Cell photoperforation system, cell photoperforation method and application of cell photoperforation system

Abstract

The present invention relates to a photoperforation system, a cell photoperforation method and the use of such a system for increasing the permeability of the cell membrane or disrupting the cell membrane for further use.

Description

Technical Field

[0001] This invention relates to photoporation systems, cell photoporation methods, and applications of such systems, with the aim of increasing cell membrane permeability or disrupting cell membranes for further applications. Background Technology

[0002] The delivery of various macromolecules into the cytosol of different cell types is essential for a wide range of applications, such as cancer immunotherapy, stem cell therapy, and other biomedical research fields. Most macromolecules cannot spontaneously cross the cell membrane because it is impermeable to many compounds. Therefore, different techniques are needed to achieve the cytosol delivery of these cell-impermeable compounds. Electroporation has been used as a standard delivery tool for many years. Electroporation has the ability to form pores in the cell membrane after the application of an electrical pulse. Cell-impermeable compounds can then migrate from the surrounding culture medium into the cell interior through these pores. Although electroporation can achieve high delivery efficiency for a wide range of cell types, several limitations have been reported, including high cytotoxicity, induction of genomic alterations, induction of phenotypic changes, and low delivery efficiency in some primary (immune) cells. Therefore, new techniques are needed that do not affect the genotype or phenotype of cells and can achieve high delivery efficiency and high viability.

[0003] One of these newer delivery methods utilizes photoperforation to create the desired pores in the cell membrane, allowing for experimentation and use on a wider range of cells. Clearly, while this is an application of great interest, development is still in its early stages in terms of methodology, particularly in terms of supporting hardware. Such methods are described in WO2022 / 189627, WO2021 / 058430, WO2020127076, and the applicant's currently unpublished patent applications PCT / EP2023 / 058509 and PCT / EP2022 / 064003.

[0004] Current optical perforation systems are primarily found in laboratory environments and are typically not dedicated systems. Instead, they are built by researchers (mostly from scratch) and are not optimized in any way for user-friendliness, efficient use of components, or increased accuracy.

[0005] The present invention aims to solve the above-mentioned problems and provide a comfortable pair of glasses that can be easily adjusted. Summary of the Invention

[0006] In a first aspect, the present invention relates to a cell photoporation system according to claim 1.

[0007] In a second aspect, the present invention relates to the application of the system according to the first aspect in photoperforation of one or more cells, and particularly to a further application for delivering cargo into said cells.

[0008] In a third aspect, the present invention relates to a method for performing a photoperforation step on one or more cells using a system according to the first aspect, preferably for delivering cargo into said cells.

[0009] Further modifications have been contemplated with respect to one or more of the foregoing aspects of the invention, as described in the dependent claims and the remainder of the specification.

[0010] The invention described provides a significant improvement over the prior art, which is lacking in already well-developed systems and associated methods and applications, thereby allowing for a more versatile integrated system for optical perforation and subsequent visualization. Attached Figure Description

[0011] Figure 1 A schematic diagram illustrating an embodiment of the system according to the present invention is shown.

[0012] Figure 2A and 2B This illustrates how the laser beam in the optical perforation is either blocked or passes through the shutter. Figure 1 The system.

[0013] Figure 3 The possible implementations and operation of the tunable power modulation device are shown.

[0014] Figure 4 It shows Figure 1 A side view of another embodiment of the system in the optically perforated laser beam propagation state.

[0015] Figure 5 It shows Figure 1 The system in which an excitation beam irradiates the sample.

[0016] Figure 6 It shows Figure 1 The system in which an optically perforated beam illuminates a sample, and the resulting emitted light is directed to a visualization device.

[0017] Figure 7 It shows Figure 1 The system in which an excitation beam illuminates a sample, and the resulting emitted light is guided to a detection device.

[0018] Figure 8 It shows Figure 1 The system in which an excitation beam illuminates a sample, and the resulting emitted light is directed to a visualization device.

[0019] Figure 9A and 9BAn embodiment of the sensor device is shown, which has a visualization device and a detection device, as well as a switching device for defining the trajectory of the returning beam. Detailed Implementation

[0020] Unless otherwise defined, all terms used in the description of this invention, including technical and scientific terms, shall have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. For a better understanding of the description of this invention, the following terms are explicitly explained.

[0021] In this document, “one” and “the” refer to the singular and plural, respectively, unless the context otherwise requires. For example, “section” means one or more sections.

[0022] When the term "about" or "approximately" is used in this document with reference to measurable quantities, parameters, durations, or moments, variations refer to referenced values ​​of about 20%, preferably about 10%, more preferably about 5%, even more preferably about 1%, and even more preferably about 0.1%, provided that such variations apply to the described invention. However, it must be understood that the values ​​of the quantities used when employing the term "about" or "approximately" are themselves specifically disclosed.

[0023] The terms “comprising,” “including,” “consisting of,” “formed by,” “equipped with,” “having,” “with,” “including,” and “containing” are synonyms and are inclusive or open terms that indicate the presence of the following and do not exclude or prevent the presence of other components, features, elements, components, or steps known or disclosed in the prior art.

[0024] The term "cell" refers to all types of biological cells, including eukaryotic and prokaryotic cells. As used herein, the terms "cell" and "biological cell" are used interchangeably.

[0025] The term "alter (altering or alteration)" refers to any way in which one or more properties of a cell are altered, at least locally, such as the cell barrier. Alteration includes, but is not limited to, inducing localized changes in the cell's composition, such as the composition of the cell barrier, by adding, removing, disrupting, or rearranging components. Alteration includes, for example, altering one or more physicochemical properties, such as viscosity, porosity, density, rigidity, elasticity, etc. Alteration also includes localized disruption or rearrangement of cell barrier components, resulting in changes in the composition and / or physicochemical properties of the cell barrier. Alteration includes other deformation, permeability, and perforation.

[0026] The terms “deformation,” “deformed,” and “distortion” refer to any way in which the spatial organization or structure of cells, particularly the spatial organization or structure of cellular barriers, is altered, at least partially, for example, at least locally. Examples of deformation include providing a porous barrier with indentations or depressions.

[0027] The terms “permeation,” “clearing,” and “permeation process” refer to any manner in which cellular permeability, particularly the permeability of cellular barriers, is altered at least partially, for example, at least locally. Examples of permeation include altering a barrier composition or structure to make it more permeable to cargo.

[0028] The term "perforate (perforating or perforation)" refers to any means of providing one or more openings, pores, or openings at least partially (e.g., at least locally) to a cell, particularly a cellular barrier. By perforating the cellular barrier, openings are formed within the barrier, thereby allowing the transport of goods through or into the barrier.

[0029] The terms “perforate,” “perforating,” or “perforation” are used interchangeably with the terms “increase its permeability,” “permeable,” “permeable,” and “make permeable.” Similarly, the terms “opening,” “pore,” and “pore” are used interchangeably in this document.

[0030] As used herein, the terms "cargo" or "reagent" refer broadly to any chemical (e.g., inorganic or organic), biochemical or biological substance, molecule or macromolecule (e.g., biological macromolecule), particle (e.g., nanoparticle), combination or mixture thereof, sample of undetermined composition, or extract made from biological material such as bacterial, plant, fungal, or animal cells or tissues. Preferred "cargo" or "reagent" includes nucleic acids, oligonucleotides, ribozymes, proteins, polypeptides, peptides, peptide mimics, peptide nucleic acids, antibodies, antibody fragments, antibody-like protein scaffolds, aptamers, photoaptamers, spiegelmers (mirror image aptamers), chemicals, lipids, sugars, polysaccharides, etc., and any combination thereof, such as gene editing systems, such as CRISPR / Cas. Depending on the context, the term "reagent" may mean a "therapeutic agent" or "medicine" that can be used or employed for the treatment, cure, prevention, or diagnosis of a disease. Cargo taught herein includes, but is not limited to, cargo in solution and cargo dried or lyophilized, such as powdered cargo.

[0031] Most preferably, "cargo" refers to nucleic acids (especially DNA (deoxyribonucleic acid) or RNA (ribonucleic acid)) and proteins.

[0032] In embodiments, the goods may comprise or consist of two or more reagents combined or chemically bonded to each other. In embodiments, the goods may comprise or consist of two or more reagents coupled to each other (e.g., covalently bonded). For example, the goods may be reagents comprising or consisting of two, three, four, five, six, or more reagents coupled to each other (e.g., covalently bonded).

[0033] The terms "photoresponse," "photosensitivity," and "photosensitization" are used interchangeably and refer to the ability to respond to electromagnetic radiation (e.g., visible light). Specifically, the term refers to the ability to respond to electromagnetic radiation generated by the optically perforated laser device of the present invention, but is not necessarily limited thereto. The "response" to electromagnetic radiation should be considered in the context of optical perforation, i.e., a response suitable for creating pores in cellular barriers (cell membranes).

[0034] Preferably, the photoresponsive particles are organic or inorganic nanoparticles. A preferred embodiment of the nanoparticles comprises metals and / or metal oxides, such as gold, titanium, and titanium oxides, such as TiO2 (titanium dioxide).

[0035] The range of numbers defined by the endpoints includes all integers, fractions, and / or real numbers between the endpoints (including those endpoints).

[0036] This invention relates to a cell photoporation system, the system comprising: a. A laser device, preferably a pulsed laser device, for generating an optically perforated laser beam having a predetermined first wavelength, preferably an optically perforated laser pulse; b. An excitation light emitting device, preferably an excitation laser device, more preferably a continuous wave laser device, for generating an excitation beam having a predetermined second wavelength, the second wavelength being different from the first wavelength; c. A sample holder configured to hold one or more containers containing cellular material; d. A sensor device configured to detect and / or visualize cellular material in the sample holder; e. Optical processing components, including: i. A first optical processing subsystem configured to guide the optically aperforated laser beam from the pulsed laser device along a first optical path to the sample holder, and configured to process the optically aperforated laser beam, the first optical processing subsystem comprising a tunable power modulation device, a first dichroic deflector, and a focusing lens along the first optical path, the tunable power modulation device for modulating the power of the optically aperforated laser beam, the first dichroic deflector being configured to further reflect light having a wavelength substantially equal to the first wavelength towards the sample holder along the first optical path, the first dichroic deflector being positioned downstream of the sample holder closer to the power modulation device along the first optical path, and the focusing lens for focusing the optically aperforated laser beam. ii. A second optical processing subsystem configured to guide the excitation beam from the excitation light emitting device along a second optical path to the sample holder; and iii. A third optical processing subsystem configured to guide the light obtained from the sample holder along a third optical path to the sensor device.

[0037] Photoperforation can be used in a variety of other applications. Photoperforation itself is used to increase permeability or to completely disrupt (or even destroy) the cell membranes of one or more cells. This permeation step is itself a precursor to further steps, such as intracellular delivery of external payloads that may or may not result in transfection (in the case of nucleic acids); release of intracellular compounds from the cell; killing cells through severe disruption / destruction of their cell membranes; and instability / permeability of intracellular organelles (e.g., nucleus, endosomes, etc.).

[0038] The system described in this invention provides a fully integrated and optimized assembly for cell photoporation, including a first laser device for generating a photoporation beam and a second laser device for generating laser beams of different wavelengths for verifying the correct functioning of the system.

[0039] The purpose of an optically perforating laser device is clear: to create (transient) pores in cell membranes by irradiating them with a laser. This can be direct (directly targeting the cell membrane, causing optical breakdown to create a pore) or indirect, such as through photosensitive nanoparticles surrounding the cell, which react with the laser irradiation, absorb the light, and heat up, thus creating a pore in the cell membrane. This requires a precise laser device capable of reliably generating high-power output.

[0040] The purpose of excitation light-emitting devices is partly as validation tools. In many applications of photoporation systems, the ultimate goal is to transfect payloads into cells, typically in experimental settings such as research and development. These payloads are often extremely difficult to produce, making them highly valuable. Particles typically consist of disparate components, requiring separate synthetic processes (and subsequent processing steps, such as purification) and eventual assembly. Furthermore, they are usually not (and are) mass-produced, meaning that production costs themselves are far from optimized, often resulting in slow and expensive processes. Additionally, the processes using such payloads are limited and relatively novel, again leading to a lack of development of production processes and facilities. Finally, payloads typically undergo extensive validation (regulatory and quality control).

[0041] Therefore, in most experiments, the system is tested using dummy payloads that are inexpensive, easy to produce, and, importantly, possess known properties that are readily available. In photoperforation, the fluorescence properties of the payloads are chosen such that they respond well to irradiation by a second-wavelength beam from the excitation light-emitting device. This allows the system to check the results of the dummy payloads before using real, expensive payloads, verifying that the photoperforation is working well and that transfection occurs as needed.

[0042] In a preferred embodiment, the optical perforation laser device is a pulsed laser device. The advantage of using pulsed laser devices is that they can generate laser beams with much higher peak power. This allows the system to effectively have a much higher power range that the operator can then define, since reducing power is a very straightforward operation, not the other way around. Therefore, unlike continuous-wave laser devices, pulsed lasers can be used to deliver energy to target materials without causing as much total heat to be generated at the target material, as the laser is only on for a short period. This is important for applications where heat input could damage the material or surrounding tissue. Additionally, it helps reduce overall heat generation in the system, as in most cases, the heat generated by the laser device will affect nearby components. In some cases, this can affect their performance, such as requiring real-time fine-tuning, adjustment, and calibration, and in other cases, even rendering them inoperable due to the extent of the heat buildup. This disadvantage can be offset by forced cooling of the laser device, but this is another cost, occupies valuable space, and can potentially adversely affect other parts of the system. Pulsed laser devices only effectively generate heat for a portion of the time, while CW laser devices do so continuously. Finally, pulsed laser devices are more precise than CW laser devices because they have very fine control over the pulse length and the interval between pulses, allowing for greater control and discrete aiming. For CW laser devices, discrete aiming requires, for example, a shutter and its very precise control.

[0043] In a particularly preferred embodiment, the pulsed laser device is a nanosecond pulsed laser device, which means that the pulse length is in the range of a few nanoseconds.

[0044] In a preferred embodiment, the pulse length of the pulse of the pulsed laser device is between 10.0 femtoseconds and 10 μs, preferably between 50 fs and 5.0 μs, more preferably between 250 fs and 2.5 μs, even more preferably between 1.0 ps and 1.0 μs, 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.

[0045] Further based on the above preferences, 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.

[0046] Further based 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.

[0047] 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.

[0048] Each laser pulse can have a velocity of 0.001 J / cm² within the focal plane of the laser. 2 Up to 100 J / cm 2 0.005 J / cm 2 Up to 50J / cm 2 0.01 J / cm 2 Up to 10 J / cm 2 Or 0.05 J / cm2 Up to 5J / cm 2 Within a range, for example, at 0.1 J / cm 2 Up to 2J / cm 2 Flux volume within the range (electromagnetic energy delivered per unit area).

[0049] The total number of laser pulses used for optical perforation can 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 can depend on the type of photoresponsive particles, cargo, and cell type.

[0050] In a preferred embodiment, the laser pulse frequency can range from less than 1 Hz to greater than 1 GHz. More preferably, the laser pulse frequency is between 10 Hz and 200 MHz.

[0051] In a preferred embodiment, the first wavelength is 532 nm, which is in the visible (green) light range. However, alternatives are of course possible.

[0052] In an alternative preferred embodiment, the optically perforated laser device is a continuous-wave laser device. CW laser devices are generally cheaper and simpler to construct. Additionally, their energy conversion is more efficient because there are no losses associated with pulse generation as in pulsed laser devices. Finally, CW laser devices typically have more stable power output, producing more constant power, which makes subsequent power control easier.

[0053] In a preferred embodiment, the optically perforated laser device includes or is provided with a cooling system for forced cooling of the laser device.

[0054] In a particularly preferred embodiment, the excitation light emitting device is an excitation laser device for generating an excitation laser beam of a second wavelength.

[0055] In an alternative embodiment, the excitation light emitting device includes one or more of the following: light-emitting diodes (LEDs), metal halide lamps, mercury vapor lamps, tungsten-halogen (incandescent) lamps, and xenon arc lamps.

[0056] 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 because it is not used for optical perforation, but rather for light / laser-induced particle excitation. In this sense, a more stable power output is preferred.

[0057] In an alternative preferred embodiment, the excitation light emitting device is a pulsed laser device.

[0058] The laser pulse that excites the laser device preferably has a pulse duration between 1 fs and 10 ns, more preferably between 10 fs and 1 ns, and more preferably between 50 fs and 500 ps. Specific preferred sub-ranges can be selected, such as 1 to 10 ps, ​​or 50 to 500 ps, ​​or between 60 fs and 200 fs, such as 70 fs or 150 fs.

[0059] The laser pulse frequency of the excitation laser device is preferably between 10 kHz and 1 GHz, more preferably between 1 MHz and 500 MHz. The preferred frequency is a multiple 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.

[0060] The power of the pulsed excitation laser device is preferably at most 10W, and ultimately depends on the pulse duration. Femtosecond lasers typically have a power of about 1 to 10W, for example 2 to 3W, while picosecond and nanosecond lasers have lower power due to the increased pulse duration, and typically have a power in the range of 100mW to 1W, for example about 500mW.

[0061] In a particularly preferred embodiment, the second wavelength is 488 nm, which is in the visible (blue) light range. However, alternatives are of course possible.

[0062] The components in the optical processing assembly allow for adjustments to the system settings, which can be adjusted depending on the sample, the desired next step (as mentioned above, destruction, transfection, destabilization, etc.), and other characteristics (such as the type of payload in the case of transfection).

[0063] As a first part, the optical processing assembly includes a first optical processing subsystem that defines a first optical path through which the laser beam from the optically perforated laser device is guided to the sample holder.

[0064] The first optical processing subsystem includes a power modulation device that allows the subsystem to reduce the power of the laser beam from the optically perforated laser device to a desired level suitable for a specific application. This power modulation device can be, for example, an acousto-optic modulator (AOM), a device that uses acoustic waves to control the properties of the medium through which light passes (involving its interaction with the light). The AOM typically comprises a transparent crystal, such as quartz or lithium niobate. When an acoustic wave is applied to the crystal, it produces a periodic change in the crystal's refractive index. This change in refractive index can be used to diffract the laser beam. By changing the intensity of the applied acoustic wave, the laser beam undergoes stronger diffraction, resulting in a reduction in the power of the laser (a portion of it) that propagates further along the first optical path.

[0065] Another possibility for power modulation devices is the 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 influence the medium and its properties. EOMs are generally faster than AOMs, but lead to more complex designs and consume more power.

[0066] However, preferably, a configuration is used in conjunction with a half-wave plate and subsequently a polarizing beam splitter. The laser beam passes through the half-wave plate, which rotates the polarization of the laser beam by 180 degrees. The laser beam then passes through the polarizing beam splitter and is transmitted only when its polarization is aligned with the transmission axis of the polarizing beam splitter, and reflected only when its polarization is aligned with the reflection axis of the polarizing beam splitter.

[0067] By rotating the half-wave plate, the polarization of the laser beam can be rotated, allowing control over the amount of light transmitted and reflected by the polarization beam splitter. Consequently, the power of the emitted laser beam can be modulated. The advantages include its simple design, low cost, and high reliability.

[0068] The first optical processing subsystem also includes a first dichroic deflector configured to further reflect light having a wavelength substantially equal to the first wavelength (the wavelength of the optically perforated laser device) along the first optical path toward the sample holder. Light having other wavelengths is transmitted (or deflected differently) such that it is directed away from the first optical path.

[0069] Dichroic mirrors are a preferred choice for dichroic deflection devices because they absorb very little incident light and can be manipulated very precisely so that they reflect only a narrow range (stopband) of wavelengths while transmitting other wavelengths. Dichroic mirrors are fabricated by building alternating layers of optical coatings with different refractive indices on a glass substrate. The interfaces between layers with different refractive indices produce phase reflection, selectively amplifying certain wavelengths of light and interfering with others. By controlling the thickness and number of layers, the frequency of the mirror's stopband can be tuned and made wide or narrow as needed. Because unwanted wavelengths are transmitted rather than absorbed, dichroic mirrors do not absorb this unwanted energy during operation and therefore do not become nearly as hot as an equivalent conventional mirror.

[0070] Therefore, one advantage is that it further filters the incident light (from the optically perforated laser device) to the desired wavelength and removes unwanted wavelengths. Another advantage is the simplicity of this mirror compared to its performance, low cost, compact construction, and high heat resistance.

[0071] The first optical processing subsystem also includes a focusing lens for focusing the optically perforated laser beam toward the sample holder. Most preferably, the focusing lens is positioned directly in front of the sample holder along the first (and second) optical paths and behind other components to ensure proper positioning of the desired focus.

[0072] In this setup, the optical processing components allow for full control over the laser beam generated by the optically perforated laser device. It can modulate the power according to the needs of a specific application, further filter the laser beam toward the desired wavelength, and focus the laser beam shortly before it reaches the sample holder.

[0073] The second optical processing subsystem is configured to guide the excitation beam from the excitation light emitting device to the sample holder along the second optical path.

[0074] The third optical processing subsystem is configured to guide the light obtained from the sample holder along the third optical path to the sensor device.

[0075] In a preferred embodiment, the first and second optical paths partially overlap or align, wherein the first and second optical processing subsystems partially overlap in terms of their included components. This allows the first and second optical processing subsystems to (re)use some of their components. Furthermore, it allows the system to be manufactured more compactly (eliminating the need for space for completely separate optical paths), and the shared components reduce cost, lower power consumption, and simplify operation.

[0076] In particular, the focusing lens is preferably a shared component that focuses the light-perforated laser beam and the excitation beam. 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, thus occupying valuable space in the system and increasing heat (and hindering the opportunity to remove said heat).

[0077] More preferably, another shared component is the first dichroic deflector. While its use of the excitation beam is limited (because it is simply transmitted, having a wavelength different from the first dichroic deflector configured for reflection), the path alignment from the sample holder to the first dichroic deflector optimizes the use of free space, especially in this case where the path is restricted, for example, at the sample holder. Essentially, this means that the second optical path will pass through the first dichroic deflector and diverge from the first optical path before that point. Sections of the first and second optical paths between the first dichroic deflector and the sample holder will be shared.

[0078] Therefore, depending on the viewing angle, the first dichroic deflection device is used to connect (or separate) the first and second optical paths.

[0079] In a preferred embodiment, the first and third optical paths partially overlap or align, wherein the first and third optical processing subsystems partially overlap in terms of the components they include. As previously described, this allows the subsystems to share components, thereby reducing the system's size, cost, power consumption, and complexity.

[0080] In particular, a shared focusing lens is preferred because the focused beam will be advantageous in both directions (towards and away from the sample holder).

[0081] The same advantages mentioned above apply to embodiments in which the first dichroic deflector is also present in both the first and third optical paths. Furthermore, the first dichroic deflector largely allows incident light arriving from the sample holder to be completely transmitted along the third optical path, as this light is a result of the fluorescence of the material irradiated at the sample holder and subsequently emitting (typically) light of different wavelengths. The first dichroic deflector reflects only the first wavelength of light and allows other wavelengths to further pass along the third optical path toward the sensor device without substantial absorption or reflection.

[0082] In a preferred embodiment, the second and third optical paths partially overlap or align, wherein the second and third optical processing subsystems partially overlap in terms of the components they include. As previously described, this allows the subsystems to share components, thereby reducing the system's size, cost, power consumption, and complexity.

[0083] In the most preferred embodiment, all three optical path portions overlap or align, wherein the first, second, and third optical processing subsystems partially overlap in terms of the components used in each of them (meaning that one or more components are used in each of the first, second, and third optical processing subsystems). Advantages arise from the combination described above.

[0084] Ideally, a single focusing lens uses minimal space around the sample holder, and three optical paths are aligned between the sample holder and the first dichroic deflector, which allows the passage of light from the excitation beam and light emitted from the sample holder due to its illumination.

[0085] The configuration of the sensor device can vary, and it can include multiple different sensors, such as imagers or detectors, at once.

[0086] In a preferred embodiment, the third optical processing subsystem further includes a beam collimator for collimating light (fluorescence, etc.) returning from the sample holder into a third optical path. Preferably, the beam collimator is positioned such that it is positioned between the movable mirror assembly and the sample holder.

[0087] In a preferred embodiment, the beam collimator is positioned along the optical path near the sample holder, preferably between the first dichroic deflector and the sample holder, but more preferably between the first dichroic deflector and the focusing lens. However, in some embodiments, the collimator is a focusing lens, wherein the focusing lens focuses the light through-hole and excitation beam along the first and second paths, and collimates the (fluorescent) light from the irradiated sample (cells) at the sample holder.

[0088] In a preferred embodiment, the second optical processing subsystem includes a second dichroic deflector located on the second and third optical paths, wherein the second dichroic deflector is configured to further reflect light having a wavelength substantially equal to the second wavelength further down the second optical path toward the sample holder, and to further transmit the resulting light returning from the sample holder having a wavelength substantially different from the (first and) second wavelengths further down the third optical path toward the sensor device.

[0089] In some variations, the second dichroic deflector acts as a bandpass filter, deflecting / reflecting all wavelengths below the second wavelength. This allows protection of components in the third path from high-energy wavelengths. There is no need to transmit lower wavelength light toward the detector, as no light should be present.

[0090] Similar to the first dichroic deflector, this is preferably in the form of a dichroic mirror. By positioning it in the second and third optical paths, it acts as a simple separator for the beams from the excitation light emitting device, ensuring that only those beams with the "correct" wavelength are further reflected along the second optical path toward the sample holder, while other beams are transmitted (to their beam collectors, etc.). Simultaneously, the light returning from the sample holder is not (or almost entirely) filtered because very little or no light with a wavelength equal to the second wavelength is reflected out of the third optical path. Thus, the returning light primarily passes through the second dichroic deflector and further along the third optical path to reach the sensor device.

[0091] In embodiments where the first, second, and third optical paths are aligned, it should also be noted that the light returning from the sample holder passes through the first and second dichroic deflectors but remains largely unchanged, and continues along the third optical path to the sensor device. Conversely, the excitation beam is reflected by the second dichroic deflector, further along the second optical path, then passes through the first dichroic deflector (further along the second optical path), and proceeds to the sample holder.

[0092] Therefore, the first optical path originates from the optically perforated laser device and is subsequently transmitted to the power modulation device, the first dichroic deflector, the focusing lens, and the sample holder. The second optical path originates from the excitation light emitting device and is subsequently transmitted to the second dichroic deflector, the first dichroic deflector, the focusing lens, and the sample holder. The third optical path originates from the sample holder and is subsequently transmitted to the first dichroic deflector, the second dichroic deflector, and finally to the sensor device.

[0093] Note that this does not preclude the presence of other components on any of the said paths.

[0094] In a preferred embodiment, the first dichroic deflection device is located in the first, second, and third optical paths, while the second dichroic deflection device is not in the first optical path (but preferably in the third optical path and the second optical path).

[0095] In this way, a portion of the optical path can extend from the first dichroic deflector to the sample holder assembly, as previously described. Conversely, it is possible to position the second dichroic deflector as a shared component in every path (where the first dichroic deflector is not in the second optical path, and preferably in the third optical path), but this results in a suboptimal setup because the more frequently used and higher-powered optically perforated laser beam passes through the second dichroic deflector each time, which is undesirable. In the first setup, the excitation beam would pass through the first dichroic deflector, but the excitation light emitting device is used at a lower frequency, with lower power, and is of less importance.

[0096] In a preferred embodiment, the power modulation device is a tunable power attenuator, which includes a half-wave plate for a first wavelength and a polarizing beam splitter located further along the first optical path than the half-wave plate. 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. In contrast to alternatives such as AOM and EOM, the above embodiment is simple, compact, low-cost, easy to calibrate, and reliable. The function of the tunable power attenuator is to regulate the power of the optically perforated laser beam reaching the sample holder.

[0097] In a preferred embodiment, the optical processing assembly includes a movable mirror assembly having at least one movable mirror for reflecting light toward a sample holder along a first or second optical path, wherein the movable mirror assembly is configured to reflect the light onto the sample holder in a (two-dimensional) scanning pattern, and the movable mirror assembly is positioned on the first, second, and third optical paths between the first dichroic deflection device and the sample holder.

[0098] In a particularly preferred embodiment, at least one movable mirror is configured to two-dimensionally change the incident position of reflected light on the sample holder in the plane of the sample holder by tilting at least one movable mirror on at least two non-parallel axes. Preferably, this is achieved by tilting each of the at least two movable mirrors in a consistent manner on at least one axis, wherein at least one axis of the at least one movable mirror is not parallel to each other.

[0099] Preferably, the optical processing assembly includes a plurality of movable mirrors, most preferably only two, each movable mirror being rotatable / tiltable on a single axis, wherein each mirror is rotatable / tiltable on different and non-parallel (preferably perpendicular) axes, thereby allowing the mirror assembly to cover a two-dimensional surface using a scanning pattern. Most preferably, it includes said plurality, preferably only two, movable mirrors shortly before the sample holder.

[0100] Alternatively, the optical processing assembly includes a movable mirror assembly with a single movable mirror for reflecting light following a first or second optical path toward the sample holder in order to scan the pattern. In this case, the single movable mirror will be tiltable / rotatable on multiple (different) axes to enable coverage of a two-dimensional region with the scan pattern. However, this is used less frequently because controlling such a single multi-axial movable mirror is more complex than using at least two, and the gain is limited (in terms of space and / or cost).

[0101] In a particularly preferred embodiment, the movable mirror is a reflecting galvanometer or a galvanometer reflecting mirror. The galvanometer mirror is operated by using a galvanometer motor to rotate the mirror portion relative to a static portion. The galvanometer motor generates torque to rotate the mirror by applying and changing a magnetic field. Galvanometer mirrors allow rotation at very high speeds and with extremely high precision (essential for manipulating light and / or laser beams), which is ideal for reflecting light and / or laser beams from a scanning pattern onto a sample holder. Furthermore, they are compact, lightweight, relatively inexpensive, and very durable.

[0102] Alternatives to the galvanometer microscope include: - Acousto-optic deflector (AOD): Excellent characteristics in terms of speed and accuracy; - Spatial light modulator (SLM): High precision, and additional features such as modulation of the properties of light and / or laser beams; - Fast Scanning Mirror (FSM): Very high speed, relatively inexpensive, and very durable; - Deformable mirror (DM): high speed, high versatility and precision.

[0103] It should be understood that when limiting statements are made, such as "only two" or "single movable mirror," this will be considered in the context of a mirror that actively participates in generating the scanning pattern. Of course, other movable mirrors may still exist in other parts of the optical path for deflecting / reflecting the beam and / or laser beam.

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

[0105] In another preferred embodiment, the power modulation device is configured to modulate the power of the optically aperforated laser beam, taking into account the incident angle of the optically aperforated laser beam on the plane of the sample holder and the related changes in the beam area of ​​the optically aperforated beam on the plane of the sample holder, so as to maintain a substantially constant flux when the optically aperforated beam is reflected with the scanning pattern.

[0106] During pattern scanning, as the laser beam's incident point on the sample holder is moved, the incident angle inevitably changes, leading to optical aberrations at the sample holder, which typically increase with distance from the axis. One of these unwanted effects is coma, a result of off-axis incident light on the (focusing) lens, causing image distortion (with a cooing tail). Another effect is that the incident area of ​​an angled laser beam increases relative to perpendicular incident light, resulting in less radiation exposure or fluence.

[0107] To address this aberration problem (especially flux reduction), the power of the laser beam can be modulated to compensate for this effect, thereby increasing the transmission power output as the incident area at the sample holder increases (and decreasing the transmission power output as the incident area decreases). In many cases, the effect of the scanning pattern on the flux at each point can be modeled / predicted and / or determined experimentally, thus enabling the cancellation of these effects through reliable power modulation.

[0108] Alternatively, instead of modulating power based on the incident angle to obtain a constant flux across the scan pattern, the sample holder can be discretely illuminated in multiple (overlapping) smaller scan regions with small flux variations to cover the total scan region formed by overlapping scan regions with constant flux. The multiple overlapping scan regions generate a mosaic representing the total scan region.

[0109] In a preferred embodiment, the first optical processing subsystem further includes a beam detection device and an optical element that partially deflects the optically perforated laser beam to the beam detection device.

[0110] In another preferred embodiment, the optical element that partially deflects the optically perforated laser beam to the beam detection device includes a polarization beam splitter along a first optical path, wherein the polarization beam splitter removes waste light from the optically perforated laser beam and guides the waste light to the beam detection device. The beam detection device is configured to detect beam information associated with the optically perforated laser beam from the waste light. This is particularly advantageous in the case of optically perforated pulsed laser devices because it allows monitoring of the pulse state and its characteristics to properly control and time the operation of other components of the optical processing assembly, such as movable mirror assemblies, and also includes power modulation devices. However, this is also advantageous for continuous-wave laser devices.

[0111] However, most importantly, the polarization beam splitter is used to clear the optically perforated laser beam at that point, thereby removing waste light and ensuring that the passing light is linearly polarized.

[0112] Therefore, the beam detection device and polarization beam splitter are preferably positioned along the first optical path (between the optically perforated laser device and the power modulation device) before the power modulation device, so that the laser beam reaching the power modulation device is already linearized and is ideal for the power modulation purpose.

[0113] Alternatively, optical elements that deflect a portion of the optically perforated laser beam into the beam detection device include non-polarized beam splitters, such as cubic beam splitters, planar beam splitters, and thin-film beam splitters.

[0114] In another preferred embodiment, the scanning pattern includes multiple discrete positions, wherein at least one movable mirror is controllably moved based on beam detection information from a beam detection device to incident reflected light from the movable mirror onto the discrete positions of the scanning pattern. It should be noted that the movable mirror can be used to perform the scanning pattern on both the excitation beam and the optically perforated laser beam. Preferably, it can also be used to guide light from the sample holder (its contents) back to the sensor device along a third optical path, ensuring that the desired area of ​​the sample holder (its contents) can be visualized / analyzed.

[0115] As mentioned above, the movable mirror assembly can have a variety of configurations, with multiple mirrors (preferably two) being a preferred option, wherein the mirrors can tilt / rotate about two different axes.

[0116] In a particularly preferred embodiment, the optically perforated laser device is a pulsed laser device. This allows for the collection of such beam information as the (desired) temporal distribution of the laser pulses.

[0117] In a possible implementation, beam information about the laser pulse can be used to time movement during pattern scanning, wherein the mirror can move continuously such that the pulse strikes the sample holder centered at the desired location. Utilizing the short pulse length, the extent to which the laser pulse reaches the sample holder is quite limited, and the interval between subsequent pulses allows the mirror to move so that the next pulse targets a subsequent location on the scan pattern.

[0118] In an alternative implementation, the movable mirror moves only during the "off time" between laser pulses striking the movable mirror. In each movement, the mirror is moved such that the next discrete location in the scan pattern is targeted by the next pulse, ensuring that only the desired area is targeted by the laser pulse and receives the same and full amount of energy, with a constant flux for each target. This results in a perceived progressive movement across the scan pattern.

[0119] In addition to scanning patterns, the system and method are also obviously applicable to "single shot" applications, where only a single location on the sample holder is targeted.

[0120] In a preferred embodiment, the focusing lens is located on the first and second optical paths between the dichroic deflection device and the sample holder, and the focusing lens can move along the second optical path to the sample holder and from the sample holder.

[0121] In a particularly preferred embodiment, the focusing lens is located between the movable mirror assembly and the sample holder. Clearly, aperture of the incident light and focusing of the excitation beam onto the sample holder can be performed more reliably and accurately, and closer to the sample holder.

[0122] In a preferred embodiment, the focusing lens is located in the first, second, and third optical paths, allowing it to focus the light propagating to the sample holder via the optically perforated laser beam and the incident excitation beam, as well as to focus the return light from the sample holder toward the sensor device.

[0123] In another preferred embodiment, the optical processing assembly further includes a preferably removable beam diameter measuring device at a measurement position on the second optical path (and in many cases also on the first or second optical path, or preferably on both, as they partially overlap / align), passing through a focusing lens, the focusing lens being movable along the second optical path to and from the measurement position, wherein the beam diameter measuring device includes a predetermined fluorescent target.

[0124] Using a predefined fluorescence target allows the system to be benchmarked in terms of focusing characteristics. The target is specifically chosen to respond well to the second wavelength of the excitation beam, ensuring reliable fluorescence emission that can be clearly visualized / analyzed, thus providing insight into the extent to which the focusing lens is positioned as needed. In this case, using a pulsed laser device for the optically perforated laser beam is particularly useful, as this allows for 2-photon excitation at the fluorescent material (with greater determinism due to the higher intensity achieved by the pulsed laser device).

[0125] In particular, combining the beam diameter measurement with information from a power output sensor (used to measure the average power of the optically perforated laser beam) allows for the determination of the flux at the sample.

[0126] Moving the focusing lens affects the beam diameter at the location of the fluorescent target. This can then be visualized or measured by determining the intensity distribution (preferably two-dimensional) of multiple relative positions of the focusing lens with respect to the target, preferably performed by a sensor device. Locations with higher peak intensity and / or narrower intensity distributions (e.g., by determining the lowest FWHM, HWHM, standard deviation, etc., or similar metrics) have smaller beam diameters, and the system can be adjusted according to specific beam diameter requirements (e.g., desired fluence).

[0127] Preferably, the position of the focusing lens relative to the fluorescent target is changed by a stepper motor or the like.

[0128] Preferably, the beam diameter measuring device is located 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 measuring device is part of a container (e.g., a tray) adapted to hold the sample and can be inserted into the sample holder.

[0129] Preferably, the relative and / or absolute distance from the focusing lens to the beam diameter measuring device is known, whether it is between the focusing lens and the sample holder, or if it is on the sample holder or a portion of a container for holding the sample, which can be placed within the sample holder. In a particularly preferred embodiment, the predetermined fluorescent target of the beam diameter measuring device comprises a fluorescent plate or a similar fluorescent target portion.

[0130] Embedding the fluorescent target in a sample holder or container is particularly advantageous because it allows for finding the relative position of the target to the ideal focusing lens (beam waist) of the beam diameter, and in this case, the position of the fluorescent target is the same as the position of the cells when placed in the sample holder.

[0131] In a preferred embodiment, the sensor device includes a visualization device for providing a visual representation of the contents of the sample holder and a detection device for determining the quantitative characteristics of the contents of the sample holder, and wherein a controllable switching device is provided for changing the third optical path to terminate at the visualization device or the detection device.

[0132] In an alternative embodiment, the sensor device includes a visualization device for providing a visual representation of the contents of the sample holder and a detection device for determining quantitative characteristics of the contents of the sample holder. The system also preferably includes an optical element at the sensor device configured to direct the resulting light along a third optical path to either the visualization device or the detection device, or both simultaneously.

[0133] Separating the two separate sensor modes is advantageous because it allows selection between qualitative representation / analysis of the sample via a visualization device and quantitative representation / analysis of the sample via a detection device. The visualization device includes an imaging system, such as a camera, adapted to convert light received at the sensor device from a third optical processing subsystem and originating from the sample holder into a visual two-dimensional or three-dimensional representation of the contents at the sample holder. Therefore, the visualization device preferably has a built-in screen, or can be connected to a visualization system such as a screen. Alternatively or additionally, internal image analysis can be performed via an integrated processing device to determine beam size and / or other relevant characteristics.

[0134] The detection device preferably includes a photomultiplier tube, more preferably a silicon photomultiplier tube (SiPM). A photomultiplier tube converts incident photons into a processable electrical signal. SiPMs operate based on the principle of SPAD (single-photon avalanche diode) and offer the advantages of providing high gain (as with conventional photomultiplier tubes, such as PMTs), but with low voltage and a very fast response time. Furthermore, SiPMs are very compact, making them ideal for this application. Alternatively, the detection device preferably includes one or more photodiodes, a thermal detector, an integrating sphere, or a calorimeter.

[0135] In a particularly preferred embodiment, the sensor device is positioned at a portion of the system relative to the optically perforated laser device in order to minimize the impact of the high-power optically perforated laser device on the sensor device.

[0136] Preferably, the switching device is a mirror adapted to deflect the light beam along the third optical path to the visualization device or the detection device.

[0137] The mirror can be moved into and out of the third optical path, causing it to deflect the light beam along the third optical path, or not to deflect the light beam, thereby causing the light beam to strike the visualization or detection device. A common example of this is the so-called flip mirror.

[0138] In another embodiment, the mirror is rotatable between at least two positions, such that in a first position, the deflected beam illuminates the visualization member, and in a second position, the deflected beam illuminates the detection member. Such a rotatable mirror could be, for example, a galvanometer mirror, but other implementations are possible. The key point of this paper is achieving accuracy and reliability in both positions during switching.

[0139] In another embodiment, the reflector can functionally switch between transmission and deflection modes (e.g., using a liquid crystal cell or electrochromic material). In transmission mode, the transmitted beam strikes the visualization or detection device, while in deflection mode, the deflected beam strikes another. This has the advantage that the switching device does not need to move and remains in position, thereby ensuring that the correct sensor device is struck at the same location each time.

[0140] Instead of using a switching device to change the third optical path to terminate at the visualization or detection device, one or both of the visualization and / or detection devices can be moved into or out of the third optical path without using a switching device.

[0141] In a particularly preferred embodiment, the system includes a power output sensor configured to measure the average power and / or flux of incident light, wherein the system includes a deflection device configured to reflect light with a wavelength substantially equal to a first wavelength from a first optical path toward the power output sensor. Alternatively or additionally, the deflection device may be configured to reflect light with a wavelength substantially equal to a second wavelength toward the power output sensor. The deflection device may be, for example, a dichroic deflection device, but alternatives are also suitable.

[0142] In a preferred embodiment, the system includes a power output sensor configured to measure the average power and / or flux of the incident light, wherein the sample holder includes a deflection device configured to reflect light having a wavelength substantially equal to a first wavelength toward the power output sensor. The primary objective is to determine the power / energy received at the sample holder from the optically perforated laser device. Based on these measurements, a power modulation device can then be adjusted to increase or decrease the emitted power, and / or the optically perforated laser device can be adjusted to increase or decrease the power of the generated optically perforated laser beam. As previously mentioned, this is particularly advantageous when combined with a beam measurement device, which allows for easy determination of fluency by combining the measured average power with the beam diameter. Alternatively or additionally, the deflection device may be configured to reflect light having a wavelength substantially equal to a second wavelength toward the power output sensor to determine the power / energy received at the sample holder from the excitation light emitting device.

[0143] The dichroic deflector can be integrated into the sample holder itself, or it can be part of a container to be placed within the sample holder. Preferably, it is integrated into the sample holder. By moving the sample holder, the system can position the dichroic deflector in the path of the light beam reaching the sample holder to deflect the beam to the power output sensor. In this way, the power output sensor is used only when testing the power output.

[0144] The output sensor preferably has a fixed position in the system to ensure that it is correctly aligned with the beam deflected by the dichroic deflector.

[0145] In a preferred embodiment, the power output sensor includes a thermopile. The thermopile is adapted to convert thermal energy from incident light into electrical energy and allows for the measurement of the energy (and power) of the incident light. The thermopile consists of several thermocouples and operates based on the thermoelectric effect, i.e., a voltage is generated when the thermocouples are exposed to a temperature difference.

[0146] In a variant, the sample holder may include a universal deflector instead of a dichroic deflector to reflect all incident light toward the power output sensor. This will further allow for the measurement of the (processed) power output of the excitation light emitting device.

[0147] In a particularly preferred embodiment, the first, second, and third optical paths are at least partially aligned between the first dichroic deflector and the sample holder. Preferably, they are fully aligned between the first dichroic deflector and the sample holder. Even more preferably, the second and third optical paths are further fully aligned between the second dichroic deflector and the sample holder. The advantages of this arrangement have been fully discussed above (reducing the volume used by employing components from multiple components in the first, second, and third optical processing subsystems).

[0148] In another aspect, the present invention relates to a cell photoporation method, which itself can form part of an overall method (e.g., cargo transfection of cells), the cargo of which has been described non-limitingly earlier in the specification.

[0149] The cell photoporation method includes the following steps: - Provide one or more samples at the sample holder, wherein the one or more samples comprise a mixture of one or more cells, one or more light-responsive particles, and cargo; - Generate an optically perforated laser beam with a predetermined first wavelength, preferably an optically perforated laser pulse; - The optically perforated laser beam is guided along the first optical path to the sample holder, and the guiding of the optically perforated laser beam further includes the following processing steps: o Modulate the power of the optically perforated laser beam; o The modulated optically perforated laser beam is reflected by a first dichroic deflector, the first dichroic deflector being configured to further reflect light having a wavelength substantially equal to the first wavelength along the first optical path toward the sample holder, and to transmit light having a wavelength substantially different from the first wavelength away from the first optical path. o Focus the reflected light through the laser beam; - Irradiate one or more samples at the sample holder with a focused optically perforated laser beam to permeate one or more cell barriers of the cells, and then allow cargo to enter the cells through the permeated cell barriers; - Generate an excitation beam having a predefined second wavelength different from the first wavelength, preferably a laser beam, and more preferably a continuous wave excitation laser beam; - The excitation beam is directed along a second optical path to the sample holder that holds the sample, wherein at least one of the cells comprises the cargo; - Irradiate the sample with the excitation beam, wherein at least one of the cells comprises the cargo; - The light emitted from the sample due to irradiation with the excitation beam is guided to the sensor device along the third optical path; - Measure one or more characteristics of the light received at the sensor device.

[0150] Integrating the optical perforation process (and cargo delivery) into subsequent analytical processes (measurement, visualization, etc.) offers a significant improvement over existing technologies, where these processes are performed separately. Furthermore, integration allows for the reuse of components, facilitates sample management processes (samples remain in one place), and provides further advantages.

[0151] It should be noted that the system used in the above method is preferably the system of the first aspect of the present invention. Therefore, any implementations or variations of the system discussed are also considered to be disclosed in relation to the method of the present invention and have not been explored. In such implementations, for example, are the characteristic ranges of the optical perforation and excitation light emitting device (power and its derivatives, such as peak power, average power, flux, pulse frequency, pulse length, pulse energy, etc.).

[0152] A specific improvement described for this system is the combination of optical paths such that all three optical paths are aligned / coincident between the first dichroic deflector and the sample holder, and the second and third optical paths preferably coincide even further between the second dichroic deflector and the sample holder. This not only makes the system more compact but also ensures that reusable components (focusing lens, collimator, dichroic deflector, etc.) do not require separate configuration, calibration, maintenance, etc., for each optical path.

[0153] Another specific improvement provides a method for (automatically) focusing a focusing lens in the system, wherein the sample holder or a container placed within the sample holder for holding cells includes a fluorescent target. In this further method, the steps include: - The focusing lens is moved continuously or gradually relative to the fluorescent target at the sample holder; - Irradiate the fluorescent target with a light beam, preferably a laser beam, preferably a single pulse, preferably an excitation beam; - The light emitted from the sample due to illumination by the beam is guided along a third optical path to the sensor device; - Measure the intensity distribution of multiple distances between the focusing lens and the fluorescent target during the movement of the focusing lens; - Determine the minimum beam diameter in the measured intensity distribution.

[0154] The intensity distribution is preferably two-dimensional (on the x and y axes in a plane perpendicular to the second optical path at the sample holder), and allows for the determination of characteristics of the distribution, such as the standard deviation σ in each dimension. x and σ y Then these properties are used to determine the position of the focusing lens relative to the target that achieves the minimum beam diameter, using the standard deviation, for example via... .

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

[0156] In a final aspect, the present invention relates to the application of a cell photoperforation system according to the first aspect, a system for permeating the cell barrier of said cells, and a system for further applications thereafter, as previously described. Specifically, such further applications include cargo transfection of cells, which have been previously described in the specification in a non-limiting manner.

[0157] WO2022 / 189627, WO2021 / 058430, WO2020127076 and the applicant’s currently unpublished patent applications PCT / EP2023 / 058509 and PCT / EP2022 / 064003 are incorporated herein by reference.

[0158] In the following description, the invention is illustrated by way of non-limiting embodiments, which are not intended to and should not be construed as limiting the scope of the invention.

[0159] Example

[0160] Figure 1Potential embodiments of the system of the present invention are described. The system includes an optically perforated laser device (1) configured to generate an optically perforated laser beam having a predetermined first wavelength, preferably pulsed. The system also includes an excitation light emitting device (2), in this case an excitation laser device, preferably a continuous-wave laser device, configured to generate an excitation beam with a second wavelength different from the first wavelength. The system further includes a sample holder (not shown, in a layer above a focusing lens), sensor devices (6, 6a, 6b), and optical processing components (3, 4, 5, 7, 8, 9, 10, 11a, 11b) to guide and process the optically perforated and excitation beams to the sample holder (not shown) and to guide the resulting light from the sample holder to the sensor devices (6, 6a, 6b).

[0161] The optical processing assembly includes a first optical processing subsystem (3, 4, 5, 8, 10, 11a, 11b) for guiding and processing the optically perforated laser beam from the optically perforated laser device (1) to the sample holder.

[0162] The first optical processing subsystem includes (in order from the optically perforated laser device): - First motion mirror (10), - An optional shutter (11a) with an integrated lens (11b) for added safety. - Second motion mirror (10'), - Power modulation device (3) for controlling the power of the optically penetrating beam that is allowed to reach the sample holder. - A first dichroic deflection device or dichroic mirror (4) is used to further reflect a light-perforated laser beam with a first wavelength 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 from the first dichroic deflector (4) toward the sample holder in a scanning pattern; - A focusing lens (5) for focusing the light beam from the movable mirror assembly (8).

[0163] The focusing lens (5) and the movable mirror assembly (8) are positioned as close as possible to the sample holder (not shown) to ensure that the optically perforated laser beam remains narrow and can be focused and properly aimed at the sample holder.

[0164] The moving mirrors (10, 10') and the mirror (11b) primarily serve to guide the light-perforated laser beam and generate the first optical path, enabling the system to be compact and efficient. However, they are not essential and their number can be reduced or increased, and / or they can be repositioned to other points in the first optical path between other components.

[0165] The shutter (11a) is also an optional feature and serves as an additional safety measure to ensure that the high-power optically perforated laser beam does not strike the sample holder when not in use.

[0166] The optical processing assembly also includes a second optical processing subsystem for guiding and processing the excitation beam from the excitation light emitting device (2) to the sample holder.

[0167] The second optical processing subsystem (sequentially starting from the excitation light emitting device) includes: - A second dichroic deflection device or dichroic mirror (7) is used to further reflect the excitation beam with a second wavelength along the second optical path and transmit light with another wavelength (away from the second optical path); - A first dichroic deflection device (4) that allows excitation beams to pass through because they have wavelengths different from the first wavelength; - A movable mirror assembly (8) for reflecting the excitation beam, which passes through the dichroic deflection device (4) in a scanning pattern, toward the sample holder; - A focusing lens (5) for focusing the light beam from the movable mirror assembly (8).

[0168] The optical processing assembly also includes a third optical processing subsystem for guiding and processing some of the light emitted from the sample holder to the sensor device (6) at the sample holder (generated by incident light on the sample held therein).

[0169] The third optical processing subsystem includes (in sequence from the sample holder): - Focusing lens (5); - Movable mirror assembly (8); - First dichroic deflection device (4), which allows the return light to pass through because they (most of them) have wavelengths different from the first wavelength; - A second dichroic deflection device (7) that allows excitation beams to pass through because they (mostly) have wavelengths different from the second wavelength; - The third moving mirror (10''') reflects the returning light that has passed through the first and second dichroic deflection devices; - A sensor device (6, 6a, 6b) comprising a detection device (6a) and a visualization device (6b), and a switching device (9) in the form of a movable flip mirror to reflect or transmit the light beam reflected by the third motion mirror (10''') to either the detection or visualization device.

[0170] Similarly, a third motion mirror (10''') is not required.

[0171] Optical path and subsystem in Figure 2A , 2B Further examples are shown in numbers 4, 5, 6, 7, 8, 9, and 10.

[0172] Figure 2A and Figure 2B This shows the shutter (11a) closed ( Figure 2A In the case of ), and when the shutter (11a) is open ( Figure 2B In the case of the light-perforated laser beam, the first optical path is followed. If closed, the light-perforated beam (20a) is deflected into another beam (20b) by the first moving mirror (10'), but stops at the shutter (11a). If open, it is reflected into another beam (20c) by the mirror (11b), and then reflected again by the second moving mirror (10') into yet another beam (20d) toward the power modulation device (3), after which the modulated beam (20e) propagates to the first dichroic deflector (4), which deflects it (or at least its component with the correct wavelength) into another beam (20f) toward the movable mirror assembly (8). After being reflected in two stages by its two mirrors, the movable mirror assembly (8) redirects the light into a first additional beam (20g) and a second additional beam (20h) toward the focusing lens (5), which ultimately transmits the final beam (20i) to strike the sample (12) in the sample holder.

[0173] The power modulation device (3) can take many forms, and Figure 3 A potential implementation is described, wherein the power modulation device includes a half-wave plate (3a) and a polarization beam splitter (3b). The half-wave plate rotates the orientation of the linearly polarized light incident (optically perforated) laser beam (30a), and then the polarization beam splitter (3b) transmits only the light having a specific (predefined) polarization orientation (30b), and deflects the remainder (30c) into a beam collector (3c).

[0174] Figure 4 A side view of the system during use of the optically perforated laser device is shown, wherein the sample holder (not shown) includes a (dichroic) deflection device (14) that deflects the light from the optically perforated laser device (1) toward a power output sensor (13) in the form of a thermopile.

[0175] Figure 5The system in use is shown, wherein an excitation light emitting device (2) generates an excitation beam (40a) along a second optical path, thereby illuminating a sample (12). The excitation beam (40a) illuminates a second dichroic deflector (7), which is configured to deflect light having a second wavelength (i.e., the wavelength of the excitation beam) along the second optical path toward a sample holder. The reflected excitation beam (40b) passes substantially entirely through the first dichroic deflector (4), as this only deflects light having a first wavelength, and arrives as another beam (40c) onto a movable mirror assembly (8), which redirects it to a focusing lens (5) in two stages (40d, 40e), and then arrives as the final excitation beam (40f) onto the sample (12).

[0176] Figure 6 The system in use is shown, wherein the optically perforated laser device (1) generates an optically perforated laser beam along a first optical path to irradiate a sample (12), and the sample (12) itself emits light generated by the irradiation, which (50a) (among other things) falls on a focusing lens (5), and the light (50a) is redirected by the focusing lens (5) as another beam (50b) to a movable mirror assembly (8), which redirects it as another beam (50c) to a first dichroic deflector (4). Since it only reflects light with the first wavelength, the additional beam (50c) is transmitted essentially entirely as the additional beam (50d) to the second dichroic deflector (7), and since the second dichroic deflector only reflects light with the second wavelength, it is again transmitted essentially entirely as the additional beam (50e) to the third motion mirror (10'''), which reflects it as the additional beam (50f) to the sensor device (6, 6a, 6b), where the switching device (9) deflects it as the additional beam (50g) to the visualization device (6b).

[0177] Figure 7 It shows the relationship with Figure 6The same diagram, but instead of the optically perforated laser device (1), an excitation light emitting device (2) is used to irradiate the sample (12), and a detection device (6a) is used to process the light obtained from irradiating the sample (12). This again causes the sample (12) itself to emit light generated by the second irradiation, which (50a) (among other things) falls on the focusing lens (5), and the light (50a) is redirected as another beam (50b) via the focusing lens (5) to the movable mirror assembly (8), which redirects it as another beam (50c) to the first dichroic deflector (4). Since this only reflects light with the first wavelength, the other beam (50c) is transmitted essentially as another beam (50d) to the second dichroic deflector (7), and again essentially as another beam (50e) to the third motion mirror (10'''), which reflects it as another beam (50f) to the sensor device (6, 6a, 6b), where the switching device (9) is not in the proper position this time, causing the beam (50f) to continue to the detection device (6a) this time.

[0178] Figure 8 It shows Figure 7 A variant thereof, wherein the visualization device (6b) is used to process the light generated by the illumination of the sample (12).

[0179] Figure 9A and Figure 9B Finally, an embodiment of the sensor device (6) is shown, which includes a visualization device in the form of a camera (6b) and a detection device (6a). Switching between the actual sensor devices is performed by a flip mirror (9). The flip mirror (9) includes a movable mirror portion that can rotate into and out of the third optical path and deflects the light (50f) returned from the sample as a deflected beam (50g) to the camera or visualization device (6b), such as... Figure 9A As shown, or allowing light (50f) returning from the sample to pass through and reach the detection device (6a), such as Figure 9B As shown.

[0180] This invention should not be construed as limited to the embodiments described above, and certain modifications or changes may be added to the described examples without having to re-evaluate the appended claims.

Claims

1. A cell photoporation system, the system comprising: a. A laser device, preferably a pulsed laser device, for generating an optically perforated laser beam having a predetermined first wavelength, preferably an optically perforated laser pulse; b. An excitation light emitting device, preferably an excitation laser device, more preferably a continuous wave laser device, for generating an excitation beam having a predetermined second wavelength, the second wavelength being different from the first wavelength; c. A sample holder configured to hold one or more containers containing cellular material; d. A sensor device configured to detect and / or visualize cellular material in the sample holder. e. Optical processing components, including: i. A first optical processing subsystem configured to guide the optically aperforated laser beam from the pulsed laser device along a first optical path to the sample holder, and configured to process the optically aperforated laser beam, the first optical processing subsystem comprising a tunable power modulation device, a first dichroic deflector, and a focusing lens along the first optical path, the tunable power modulation device for modulating the power of the optically aperforated laser beam, the first dichroic deflector being configured to further reflect light having a wavelength substantially equal to the first wavelength towards the sample holder along the first optical path, the first dichroic deflector being positioned downstream of the sample holder closer to the power modulation device along the first optical path, and the focusing lens for focusing the optically aperforated laser beam. ii. A second optical processing subsystem configured to guide the excitation beam from the excitation light emitting device along a second optical path to the sample holder; as well as iii. A third optical processing subsystem configured to guide the light obtained from the sample holder along a third optical path to the sensor device.

2. The system according to claim 1, wherein the second optical processing subsystem includes a second dichroic deflector positioned on the second optical path and the third optical path, wherein the second dichroic deflector is configured to further reflect light having a wavelength substantially equal to the second wavelength along the second optical path toward the sample holder, and to further transmit light returning from the sample holder having a wavelength substantially different from the second wavelength along the third optical path toward the sensor device.

3. The system according to any one of claims 1 or 2, wherein, The first dichroic deflection device is positioned on the first, second, and third optical paths, and wherein the focusing lens is also configured to focus the excitation beam.

4. The system according to any one of claims 1 to 3, wherein, The power modulation device is a tunable power attenuator, which includes a half-wave plate for the first wavelength and a polarization beam splitter located further along the first optical path than the half-wave plate, wherein the relative orientation of the half-wave plate and the polarization beam splitter is tunable.

5. The system according to any one of claims 1 to 4, wherein, The optical processing assembly includes a movable mirror assembly, which includes at least one movable mirror for reflecting light along the first optical path or the second optical path toward the sample holder. The movable mirror assembly is configured to reflect the light onto the sample holder in a scanning pattern. The movable mirror assembly is positioned on the first optical path, the second optical path, and the third optical path between the first dichroic deflector and the sample holder. The movable mirror is preferably a mirror galvanometer.

6. The system according to claim 5, wherein, The at least one movable mirror is configured to change the position of the reflected light incident on the sample holder in two dimensions in the plane of the sample holder by tilting the at least one movable mirror on at least two non-parallel axes, preferably by tilting each of the at least two movable mirrors on at least one axis, wherein the at least one axis of the at least one movable mirror is not parallel to each other.

7. The system according to claim 5 or 6, wherein, The power modulation device is configured to modulate the power of the optically aperforated laser beam, taking into account the incident angle of the optically aperforated laser beam on the plane of the sample holder and the related changes in the beam area of ​​the optically aperforated beam on the plane of the sample holder, so as to maintain a substantially constant flux when the optically aperforated beam is reflected in the scanning pattern.

8. The system according to any one of claims 1 to 7, wherein, The first optical processing subsystem further includes a beam detection device and an optical element, wherein the optical element partially deflects the optically perforated laser beam to the beam detection device.

9. The system according to claim 8 and any one of claims 5 to 7, wherein, The scanning pattern includes multiple discrete positions, wherein at least one movable mirror is controllably moved based on the beam detection information from the beam detection device to incident the reflected light onto the discrete positions of the scanning pattern.

10. The system according to any one of claims 1 to 9, wherein, The focusing lens is positioned on the first and second optical paths between the dichroic deflection device and the sample holder, preferably between the movable mirror assembly and the sample holder. The focusing lens is capable of moving along the second optical path toward the sample holder and moving away from the sample holder.

11. The system according to claim 10, wherein, The optical processing assembly further includes, preferably, a removable beam diameter measuring device at a measurement position on the second optical path passing through the focusing lens, preferably between the focusing lens and the sample holder or at the sample holder, wherein the focusing lens is movable along the second optical path toward and away from the measurement position, and wherein the beam diameter measuring device includes a predetermined fluorescent target, preferably a fluorescent plate.

12. The system according to any one of claims 1 to 11, wherein, The sensor device includes a visualization device for providing a visual representation of the contents of the sample holder and a detection device for determining the quantitative characteristics of the contents of the sample holder, the detection device preferably being a photomultiplier tube, and wherein a controllable switching device is provided for changing the third optical path to terminate at the visualization device or the detection device.

13. The system according to any one of claims 1 to 11, wherein, The sensor device includes a visualization device for providing a visual representation of the contents of the sample holder and a detection device for determining quantitative characteristics of the contents of the sample holder. The detection device is preferably a photomultiplier tube and includes optical elements configured to guide the obtained light along the third optical path to the visualization device or the detection device, or both.

14. The system according to any one of claims 1 to 13, wherein, The system includes a power output sensor, preferably a thermopile, configured to measure the average power and / or flux of incident light, and a deflection device, preferably a dichroic deflection device, configured to reflect light with a wavelength substantially equal to the first wavelength and / or the second wavelength from the first optical path toward the power output sensor.

15. The system according to any one of claims 1 to 14, wherein, The first optical path, the second optical path, and the third optical path are at least partially and preferably completely aligned between the first dichroic deflection device and the sample holder.

16. The application of the system according to any one of claims 1 to 15 in cell photoporation.

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