the photoporation laser device is a pulsed laser device. This allows such beam information to be gathered as the (expected) time profile of the laser pulses. In a possible embodiment, the beam information on the laser pulses can be used to time the movement during the scanning pattern, wherein the mirrors are movable in a continuous fashion, such that the pulses impinge at the sample holder centered on the desired positions. Taking advantage of the short length of the pulses, the spread of the area on which the laser pulse arrives at the sample holder is fairly limited, and the intervals between subsequent pulses allow the mirrors to move such that the next pulse is targeted at a subsequent position on the scanning pattern.

[0117] In an alternative embodiment, the movable mirrors are only moved during the "off- time" between laser pulses impinging on the movable mirrors. In each of said movements, the mirrors are moved such that the 'next' discrete position in the scanning pattern is targeted with the next pulse, ensuring that only the desired area is targeted with the laser pulse, and receives the same and full amount of energy, and with a constant fluence for each 'target'. This leads to a perceived stepwise movement across the scanning pattern.

[0118] Aside from the scanning pattern, the system and methodology is obviously also applicable for 'single shot' applications, wherein only a single position on the sample holder is targeted.

[0119] In a preferred embodiment, the focusing lens is positioned on the first and second optical path between the dichroic deflection means and the sample holder, the focusing lens being movable along the second optical path to and from the sample holder.

[0120] In a particularly preferred embodiment, the focusing lens is positioned between the movable mirror assembly and the sample holder. It is clear that focusing the incident photoporation and excitation light beams on the sample holder, can be performed more reliably and accurately closer to the sample holder.

[0121] In a preferred embodiment, the focusing lens is positioned on both the first, second and third optical path, allowing it to be used for focusing photoporation laser beams and incident excitation light beams propagating to the sample holder, as well as for focusing returning light from the sample holder, towards the sensor means.

[0122] In a further preferred embodiment, the optical processing assembly further comprises a, preferably removable, beam diameter measurement means at a measurement position on the second optical path (and in many cases also on the first or second, or preferably both, optical path, as they coincide / align partially) past the focusing lens, the focusing lens being movable along the second optical path to and from the measurement position, wherein the beam diameter measurement means comprises a predefined fluorescent target.

[0123] Using a predefined fluorescent target allows the system to benchmark its operation in terms of focusing characteristics. The target is specifically chosen to respond well to the second wavelength of the excitation light beam, ensuring a reliable fluorescent emission that can be clearly visualized / analyzed, providing an insight on the extent to which the focusing lens is positioned as desired. In this light, the use of a pulsed laser device for the photoporation laser beams is particularly useful, as this allows for 2-photon excitation at the fluorescent material (with a higher certainty due to the higher intensity achieved by pulsed laser devices).

[0124] In particular, combining the beam diameter measurements along with information from a power output sensor (for measuring average power of the photoporation laser beams), allows the fluence at the sample to be determined.

[0125] Moving the focusing lens impacts the beam diameter at the position of the fluorescent target. This can then be visualized or measured by determining the intensity profile (preferably two-dimensionally) for multiple relative positions of the focusing lens to the target, which is preferably performed by the sensor means. By looking at positions with a higher value of the peak intensity and / or a narrower intensity profile (for instance, by determining the lowest FWHM, HWHM, standard deviation, etc., or a similar metric), the beam diameter is smaller, and the system can be adjusted according to specific requirements (for instance, desired fluence) for the beam diameter.

[0126] Preferably, the position of the focusing lens with respect to the fluorescent target is changed via a stepper motor or the likes thereof.

[0127] Preferably, the beam diameter measurement means is positioned between the focusing lens and the sample holder or at the sample holder. More preferably it is positioned at the sample holder, i.e., at the focal plane. Even more preferably, the beam diameter measurement means forms part of a receptacle, such as a tray, which is suitable for holding samples and for being insertable in the sample holder.

[0128] Preferably, the relative and / or the absolute distance from the focusing lens to the beam diameter measurement means is known, whether if it is between the focusing lens and the sample holder, or if it is on the sample holder or part of a receptacle for holding the samples, which receptacle can be placed in the sample holder. In a particularly preferred embodiment, the predefined fluorescent target of the beam diameter measurement means comprises a fluorescent plate or similar fluorescent target section.

[0129] Embedding the fluorescent target in the sample holder or in the receptacle is particularly advantageous, as this way, the relative position of the target to the focusing lens for which the beam diameter is ideal (the beam waist) can be found, and in that situation, the position of the fluorescent target is the same as the position of the cells when placed in the sample holder.

[0130] In a preferred embodiment, the sensor means comprises a visualization means for providing a visual representation of contents of the sample holder and a detection means for determining a quantitative characteristic of contents of the sample holder, and wherein a controllable switching means is provided for changing the third optical path to terminate on the visualization means or on the detection means.

[0131] In an alternative embodiment, the sensor means comprises a visualization means for providing a visual representation of contents of the sample holder and a detection means for determining a quantitative characteristic of contents of the sample holder. The system further comprises, preferably at the sensor means, an optical element configured for directing resultant light along the third optical path to either the visualization means or to the detection means, or to both simultaneously.

[0132] The split between two separate sensor modes is advantageous as it allows for a choice between a qualitative representation / analysis of a sample, via the visualization means, and a quantitative representation / analysis of the sample, via the detection means. The visualization means comprises an imaging system, such as a camera, suitable for converting the received light at the sensor means from the third optical processing subsystem, and originating from the sample holder, into a visual two- or three- dimensional representation of contents at the sample holder. As such, the visualization means preferably has an inbuilt screen, or is connectable to a visualization system such as a screen. Alternatively or additionally, internal image analysis, via integrated processing means, can be performed to determine the beam size and / or other relevant features.

[0133] The detection means preferably comprises a photomultiplier, more preferably a silicon photomultiplier (SiPM). The photomultiplier converts incident photons into a processable electrical signal. SiPMs operate based on the principle of a SPAD (Single-photon Avalanche Diode) and have the advantage of providing high gain (as do 'regular' photomultipliers, such as PMTs), but with low voltage and very fast response times. Additionally, SiPMs are very compact, making them ideal for the present application. Alternatively, the detection means preferably comprises one or more photodiodes, thermal detectors, integrating spheres, calorimeters.

[0134] In a particularly preferred embodiment, the sensor means are positioned at an section of the system oppositely positioned with respect to the photoporation laser device, in order to minimize effects the high-power photoporation laser device could have on the sensor means. Preferably, the switching means is a mirror suitable for deflecting a beam along the third optical path to the visualization means or to the detection means.

[0135] The mirror can be movable in and out of the third optical path, such that it either deflects the beams along said third optical path, or does not deflect said beams, leading the beam to impinge on either the visualization means or on the detection means. A common example of this is a so-called flip mirror.

[0136] In another embodiment, the mirror is rotatable between at least two positions, such that in a first of the two positions, the deflected beam impinges on the visualization means, and in a second of the two positions, the deflection beam impinges on the detection means. Such a rotatable mirror can for instance be a galvo mirror, although other realization are possible as well. Crucial herein is the accuracy and reliability with which the two positions are reached when switched.

[0137] In another embodiment, the mirror can be functionally changed between transmission mode and deflection mode (for instance, using a liquid-crystal cell, electrochromic materials). In the case of transmission mode, the transmitted beam impinges on either the visualization means or on the detection means, in the case of deflection mode, the deflected beam impinges on the other. This has the advantage the switching means does not need to be moved, and maintains its position, ensuring the 'right' sensor means is hit at the same position every time.

[0138] Alternative to using a switching means to change the third optical path to terminate on the visualization means or on the detection, one or both of the visualization and / or detection means can be moved in or out of the third optical path, without employing a switching means.

[0139] In a particularly preferred embodiment, the system comprises a power output sensor, configured for measuring an average power and / or fluence of incident light, wherein the system comprises a deflection means configured for reflecting light with a wavelength substantially equal to the first wavelength from the first optical path towards the power output sensor. Alternatively or additionally, the deflection means may be configured for reflecting light with a wavelength substantially equal to the second wavelength towards the power output sensor. The deflection means can for instance be a dichroic deflection means, although alternatives are suitable as well.

[0140] In a preferred embodiment, the system comprises an power output sensor, configured for measuring an average power and / or fluence of incident light, wherein the sample holder comprises a deflection means configured for reflecting light with a wavelength substantially equal to the first wavelength towards the power output sensor. The primary goal is to ascertain the power / energy received at the sample holder from the photoporation laser device. Based on these measurements, the power modulation means can then be adjusted to increase or decrease transmitted power, and / or the photoporation laser device can be adjusted to increase or decrease power of the generated photoporation laser beams. As mentioned earlier, this is particularly advantageous in combination with the beam measurement means, which allows the fluency to be easily determined combining the measured average power, along with the beam diameter. Alternatively or additionally, the deflection means may be configured for reflecting light with a wavelength substantially equal to the second wavelength towards the power output sensor, in order to ascertain the power / energy received at the sample holder from the excitation light emitting device.

[0141] The dichroic deflection means can be integrated in the sample holder itself, but can be part of a receptacle to be placed in the sample holder. Preferably, it is integrated in the sample holder. By moving the sample holder, the system can position the dichroic deflection means in the path of the beams arriving at the sample holder, to deflect said beams to the power output sensor. This way, the power output sensor is only used when the power output is tested.

[0142] The output sensor preferably has a fixed position in the system, to ensure that it is properly aligned with the beams deflected by the dichroic deflection means.

[0143] In a preferred embodiment, the power output sensor comprises a thermopile. The thermopile is suitable for converting thermal energy from the incident light into electrical energy and allows measuring the energy (and power) of the incident light. Thermopiles are composed of several thermocouples, and operate based on the thermoelectric effect, i.e., generating a voltage when the thermocouples are exposed to a temperature difference.

[0144] In a variation, the sample holder can comprise a general deflection means instead of a dichroic deflection means, for reflecting all incident light towards the power output sensor. This would further allow to measure the (processed) power output of the excitation light emitting device.

[0145] In a particularly preferred embodiment, the first, second and third optical path at least partially align between the first dichroic deflection means and the sample holder. Preferably, they entirely align between the first dichroic deflection means and the sample holder. Even more preferably, the second and third optical path are further entirely aligned between the second dichroic deflection means and the sample holder. The advantages of this setup (reduction of used volume, using components in multiple of the first, second and third optical processing subsystems) have been amply discussed before.

[0146] In a further aspect, the invention relates to a method for photoporation of cells, which in itself may form part of an overarching method, such as cargo transfection of cells, the cargo having been described, non-limitatively, earlier in the description.

[0147] The method for photoporation of cells comprises the following steps: providing one or more samples at a sample holder, wherein said one or more samples comprise a mixture of one or more cells, one or more photoresponsive particles and a cargo;

[0148] - generating photoporation laser beams, preferably photoporation laser pulses, with a predefined first wavelength;

[0149] - directing said photoporation laser beams along a first optical path towards the sample holder, the directing said photoporation laser beams further comprising the following processing steps: o modulating power of the photoporation laser beams; o reflecting the modulated photoporation laser beams with a first dichroic deflection means configured for reflecting light with a wavelength substantially equal to the first wavelength further along the first optical path towards the sample holder and transmitting light with a wavelength substantially different from said first wavelength away from the first optical path; o focusing the reflected photoporation laser beams; irradiating the one or more samples at the sample holder with the focused photoporation laser beams, thereby causing permeabilization of one or more cell barriers of the cells and subsequent passage of the cargo through the permeabilized cell barriers, into the cell; generating excitation light beams, preferably laser beams, and more preferably continuous wave excitation laser beams, with a predefined second wavelength, different from the first wavelength;

[0150] - directing said excitation light beams along a second optical path towards the sample holder holding the sample wherein at least one of the cells comprises the cargo; irradiating the sample wherein at least one of the cells comprises the cargo with the excitation light beams; directing emitted light from the sample as a result of irradiation with the excitation light beams along a third optical path to a sensor means; measuring one or more characteristics of the light received at the sensor means.

[0151] The integration of the photoporation process (and cargo delivery) into that of the subsequent process of analysis (measuring, visualizing, etc.) provides for clear improvement over the prior art, where the processes are performed separately. Additionally, the integration allows re-using components, facilitates the process of sample management (the samples stay in one place), along with further advantages.

[0152] It should be noted that the system used for the above method is preferably that of the first aspect of the invention. As such, any embodiment or variation discussed with respect to the system is considered as disclosed as such for the method of the invention as well, without having been explicited. Amongst such embodiments are for instance the ranges in characteristics of the photoporation and excitation light emitting devices (power and derivatives thereof such as peak power, average power, fluence, pulse frequency, pulse length, pulse energy, etc.).

[0153] A particular improvement described for the system is that of the combination of the optical paths, such that all three optical paths align / coincide between the first dichroic deflection means and the sample holder, and with the second and third preferably coinciding even further, between the second dichroic deflection means and the sample holder. Not only does this make a system more compact, it furthermore ensures that the re-used components (focusing lens, collimator, dichroic deflection means, etc.) do not need to be configured, calibrated, maintained, etc., separately for each optical path.

[0154] Another particular improvement provides for a method of (auto)focusing a focusing lens in such a system, wherein the sample holder or a receptacle for holding the cells that is placed in the sample holder, comprises a fluorescent target. In such a further method, steps are comprised of: moving the focusing lens continuously or stepwise relatively to the fluorescent target at the sample holder; irradiating the fluorescent target with a light beam, preferably a laser beam, preferably a single pulse, preferably with excitation light beams; directing emitted light from the sample as a result of irradiation with the light beam along a third optical path to a sensor means; measuring an intensity distribution for multiple distances between the focusing lens and the fluorescent target during the moving of the focusing lens; determining a minimal beam diameter in said measured intensity distributions. The intensity distributions are preferably two-dimensional (x and y axis in the plane perpendicular to the second optical path at the sample holder), and allows to determine characteristics on the distribution such as the standard deviations in each dimension, oxand oy. These characteristics are then used to determine the position of the focusing lens with respect to the target for which a minimal beam diameter is achieved, in the case the standard deviations are used, for instance via

[0155] Based on this, not only (auto)focusing and (auto)calibration is possible, but further corrections can be performed, such as adjusting power to achieve a certain desired fluence (energy per unit area).

[0156] In a final aspect, the invention pertains to a use of the system for photoporation of cells according to the first aspect, for permeabilization of cell barriers of said cells, as well as for further applications thereafter as described earlier. Specifically, such further applications comprise cargo transfect of cells, the cargo having been non-limitatively described earlier in the description.

[0157] WO2022 / 189627, W02021 / 058430, W02020127076, and applicant's presently unpublished patent applications PCT / EP2023 / 058509, PCT / EP2022 / 064003 are hereby incorporated by reference.

[0158] In what follows, the invention is described by way of non-limiting examples illustrating the invention, and which are not intended to and should not be interpreted as limiting the scope of the invention.

[0159] EXAMPLES

[0160] Fig. 1 describes a potential embodiment of the system of the invention. The system therein comprises a photoporation laser device (1), configured for generating photoporation laser beams, preferably pulses, with the predefined first wavelength. The system further comprises a excitation light emitting device (2), in this case an excitation laser device, which is preferably a continuous wave laser device, configured for generating excitation light beams of a second wavelength, different from the first wavelength. The system further comprises a sample holder (not shown, in a layer above the focusing lens), a sensor means (6, 6a, 6b) and an optical processing assembly (3, 4, 5, 7, 8, 9, 10, Ila, 11b), to guide and process the photoporation and excitation light beams to the sample holder (not shown), and guide resulting light from the sample holder to the sensor means (6, 6a, 6b). The optical processing assembly comprises a first optical processing subsystem (3, 4, 5, 8, 10, Ila, 11b), which is used for guiding and processing the photoporation laser beams from the photoporation laser device (1) to the sample holder.

[0161] The first optical processing subsystem comprises (sequentially from the photoporation laser device): a first kinematic mirror (10),

[0162] - an (optional) shutter (Ila) for additional safety with integrated mirror (11b), a second kinematic mirror (10'),

[0163] - a power modulation means (3) to control the power of the photoporation beam that is allowed to reach the sample holder, a first dichroic deflection means or dichroic mirror (4), to reflect the photoporation laser beams with the first wavelength further along the first optical path and transmit light with another wavelength (away from the first optical path), a movable mirror assembly (8) for reflecting light coming from the first dichroic deflection means (4) in a scanning pattern towards the sample holder; a focusing lens (5) for focusing the beams from the movable mirror assembly (8).

[0164] The focusing lens (5) and movable mirror assembly (8) are positioned as close as possible to the sample holder (not shown), to ensure that the photoporation laser beams remains narrow and can be focused and correctly targeted on the sample holder.

[0165] The kinematic mirrors (10, 10'), as well as the mirror (11b) primarily serve the function of guiding the photoporation laser beam and creating a first optical path such that the system can be made compact and efficient. They are however not essential, and may be reduced or increased in number, and / or may be repositioned to other points in the first optical path between other components.

[0166] The shutter (Ila) likewise is an optional feature, and serves as an additional safety measure for guaranteeing the high-power photoporation laser beams can't impinge on the sample holder when this is not desired.

[0167] The optical processing assembly further comprises a second optical processing subsystem, which is used for guiding and processing the excitation light beams from the excitation light emitting device (2) to the sample holder.

[0168] The second optical processing subsystem comprises (sequentially from the excitation light emitting device): a second dichroic deflection means or dichroic mirror (7), to reflect the excitation light beams with the second wavelength further along the second optical path and transmit light with another wavelength (away from the second optical path); - the first dichroic deflection means (4), which allows the excitation beams to pass through as they have a wavelength different from the first wavelength;

[0169] - the movable mirror assembly (8) for reflecting the excitation beams that passed through the dichroic deflection means (4) in a scanning pattern towards the sample holder; a focusing lens (5) for focusing the beams from the movable mirror assembly (8).

[0170] The optical processing assembly further comprises a third optical processing subsystem, which is used for guiding and processing (some of the) light emitted at the sample holder (by samples held therein, resulting from impinging light), from the sample holder to the sensor means (6).

[0171] The third optical processing subsystem comprises (sequentially from the sample holder):

[0172] - the focusing lens (5);

[0173] - the movable mirror assembly (8);

[0174] - the first dichroic deflection means (4), which allows the returning light to pass through as they (mostly) have a wavelength different from the first wavelength;

[0175] - the second dichroic deflection means (7), which allows the excitation beams to pass through as they (mostly) have a wavelength different from the second wavelength; a third kinematic mirror (10"'), reflecting the returning light that has passed through the first and second dichroic deflection means; a sensor means (6, 6a, 6b) comprising a detection means (6a) and a visualization means (6b), and a switching means (9) in the form of a movable flip mirror to reflect or pass the beam reflected by the third kinematic mirror (10"') towards either one of the detection or visualization means.

[0176] Again, the third kinematic mirror (10'") is not essential.

[0177] The optical paths and subsystems are further shown in Figs. 2A, 2B, 4, 5, 6, 7, 8, 9 and 10.

[0178] Fig. 2A and 2B show the first optical path followed by a photoporation laser beam, in the case of the shutter (Ila) being closed (Fig. 2A), and in the case of the shutter (Ila) being open (Fig. 2B). If closed, the photoporation beam (20a) is deflected by the first kinematic mirror (10') into a further beam (20b), but is stopped at the shutter (Ila). If open, it is reflected by the mirror (11b) as a further beam (20c), and reflected again by the second kinematic mirror (10') as yet another further beam (20d) towards the power modulation means (3), after which a modulated further beam (20e) propagates to the first dichroic deflection means (4), which deflects it (or at least, the components thereof with the 'right' wavelength) towards the movable mirror assembly (8) as a further beam (20f). The movable mirror assembly (8) redirects it in two phases, after reflection with both its two mirrors as first further beam (20g) and a second further beam (20h), towards the focusing lens (5), which finally transmits a final beam (20i) to impinge at the sample (12) in the sample holder.

[0179] The power modulation means (3) can take a number of forms, and is described in a potential embodiment in Fig. 3, wherein the power modulation means comprises a halfwave plate (3a) and a polarizing beam splitter (3b). The halfwave plate rotates the orientation of the linearly polarized light of the incoming (photoporation) laser beam (30a), with the polarizing beam splitter (3b) then transmitting only light with a specific (predefined) polarization orientation (30b), and deflecting the rest (30c) into a beam dump (3c).

[0180] Fig. 4 shows a sideways view of the system during use of the photoporation laser device, wherein the sample holder (not shown) comprises a (dichroic) deflection means (14), deflecting light from the photoporation laser device (1) towards a power output sensor (13) in the form of a thermopile.

[0181] Fig. 5 shows the system in use, where the excitation light emitting device (2) produces a excitation light beam (40a) along the second optical path, irradiating the sample (12). The excitation light beam (40a) impinges on the second dichroic deflection means (7), which is configured for deflecting light with the second wavelength, i.e., that of the excitation light beams, towards the sample holder along the second optical path. The reflected excitation light beam (40b) passes substantially entirely through the first dichroic deflection means (4), as this only deflects light with the first wavelength, as a further beam (40c) onto the movable mirror assembly (8), which redirects it in two stages (40d, 40e) towards the focusing lens (5), and then as a final excitation light beam (40f) onto the sample (12).

[0182] Fig. 6 shows the system in use, where the photoporation laser device (1) produces a photoporation laser beam along the first optical path, irradiating the sample (12), and with the sample (12) itself emitting light resulting from said irradiation, which light (50a) falls (amongst others) upon the focusing lens (5), via which it is redirected as a further beam (50b) to the movable mirror assembly (8), which redirects it as a further beam (50c) to the first dichroic deflection means (4). As it only reflects light with the first wavelength, the further beam (50c), substantially entirely, passes on to the second dichroic deflection means (7) as a further beam (50d), and is again, substantially entirely, since the second dichroic deflection means only reflects light with the second wavelength, passed as a further beam (50e) to the third kinematic mirror (10"'), which reflects it as further beam (50f) to the sensor means (6, 6a, 6b), where the switching means (9) deflects it as further beam (50g) to the visualization means (6b).

[0183] Fig. 7 shows the same as Fig. 6, but instead of the photoporation laser device (1), the excitation light emitting device (2) is used to irradiate the sample (12), and the detection means (6a) is used to process the resulting light from the irradiation of the sample (12). This again results in the sample (12) itself emitting light resulting from said irradiation, which light (50a) falls (amongst others) upon the focusing lens (5), via which it is redirected as a further beam (50b) to the movable mirror assembly (8), which redirects it as a further beam (50c) to the first dichroic deflection means (4). As this only reflects light with the first wavelength, the further beam (50c), substantially entirely, passes on to the second dichroic deflection means (7) as a further beam (50d), and is again, substantially entirely, passed as a further beam (50e), to the third kinematic mirror (10"'), which reflects it as further beam (50f) to the sensor means (6, 6a, 6b), where the switching means (9) this time is not in place, resulting the beam (50f) to continue to the detection means (6a) this time.

[0184] Fig. 8 shows a variation of Fig. 7, where the visualization means (6b) is used to process the resulting light from the irradiation of the sample (12).

[0185] Fig. 9A and Fig. 9B finally show an embodiment of the sensor means (6), which comprises a visualization means in the form of a camera (6b), and a detection means (6a). Switching between which sensor means is actually used, is performed by a flip mirror (9). The flip mirror (9) comprises a movable mirror part, which can be rotated in and out of the third optical path, and either deflects the light (50f) returning from the sample as a deflected beam (50g), to the camera or visualization means (6b) as in Fig. 9A, or allows passage of the light (50f) returning from the sample, to the detection means (6a) as in Fig. 9B.

[0186] The present invention should not be construed as being limited to the embodiments described above and certain modifications or changes may be added to the examples described without having to re-evaluate the appended claims.

Claims

CLAIMS1. System for photoporation of cells, said system comprising: a. a laser device, preferably a pulsed laser device, for generation of photoporation laser beams, preferably photoporation laser pulses, with a predefined first wavelength; b. an excitation light emitting device, preferably an excitation laser device, more preferably a continuous wave laser device, for generation of excitation light beams with a predefined second wavelength, the second wavelength different from the first wavelength; c. a sample holder configured for holding one or more receptacles with cell material; d. a sensor means, configured for detection and / or visualization of cell material in the sample holder, e. an optical processing assembly, comprising: i. a first optical processing subsystem configured for directing the photoporation laser beam from the pulsed laser device along a first optical path to the sample holder, and configured for processing said photoporation laser beam, the first optical processing subsystem comprising along the first optical path a tunable power modulation means for modulating power of the photoporation laser beam, a first dichroic deflection means configured for reflecting light with a wavelength substantially equal to the first wavelength further along the first optical path towards the sample holder, the first dichroic deflection means being positioned downstream along the first optical path more proximal to the sample holder than the power modulation means, and a focusing lens for focusing the photoporation laser beam, ii. a second optical processing subsystem configured for directing the excitation light beam from the excitation light emitting device along a second optical path to the sample holder; and iii. a third optical processing subsystem configured for directing resultant light from the sample holder along a third optical path to the sensor means.

2. System according to the preceding claim 1, the second optical processing subsystem comprising a second dichroic deflection means positioned on the second and third optical path, wherein the second dichroic deflection means isconfigured for reflecting light with a wavelength substantially equal to the second wavelength further down the second optical path towards the sample holder, and for transmitting resultant light returning form the sample holder with a wavelength substantially different from the second wavelength further down the third optical path towards the sensor means.

3. System according to any one of the preceding claims 1 or 2, wherein the first dichroic deflection means is positioned on both the first, the second and the third optical path, and wherein the focusing lens is also configured for focusing the excitation light beam.

4. System according to any one of the preceding claims 1 to 3, wherein the power modulation means is a tunable power attenuator, the tunable power attenuator comprising a half-wave plate for the first wavelength, and a polarizing beam splitter further along the first optical path than the half-wave plate, wherein the relative orientation of the half-wave plate and the polarizing beam splitter is tunable.

5. System according to any one of the preceding claims 1 to 4, wherein the optical processing assembly comprises a movable mirror assembly comprising at least one movable mirror for reflecting light following the first or second optical path towards the sample holder, wherein the movable mirror assembly is configured to reflect said light onto the sample holder in a scanning pattern, the movable mirror assembly being positioned on the first, the second and the third optical path, between the first dichroic deflection means and the sample holder, the movable mirror preferably being a mirror galvanometer.

6. System according to the preceding claim 5, wherein the at least one movable mirror is configured for changing the position of incidence of the reflected light on the sample holder two-dimensionally in the plane of the sample holder, by tilting the at least one movable mirror over at least two non-parallel axes, preferably by tilting each at least two movable mirrors over at least one axis, the at least one axis of the at least movable mirrors being mutually non-parallel.

7. System according to the preceding claim 5 or 6, wherein the power modulation means is configured for modulating the power of the photoporation laser beam to maintain a substantially constant flux as the photoporation beam are reflected in the scanning pattern, taking into account the angle of incidence of thephotoporation laser beam on the plane of the sample holder and an associated change in beam area of the photoporation beam on the plane of the sample holder.

8. System according to any one of the preceding claims 1 to 7, wherein the first optical processing subsystem further comprises a beam detection means and an optical element which partially deflects the photoporation laser beam to the beam detection means.

9. System according to claim 8 and any one of the preceding claims 5 to 7, wherein the scanning pattern comprises a plurality of discrete positions, wherein the at least one movable mirror is controllably moved based on the beam detection information from the beam detection means for incidence of the reflected light on the discrete positions of the scanning pattern.

10. System according to any one of the preceding claims 1 to 9, wherein the focusing lens is positioned on the first and second optical path between the dichroic deflection means and the sample holder, preferably between the movable mirror assembly and the sample holder, the focusing lens being movable along the second optical path to and from the sample holder.

11. System according to the preceding claim 10, wherein the optical processing assembly further comprises a, preferably removable, beam diameter measurement means at a measurement position on the second optical path past the focusing lens, preferably between the focusing lens and the sample holder or at the sample holder, the focusing lens being movable along the second optical path to and from the measurement position, wherein the beam diameter measurement means comprises a predefined fluorescent target, preferably a fluorescent plate.

12. System according to any one of the preceding claims 1 to 11, wherein the sensor means comprises a visualization means for providing a visual representation of contents of the sample holder and a detection means for determining a quantitative characteristic of contents of the sample holder, said detection means preferably being a photomultiplier, and wherein a controllable switching means is provided for changing the third optical path to terminate on the visualization means or on the detection means.

13. System according to any one of the preceding claims 1 to 11, wherein the sensor means comprises a visualization means for providing a visual representation of contents of the sample holder and a detection means for determining a quantitative characteristic of contents of the sample holder, said detection means preferably being a photomultiplier, and comprising an optical element configured for directing resultant light along the third optical path to either the visualization means or to the detection means, or to both simultaneously.

14. System according to any one of the preceding claims 1 to 13, wherein the system comprises an power output sensor, preferably a thermopile, configured for measuring an average power and / or fluence of incident light, further comprising a deflection means, preferably dichroic, configured for reflecting light with a wavelength substantially equal to the first wavelength and / or the second wavelength from the first optical path towards the power output sensor.

15. System according to any one of the preceding claims 1 to 14, wherein the first, second and third optical path at least partially, and preferably entirely, align between the first dichroic deflection means and the sample holder.

16. Use of the system according to any one of the preceding claims 1 to 15 for photoporation of cells.

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