Microscope device, microscope system and methods for use thereof

Abstract

A microscope device according to the invention relates to a bottom-up brightfield microscope device for examining samples illuminated by visible light. The optical path of the microscope device is configured to be independent of the environment lighting and provides constant exposure in light and dark conditions. A microscope system comprises a microscope device according to the invention and at least one robotic manipulator. The microscope system can be used to implement Assisted Reproductive Technology (ART) automatically. The invention relates to methods with which it is possible to examine imaging subjects, such as biological samples or non-biological sample.

Description

[0001]Microscope device, microscope system andmethods for use thereofDESCRIPTIONTECHNICAL FIELDThe present invention relates to a microscope device, a microscope system, and methods for usethereof, and more particularly the present invention relates to specific bottom-up brightfieldmicroscope solutions for examining samples illuminated by visible light.BACKGROUND ARTThe US document No. 11037293 B2 describes a cell observation system for evaluating thequality of the cultured cells. The cell observation system comprises an image acquiring unit housedin an incubator for acquiring images over time of the cells being cultured in a culture container; animage analysing unit for quantitatively analysing a culture state of the cultured cells based on each ofthe images acquired by the image acquiring unit, wherein the communication between the imageacquiring unit and the image analysing unit is performed wirelessly; a statistical analysis unit forstatistically analysing the data quantitatively analysed by the image analysis unit; and a display unitfor displaying the proliferation curves representing a temporal change in the number of the cellswithin each of the culture periods.In a preferred embodiment according to the cell observation system, the image acquiringunit further comprises a light source below the culture container that has a ring-like shape aroundthe objective. The illumination light from the light source is transmitted through the bottom surfaceof the culture container from the bottom upward and is reflected by the top surface of the culturecontainer or a top reflecting member and radiates the cells obliquely from above. Among theillumination light radiated on the cells, the transmitted light of the illumination light transmittedthrough the cells passes through the bottom surface of the culture container from the top downwardand is incident on an objective lens of the image acquiring unit.In this case, the emitted and reflected light are directed along different optical paths, whereinoblique radiation angles of the illumination light are required to pass through the objective lens inorder to imagine the structures of the cells. Furthermore, the image acquiring unit is not enclosed ina light-excluding house to prevent outside light, and the cells to be obtained need to be stored in aculture container for observation.The WO document No. 2011090792 A1 describes a fully automated cell culture monitoringsystem for remotely monitoring the health and viability of cells and the status of a cell culture. Thesystem comprises one or more imaging sensor pods designed to fit inside a standard biologicalsample incubator and positioned below the samples, such that a camera can take images of thesamples; a management control unit, such as a computer, which is adapted to receive the transmittedcell data and cell images wirelessly; and a monitor for viewing the cell data and cell images.The cell culture monitoring system according to the invention is adapted to capture images ofthe vessel to determine the status of samples stored in the vessel based on the captured images,however, the present invention is not capable of any other type of application beyond theexamination of the status of cell cultures.The WO document No. 2017087698 A1 describes an incubator insert system comprises arobotic pipettor above the microscope module attached to a support platform for inserting a fluidinto one or more fluid containers; a perfusion system attached to support platform and in fluidcommunication with robotic pipettor; and a miniaturized brightfield microscope module attached tosupport platform and configured to capture images of samples from below, wherein the microscopemodule has external LEDs which illuminate the sample from above; a chip holder module, which is aplatform for holding organ chips and associated reservoirs (as well as well plates and pipette boxes),and a software module for integrating the hardware modules. The incubator insert system is suppliedvia a power cable.The present incubator insert system is suitable for automated culture experimentation or forex vivo studies with cultured small embryos, living tissues, or organ explants, however the roboticpipettor is designed to move along 3 axes, but cannot to rotate or tilt, therefore does not allow precisecapture of a single cell.The WO document No. 2021014461 A1 describes a system for automated intracytoplasmicsperm injection. The system comprises a first syringe connected to a first micropipette; a secondsyringe connected to a second micropipette; a processing unit to store a first pressure value forcollecting a sperm into the second micropipette, a second pressure value for holding an oocyte at atip of the first micropipette, a third pressure value for puncturing a second layer of the oocyte via thesecond micropipette, and a fourth pressure value for depositing the collected sperm into the oocyte;a first pressure sensor connected between the first micropipette and the first syringe, where the firstpressure sensor is to measure, in real-time, the second pressure value; a second pressure sensorconnected between the second micropipette and the second syringe, where the second pressuresensor is to measure, the first pressure value, the third pressure value, and the fourth pressure value;and an actuator coupled to the processing unit, the first syringe, and the second syringe.. The systemfurther comprises an imaging unit movably arranged above the sperm injection region which can beused to position the micropipettes relative to the sperm and oocyte.The integration of the data into the intracytoplasmic sperm injection visualization softwarefor real-time monitoring allows the user to easily determine the number of oocytes injected withsperms, and the pressures applied at holding and injection needle sides with respect to time. Thereal-time monitoring of the oocyte holding pressure may allow for avoiding inter-user dependentvariations in applying holding pressure for holding the oocyte; and may avoid human errors,real-time pattern recognition of oocyte biomechanical properties during sperm deposition, whichallows the measurement of oocyte quality. The system according to the invention is capable ofmonitoring injection pressure patterns and predicting oocyte quality after the intracytoplasmicsperm injection but cannot monitor or determine the quality or status of sperms and oocyte beforethe injection.None of the above-mentioned solutions can provide a compact microscope system forautomatic observing, imaging, optionally manipulating samples, as well as automatic analysis ofsample data. The present invention has been conceived in light of the above-describedcircumstances, and an object thereof is to provide a microscope device, a microscope system andmethods with which it is possible to examine imaging subjects, such as biological samples ornon-biological samples, with visible light, in particular automatically perform assisted reproductivetechnology. DEFINITION Some of the features of the invention and related definitions are described below. Thefeatures of the invention should be interpreted based on the knowledge of the skilled person.A microscope device according to the invention comprises a bottom-up brightfieldmicroscope module enclosed by a light-excluding house, which has an opening around themicroscope objective. The external light is excluded from the optical path of the microscope deviceby the light-excluding house, so the detector unit can detect the light reflected directly from thesample. An examination surface of a microscope device is an open, large, flat, horizontal, opticallytransparent examination area without holders onto which multiple samples in different opticallytransparent containers or by themself can be placed in any desired arrangement in x-y directions dueto the bottom-up imaging arrangement which illuminates the sample and captures digital images of itfrom the bottom through a transparent surface. Any desired arrangement of the sample containers orthe samples themselves means that the examination surface does not include any container holdersor other means for receiving and securing the containers of the samples, in other words, the samplecontainers are placed in non-fixed positions on the examination surface. Therefore, on the one hand,the arrangements of the sample can be determined freely and independently, on the other hand,given samples, i.e., non-biological solid samples can be placed directly on the examinationsurface. Optionally, the examination surface can be provided with corresponding holders forreceiving and securing corresponding sample containers, wherein said holders can be opticallytransparent holders and / or holders, which are opened from at least the bottom, for example a framefor receiving sample containers. In this case, the holders can also be placed in any desired positionson the examination surface, i.e., in regular rows, or in places designated by a user, so the placementof the samples can be changed depending on the experimental purposes. In all cases, it is essentialthat the samples can be illuminated from below with an illumination unit.The bottom-up brightfield microscope module can be included at least one of the followingelements: lens, tube, dichroic mirrors. The number of these elements depends on the experimentalpurpose, in other words, these additional elements are used to modify the optical path according tothe examination criteria of given samples. The arrangement of these additional elements is obviousto a skilled person in the art, therefore, the technical background of the elements of a brightfieldmicroscope is not detailed in this description.Any sample to be examined with the microscope device according to the invention is suitablefor observation in visible light. The samples can be biological samples, i.e., live or fixedcells / microorganisms, tissue, fungi, bacteria etc, of unlabelled living or fixed samples or sampleslabelled with such markers that are visible in natural light, or non-biological samples, such as organicsolid materials e.g., wood, paper, plastics, textiles, rubber, inorganic solid materials e.g.,carbon-hydrogen compounds: metals, minerals, ceramics, glasses, and semiconductors, crystallinematerials e.g. diamond, quartz, salt, and various metals like gold and silver, amorphous solidmaterials e.g. polymers, composite materials e.g. fiberglass (glass fibers embedded in a polymermatrix), carbon fiber composites, and reinforced concrete, nanostructured materials e.g.nanoparticles, nanotubes, and nanocomposites etc.Examining samples comprises observation, imaging, data analysis based on observationand / or imaging and storing raw and / or processed data related to the samples.The inside of the microscope housing is coated a cooling surface, for example metal surface,on at least connecting surfaces of the microscope device, wherein the connecting surfaces areessentially connection areas between the inside of the microscope house and the individualconnected and secured elements of the microscope device. Instead, or in addition, the microscopedevice can be provided with a cooling unit, which can be a conventional cooling unit. The purpose ofthe above-mentioned elements is cooling of the heat-generating units.The microscope device according to the invention comprises a control and processing unitarranged inside the microscope house and an external processing and monitoring system wirelesslyconnected to the control and processing unit. The control and processing unit and the externalprocessing and monitoring system are also suitable for receiving data, analysing data and storingdata. Typically, the control and processing unit is used to receive data, i.e., digital images and / orvideos from the detector unit, transmit data to the external processing and monitoring unit.Additionally, the control and processing unit can be used to store raw data and / or process raw dataand store them. Normally, this mentioned additional function can be used when data cannot betransmitted to the external processing and monitoring system due to a wireless connection or othererror, i.e., the data storing is usually short-term. The external processing and monitoring system canbe a conventional PC, similar computation-capable device or cloud service and is configured toremotely control the control and processing unit, as well as store and process digital images and / orvideos transmitted from the control and processing unit, wherein the data storage is a local and / orcloud-based storage, and usually long-term storage.The operation of the microscope device means that the microscope device according to theinvention can be controlled to observe samples placed on the examination surfaces illuminated bybottom-up light, image sample, process data, store raw and / or processed image data, transmit imagedata, and analyse raw and / or processed data using deep learning algorithms, machine learningand / or image-processing.SUMMARY OF THE INVENTIONIn order to achieve the above-described object, the present invention provides the followingsolutions. An aspect of the present invention provides a microscope device including at least thefeatures of claim 1, optionally the features of the dependent claims thereof. Essentially, a microscopedevice according to the invention relates to a bottom-up brightfield microscope device for examiningsamples illuminated by visible light. The optical path of the microscope device is configured to beindependent of the environment lighting and provides constant exposure in light and darkconditions, without the need for an additional compartment such as a lid or an enclosing element toblock the light entering from the outside. The size of the microscope device can be reduced due tothe bottom-up illumination and the lighting independent of an environment light, thereby improvingspace utilization, for example it can be placed in an CO2 incubator. Among other things, themicroscope device comprises an examination surface for placing samples in any desiredarrangement, thereby reducing the time of placing the samples on the examination surface, andallows any type of container to be placed, or specific samples can be placed directly on theexamination surface.Another aspect of the present invention provides a microscope system according to claim 9.Another aspect of the present invention relates to the use of a microscope device accordingto the invention, which provides a method according to claim 25.Another aspect of the present invention relates to the use of a microscope system accordingto the invention, which provides a method according to claim 35.Other aspects and advantages of the present invention will be apparent from the followingdetailed description of the preferred embodiments and the accompanying drawing figures.BRIEF DESCRIPTION OF THE DRAWINGSHereinafter, preferred exemplary embodiments of the invention are described with the helpof the accompanying drawing. In the drawing:Figure 1 is a schematic side cross-sectional view illustrating an embodiment of themicroscope device.Figure 2 is a schematic side cross-sectional view illustrating an exemplary embodiment of amicroscope system having a robotic manipulator.Figure 3 is a perspective view illustrating a moving unit with a light-excluding house of themicroscope device according to the invention.Figure 4 is a perspective view of an embodiment of a microscope system having two roboticmanipulators. Figure 5A is a schematic side view illustrating a simple optical path of an embodiment of themicroscope device according to the invention.Figure 5B is a schematic side view illustrating an optical path of an embodiment of themicroscope device including four lenses.Figure 5C is a schematic side view illustrating a U-shape optical path of an embodiment ofthe microscope device including with two mirrors.Figure 5D is a schematic side view illustrating a U-shape optical of an embodiment of themicroscope device including four lenses and two mirrors.Figure 5E is a schematic side view illustrating an inverted S-shape optical path of anembodiment of the microscope device including four mirrors.Figure 5F is a schematic side view illustrating an inverted S-shape optical path of anembodiment of the microscope device including four lenses and four mirrors.Figure 5G is a schematic side view illustrating an optical path of an embodiment of themicroscope device including three lenses and two tubes.Figure 5H is a perspective side view illustrating an optical path of a microscope deviceaccording to Figure 5G.Figures 6A-6J are a top view illustrating examples of arrangement of sample containers andsamples arranged on the examination surface.Figure 7 is a block diagram of an exemplary method for using the microscope deviceaccording to the invention.Figure 8 is a block diagram of an exemplary method for using the microscope systemaccording to the invention.DETAILED DESCRIPTIONFigure 1 schematically shows a side cross-sectional view of an embodiment of a microscopedevice 100. The microscope device 100 according to the invention is configured to examine samples201 illuminated by visible light. The microscope device 100 comprises a microscope housing 110 thatsurrounds the microscope against the external negative effects. The microscope housing 110 has aframe 111, which provides structural rigidity of the microscope device 100 and positions thecomponents. The inside of the microscope housing 110 can be coated a cooling surface on at leastconnecting surfaces of the microscope device 100 and / or the microscope housing 110 may providewith a cooling unit. The cooling surface is ideally made of metal e.g., steel or aluminium, in whichcase it is suitable to absorb excess heat and distribute it to its large surface such that it does notinfluence the temperature of its environment significantly. The microscope device 100 does notincrease humidity, emits chemicals or gases, and does not interfere with the pH level of itssurroundings. The microscope housing 110 can be constructed from steel plates as a single unit, forexample by welding, but also as an assembled unit, for example by using fasteners.The microscope device 100 comprises a flat examination surface 125 that is arranged on thetop of the frame 111 horizontally of the microscope housing 110. The examination surface 125 ismade of optically transparent material such as glass, plastic, or any similar material provided it doesnot cause such a diffraction of light that it would significantly disturb the illuminating light beamreaching a sample 201 or the reflected light beam reaching a detector unit 134. The examinationsurface 125 is configured to hold at least one sample 201 visible in natural light.In a preferred embodiment, the examination surface 125 can be configured as a flat surfacewithout holders, so that the at least one sample 201 containing in a container 200 visible in naturallight can be placed on the examination surface 125 in any non-fixed, non-predeterminedarrangement. In an optional embodiment, the examination surface 125 can be provided at least oneadditional fixed optically transparent holder and / or a bottom-opened holder arranged at a desiredposition of the examination surface 125 for securing the one or more optically transparent containers200 on the examination surface 125. In this embodiment, the at least one holder can be fixed on theexamination surface 125 in order to receive and fix a predetermined position of a sample container200, such as multi-well micro-titer plates, flasks, dishes, slides, chambers or any other samplecontainer or vessel. The holder enables quick navigation across sample holders and allows easymanagement of multiple container 200 and / or multiple users parallelly.One or more optically transparent sample containers 200 each comprising a sample 201visible in natural light can be placed on the examination surface 125. The one or more opticallytransparent 200 containers can be selected from the group essentially comprising opticallytransparent multi-well micro-titer plates, flasks, dishes, slides, chambers, and / or any other opticallytransparent sample containers or vessels.Figures 6A-6J are a top view illustrating examples of arrangement of sample containers 200and samples 201 arranged on the examination surface 125. Figure 6A shows six standard multi-wellplates, i.e., here 96-well plates. Figure 6B shows Petri dishes, here nine Petri dishes in 3x3arrangement. Figure 6C shows flasks and a multi-well plate, here three larger T75 flasks and twosmaller T25 flasks, as well as 96-well plates. Figure 6D shows 33 standard microscope slides. Figure 6Eshows three larger T75 flasks and two 96-well plates. Figure 6F shows three larger T75 flasks and fivesmaller T25 flasks. Figure 6G shows three 96-well plates, three standard microscope slides and threePetri dishes. Figure 6H shows a larger T75 flask and a non-biological sample 201 placed directly onthe examination surface 125. Figure 6I shows a larger T75 flask, two smaller T25 flasks, two standardslides, a Petri dish and a 96-well plate in a non-regular arrangement. Figure 6J shows 11 smaller T25flasks in a non-regular arrangement. According to Figure 6A-6J, any type of containers 200 can beplaced in the examination surface 125 in regular or non-regular arrangements, and at the same time,non-biological samples 201 can also be placed on the examination surface 125. Biological samples 201need to be stored in a container 200 for proper and high-quality examination.The containers 200 can be placed on the examination surface 125 with or without holders.Preferably, container 200 arrangements shown in Figures 6A, 6B, 6D and 6F are placed in standardholders mounted on the examination surface 125. Preferably, container 200 arrangements shown inFigures 6C, 6E and 6G are placed in customized holders mounted on the examination surface 125.Preferably, container 200 arrangements shown in Figure 6H, 6I and 6J are placed on the examinationsurface 125 without holders.Each sample 201 stored in the optically transparent containers 200 and illuminated by visiblelight can be a biological sample and selected from the group essentially comprising live-cell cultures,bacteria, fungi, blood or pap smears, histopathology tissues or similar entities, and subcellularstructures such as nucleoli or similar entities, unlabelled living or fixed samples, or samples labelledwith such markers that are visible in natural light. It is necessary to contain the biological samples201 in container 200 to provide suitable conditions for these samples 201. The samples 201 visible innatural light can also be non-biological samples such as organic solid materials, inorganic solidmaterials, crystalline materials, amorphous solid materials, composite materials, ornanostructured materials. Non-biological samples can be stored in containers 200 or placed directlyon the examination surface 125 without containers 200.The microscope device 100 comprises a bottom-up brightfield microscope module 130arranged under the examination surface 125. The bottom-up brightfield microscope module 130 ismovable along x -y-z axes.The brightfield microscope module 130 includes an illumination unit 131 parallel to the examinationsurface 125 for emitting visible light upward from below the sample 201. The illumination unit 131 isa single or multiple LED(s). The colour of the LED(s) preferably is white, but other colours can also beused. The illumination unit 131 preferably emits visible light with wavelengths between 380 and 700nanometres to examine samples 201.The brightfield microscope module 130 includes a microscope objective 132, which ispositioned vertically with respect the examination surface 125 and directly under said examinationsurface 125 for magnifying the sample 201. The microscope objective 132 can be configured to allowthe observation of the samples 201 with suitable examination magnification such as1x,2x,4x,10x,20x,40x,60x,63x,100x or 150x magnification. Similarly, an objective with achromatic,apochromatic, semi-plan, plan or other, similarly defined objective properties, from anymanufacturer or vendor may suffice as the microscope objective in the device.The brightfield microscope module 130 further includes a detector unit 134, e.g., CMOSsensor, for detecting light reflected from the sample 201 and capturing digital image(s) and / orvideo(s) of the sample 201. The detector unit 134 can be adapted to a focus capability for imagingstructures of the samples 201 visible in natural light. Preferably, the detector unit 134 preferably is adigital camera. However, any sensor with suitable resolution and suitable sensor size manufacturedby any manufacturer may be applicable.The brightfield microscope module 130 includes a beam splitter 133, which is essentially inline with the illumination unit 131 and positioned between the illumination device 131 and themicroscope objective 132 for controlling and directing light along an optical path. The beam splitter133 is arranged to create the optical path by directing visible light from the illumination unit 131through the microscope objective 132 and then transmitting light essentially directly reflected fromthe sample 201 to the detector unit 134, wherein the light is emitted and reflected along the sameoptical path between the sample 201 and the detector unit 134. Exemplary optical paths are describedin Figure 5A-5H.Figure 5A is a schematic side view illustrating a simple optical path of an embodiment of themicroscope device 100. In this case, the brightfield microscope module 130 comprises theillumination unit 131, the microscope objective 132, the beam splitter 133 and the detector unit 134,wherein these units are arranged as described above and in claim 1, and the optical path presents asdescribed above and in claim 1.Preferably, the bottom-up brightfield microscope module 130 further includes at least one ofthe following elements: lens 136, tube 138, dichroic mirrors 137. Exemplary arrangements of theseelements are described in Figure 5B-5H.The bottom-up brightfield microscope module 130 is provided with at least one lens 136along the optical path for focusing visible light emitted from the illumination unit 131 or controllingthe amount of visible light along the optical path. Figure 5B is a schematic side view illustrating anoptical path of an embodiment of the microscope device 100 including four lenses 136. The lens 136arranged adjacent to the illumination unit 131 enables the visible light emitted from the illuminationunit 131 to be focused. The other three lenses arranged along the optical path enables the amount ofvisible light along the optical path to be controlled.The bottom-up brightfield microscope module 130 is provided with at least one pair ofdichroic mirrors 137 for reflecting light between the beam splitter 133 and the detector unit 134. Thepurpose of dichroic mirrors 137 is to extend the path of light along the optical path. Figure 5C is aschematic side view illustrating a U-shape optical path of an embodiment of the bottom-upbrightfield microscope module 130 including with two mirrors 137. The dichroic mirrors 137 arearranged between the beam splitter 133 and the detector unit 134.Figure 5D is a schematic side view illustrating a U-shape optical path of an embodiment ofthe microscope device including four lenses and two mirrors. The embodiment according to Figure5D comprises the features of the embodiments of Figures 5B and 5C.Figure 5E is a schematic side view illustrating an inverted S-shape optical path of anembodiment of the microscope device including four mirrors 137, i.e., two pairs of dichroic mirrors137 arranged between the beam splitter 133 and the detector unit 134. The greater the number ofmirrors 137 used in a suitable, reflective arrangement, the longer the path of the light can be, whichis expedient in certain examination cases.Figure 5F is a schematic side view illustrating an inverted S-shape optical path of anembodiment of the microscope device 100 including four lenses 136 and four mirrors 137. Theembodiment according to Figure 5F comprises the features of the embodiments of Figures 5B and 5E.The bottom-up brightfield microscope module 130 is provided with at least one tube 138arranged along the optical path for encapsulating the visible light beam from the illumination unit131 to the detector unit 134. Figure 5G is a schematic side view illustrating an optical path of anembodiment of the microscope device 100 including three lenses 136 and two tubes 138. One of thetubes 138 is arranged between the beam splitter 133 and the microscope objective 132, and anothertube 138 is arranged between the beam splitter 133 and the detector unit 134. The purpose of thetubes 138 is to encapsulate the path of light along the optical path.Figure 5H is a perspective side view illustrating an optical path of a microscope device 100 accordingto Figure 5G. The tubes 138 are cylindrical to guide the optical path between the units of thebottom-up brightfield microscope module 130.The bottom-up brightfield microscope module 130 is enclosed by a light-excluding house 135having an opening around the microscope objective 132, so the light may pass through said openingand the microscope objective 132 to create an optical path. Practically, the light-excluding house 135may include other openings, holes and / or slots for electrical lines or wires. Essentially, thelight-excluding house 135 is enclosed all compartments including non-essential compartmentssuch as lenses, tubes, or mirrors, of the bottom-up brightfield microscope module 130 to excludeexternal light, i.e., non-illuminated light from the illumination unit 131. Since the light-excludinghouse 135 has an opening around the microscope objective 132, it is possible that external light canpass through it, but essentially the optical path between the sample 201 and the detector unit 134 isnot affected by this amount of external light. The light-excluding house 135 is shown in Figure 3.The microscope device 100 further comprises a moving unit 140 connected to the frame 111and configured to position the bottom-up brightfield microscope module 130 enclosed in thelight-excluding house 135 in any of the x-y-z directions. An embodiment of the moving unit 140 isdescribed in Figure 3.In Figure 3, the moving unit 140 is attached to the frame 111 of the microscope device 100between the bottom part of the microscope housing 110 and the examination surface 125 to enableprecise movement along the given axis. Precise movement is defined such that in x-y-z it is possibleto move the field of view to at least such a distance that based on the details of the captured digitalimage corresponding to the direction of the movement the overlapping region can be recognised onboth images before and after the movement. The order of magnitude for the precision of movementin x-y may be approximately 0.1,1,10,100,1000 μm, or any precision suitable to observe samples 201,and similar or even smaller in z to enable precise focusing.The microscope device 100 further comprises a control and processing unit 150 forcontrolling operation of the microscope device 100 and receiving, storing and processing digitalimage(s) and / or video(s) from the detector unit 134.The microscope device 100 comprise an external processing and monitoring system 500wirelessly connected to the control and processing unit 150. The external processing and monitoringsystem 500 is arranged outside the microscope house 110 and configured to remotely control thecontrol and processing unit 150, as well as store and process digital image(s) and / or video(s)transmitted from the control and processing unit 150. Essentially, the external processing andmonitoring system 500 can indirectly control the microscope device 100 via the control andprocessing unit 150.The control and processing unit 150 and / or the external processing and monitoring system500 can perform at least the following functions: controls the movements of the moving unit 140 ofthe microscope device 100 to change the position of the field of view acquired by the detector unit134; controls the focusing of the bottom-up brightfield microscope module 130, i.e., autofocus ormanual focus, wherein the autofocus may be realized with any suitable physical apparatus with orwithout specific software function to perform the focusing or merely with software function.Manages the power supply and at least one integrated battery 160; controls the illumination unit 131;controls the wireless communication; controls the detector unit 134; handles the raw digital imagedata acquired by the detector unit 134; processes the raw image data; analyses raw and processedimage data and stores raw, processed image data, as well as information regarding analyses, but otheradditional functions can also be performed.The microscope device 100 comprise an integrated battery 160 arranged in the microscopehousing 110. The integrated battery 160 can power the microscope device 100 in a completelywireless manner. The integrated battery 160 is configured to power the microscope device 100 forseveral days, but at least approximately 72 hours, preferably about 1-7 days. The standby or operatingtime of the integrated battery 160 is basically dependent on the operating time of the microscopedevice 100. For example, in the case of repeated, short-term operation, the microscope device 100can be powered by the integrated battery 160 for several weeks or months. Even with continuousoperation of the microscope device 100, the integrated battery 160 can operate the microscope device100 for several days, approximately 1-7 days. The battery charge level of the integrated battery 160can be monitored by the control and processing unit 150 and / or the external processing andmonitoring system 500.Optionally, the microscope device 100 further comprise at least one additional integratedbattery 160, each configured to power the microscope device 100 for several days such as about 1-7days. Due the at least one additional integrated battery 160, the continuous operation of themicroscope device 100 can be extended, thereby this embodiment of the microscope device 100 issuitable for long-term continuous examination, even for several weeks.In an optional embodiment, the microscope device 100 comprises a non-transparent lid toenclose the examination surface 125 for protecting the samples 201. On the one hand, thenon-transparent lid protects the samples 201 from external effects, on the other hand, thenon-transparent lid excludes external light. The non-transparent lid is not shown in the Figures.As shown in Figure 1, the microscope device 100 can be placed in an incubator 400,preferably in a CO2 incubator. The microscope device 100 is operated via integrated battery 160 andfully wirelessly, so the samples 201 in the incubator 400 are not disturbed at all. Thus, imaging of e.g.,live cell experiments and cell growth is possible with the microscope device 100 inside the incubator400 while operation is remotely handled by a user.Figure 2 schematically shows a side cross-sectional view of an embodiment of a microscopesystem 10. The microscope system 10 comprises an embodiment of the microscope device 100according to the invention and at least one robotic manipulator 300 is mounted on an externalsurface of an upper part of the microscope housing 110. The at least one robotic manipulator 300 canbe controlled wirelessly by the control and management unit 150 and / or the processing andmonitoring system 500. Preferably, the at least one robotic manipulator 300 can be a robotic armhaving tilting parts and is configured to allow movement in a direction of x-y-z axes and rotationaround the x axis. The at least one robotic manipulator 300 can be configured to connect a respectiveexperimental tool such as a pipette, needle, syringe. Optionally, the robotic manipulator 300 has anintegrated rechargeable battery for power supply. In other optional embodiment, the roboticmanipulator 300 is configured to power by the integrated battery 160 of the microscope device 100.When multiple robotic manipulators 300 are mounted on the microscope device 100, all of them arecontrolled independently by a user, and the robotic manipulators 300 do not communicate with eachother. The operation height of the robotic manipulator 300 i.e., the maximal z distance it can bemoved to upwards from the examination surface 125 should be high enough for the pipette or otherexperimental tool to be moved above the highest dimension of the sample container 200.The microscope system 10 can be used to implement Assisted Reproductive Technology(ART) automatically. In Figure 4, a preferred embodiment of the microscope system 10 is shown,wherein the microscope system 10 comprises two robotic manipulator 300A, 300B which aremounted on the external surface of the upper part of the microscope housing 110, wherein one of therobotic manipulators 300A is positioned on one side of the microscope housing 110, and the secondrobotic manipulator 300B is positioned on the opposite side of the microscope housing 110. In thecase of implementation of ART, one of the robotic manipulators 300 is a pipetting injector unit forinjecting sperms, and another of the robotic manipulators 300 is a pipetting needle unit for holdingan oocyte. The containers 200 for containing sperms and oocyte are not shown in this Figure.Preferably, these containers 200 are received in holder placed on the examination surface 125 to fixthe position of the containers 200 during performing ART.The microscope system 10 according to the invention can be placed in an incubator 400,preferably in a CO2 incubator, where a live-cell experiment may occur.Hereinafter, the method for using the microscope device 100 for examining samples 201illuminated by visible light will be described with reference to Figure 7.In step S10, the illumination unit 131 is emitting visible light.In step S20 the beam splitter 133 is transmitting emitted light through a microscope objective132 to a given sample 201.In step S30 the transmitted emitted light is reflecting directly off the given sample 201.In step S40 the detector unit 134 is detecting light reflected essentially directly from the givensample 201 through the microscope objective 132 and the beam splitter 133.This step essentially means screening or observation of a given sample by the detector unit134. The observation can be a continuous observation of a sample e.g., monitoring of cell growth orbiological phenomenon such as mitosis or any other process which lasts at least as long as multipleframes of the field of view can be captured is possible with the detector unit 134.Furthermore, the observation can also be scheduled time point-based digital imagedetection, which allows the sample to be observed regularly at predetermined time intervals, such asonce every 6 hours or any desired number of times in any desired time period, e.g., to observe theeffect of compounds to cell viability, motility, morphology or any other characteristic of the sampleand its change in time to any applied perturbation in the experiment.In step S40, the screening or observation can be performed as a quick screening to check theactual status of the sample 201 very quickly, select which container 200 or sample 201, which region(well in case of plate), which sub-region to screen. The screening or observation can be performed asa full screening to screen entire container 200, such as plates, flasks, dishes, slides or other vessels, orentire sample 201. Furthermore, the screening or observation can be performed as a customscreening to select which container 200, which region (well in case of plate), which sub-region toscreen, and combine selections in any desired configuration.In step S50 the detector unit 134 is capturing at least one digital image, preferably a pluralityof digital images and / or video, of the detected light reflected directly from the given sample 201.For example, in the case of a continuous observing according to an above-mentionedexemplary step S40, time-lapse recordings can also be captured or sample 201 can be observed in alive view mode.In addition, in the case of scheduled time point-based image detection according to anotherabove-mentioned example of step S40, the detector unit 134 can be controlled to select such a uniquecustom schedule for imaging to take place as well as the selection of which field of view to capture.The schedule selection can be saved such that a user can see already scheduled imaging eventscreated by himself / herself or other users, the latter of which is displayed in a visually easilydistinguishable manner.In addition, the detector unit 134 can be used to screen a specific region or larger region orphysical container-defined sample part such as a well of a multi-well micro-titer plate or a smallerregion of it or an entire sample. Screening is the process of capturing multiple digital images of thesample with the microscope device 100. Illumination settings and focus can be modified by a userduring steps S40 and / or S50.In step S60 the microscope device 100 is processing the captured at least one digital image,preferably a plurality of digital images and / or video. The processing step S60 comprising at least oneof the following: quality improvement of the at least one digital image of the samples 201; confluencycalculation of the at least one digital image of biological samples 201; segmentation, detection,counting of given objects of the 201 samples based on at least one digital image of the samples 201;classification of given objects and / or samples 201 based on at least one digital image of the samples201; statistical analysis of quantitative features of the at least one digital image of the samples 201; ora combination thereof.In step S60, digital image quality improvement including but not limited to unevenillumination correction and any other operation which improves the quality of the raw digital imagesacquired. The processing step S60 can be a confluency calculation to determine confluence of thebiological sample 201 e.g., cell culture or any other type of sample suitable to be observed in themicroscope device 100 to which the calculation of confluence bears a scientific or othercharacteristic. Confluence is the area covered by e.g., cells related to the entire surface area of theculture vessel or container 200 possible for cells or other entities to adhere to. This relative area isdetermined by a processing step detecting the total extension of all cells in the sample 201 divided bythe entire surface area of the culture vessel or container 200.The processing step S60 can be performed by detecting or counting the objects of a sample201. The number of individual objects in the sample 201, e.g., cells in live-cell assays, is determinedby detecting each object separately, and also retrieves the total count of the objects. The extension ofthe object on the 2D digital image i.e., size and position are also determined, which may berepresented as a bounding box around the object or any other representative manner.The processing step S60 may be performed by instance segmentation of objects in a sample201. Each pixel of the digital image either corresponds to the foreground of objects - in case ofphenotyping, only interesting or relevant objects - or the background of everything else on the imagee.g., surrounding tissue structure, dust, artefacts, culture media, parts of the vessel etc. The result ofthis processing step may be a so-called instance segmentation mask which is object-aware and maybe represented as a multi-level grayscale digital image where each different gray intensity in e.g.,0-255 represents the set of pixels that either belong to one object or the background, or any other waythat represents the instances (objects).The processing step S60 may be performed by classification or clustering of objects in asample 201. The classification can be determined by different predetermined parameters and / orfeatures. The processing step S60 may be performed by calculate statistics and create plots ofmeasurements or other pieces of analysis. Any measurement or piece of analysis may becharacterized with basic statistics such as mean, median, standard deviation etc. or more complexstatistical methods, as well as such measurements or pieces of analysis may be plotted on a graph tobetter visualize the measurement or to provide a graphical representation to the user for furtherutilization such as to display the graph in a presentation, publication, education material and / or anyother way.The processing step S60 can be performed automatically using deep learning algorithms,machine learning and / or image-processing.In step S70 the microscope device 100 is storing the captured and / or the processed at leastone digital image, preferably a plurality of digital images and / or video. The S70 storing step isperformed by local storing and / or cloud-based storing.A preferred method according to the invention can comprises additional steps, which are notshown in Figure 7. In this preferred method, after the step S10, the method comprising further:- at least one focusing step of focusing emitted visible light from the illumination unit 131along an optical path by at least one lens 136 in connection to any of steps S20-S40, or a combinationthereof, and / or- at least one controlling step of controlling the amount of visible light along the optical pathby the at least one lens 136 in connection to any of steps S20-S40, or a combination thereof, and / or- at least one guiding step of guiding light along the optical path by at least one tube 138 inconnection to any of steps S20-S40, or a combination thereof.Furthermore, a preferred method according to the invention can comprises the followingoptional step: after step S30, the method comprising:- an additional reflecting step of reflecting visible light between the beam splitter 133 and thedetector unit 134 by at least one pair of dichroic mirrors 137.The aforementioned, preferred methods can be performed with a microscope device 100which, depending on the additional step(s) of the method, has at least one lens 136, at least onemirror 137 and / or at least one tube 138.Preferably, the microscope device 100 is repeating at least S10-S40 steps, preferably S10-S50steps for each sample 201 to be examined, thereby all samples 201 arranged on the examinationsurface 125 can be examined.Hereinafter, the preferred method for using the microscope system 10 for automatedimplementation of Assisted Reproductive Technology (ART) will be described with reference toFigure 8. This method according to the invention can be performed entirely automatically, withouthuman intervention, based on live digital image capture by the microscope device 100.In step S100 of said method, providing a first optically transparent container 200 comprisingliving sperms.In step S200 providing a second optically transparent container 200 comprising living oocyte.In step S300 automatically detecting and imaging moving sperms in the first container 200 inreal time.In step S400 automatically analysing digital image(s) and / or video(s) of the detected movingsperms based on predetermined features to select at least one desired sperm for injection. In step 400the method allows the detection, scoring and selection of sperm in real time in order to select one ormore desired sperms for injection.In step S500 a pipetting injector unit is tracking the at least one selected sperm in real timeand collecting the at least one selected sperm using a negative pressure.In a preferred embodiment of the method according to Figure 8, in step S510 the microscopesystem 10 is confirming the step S500 based on digital imaging performed by the microscope system10. In step S600 the pipetting needle unit is holding the oocyte.In step S700 the pipetting injector unit is penetrating the oocyte to enter the cytoplasm of theoocyte. In step S800 step the pipetting injector unit is collecting a part of the cytoplasm of the oocyteusing a negative pressure.In step S900 step the microscope system 10 is changing the negative pressure of the pipettinginjector unit to positive pressure in order to insert the collected sperm and the collected part of thecytoplasm of the oocyte into the oocyte.In the preferred embodiment of the method according to Figure 8, in the S910 step themicroscope system 10 is confirming the S900 step based on digital imaging performed by themicroscope system 10.In step S1000 the microscope system 10 is removing the pipetting injector unit and thepipetting needle unit from the ooctyte.In step S1100 the microscope system 10 is real-time monitoring and qualifying selectedfeatures of the fertilized oocyte continuously or at predetermined time based on digital imagingperformed. In step S1200 the microscope system 10 is determining the quality of the fertilized oocyte bycomparing qualified features of step S1100 with predetermined features. For example, the qualitycontrol of the fertilized oocyte can be performed by capturing raw digital images either in real timeor on a time-point based manner.In another preferred embodiment of the method according to the invention, the steps of themethod for using the microscope system 10 for automated implementation of Assisted ReproductiveTechnology (ART) are performed using deep learning algorithms, machine learning and / orimage-processing. The advantage of the microscope device according to the invention is that it is suitable for theexamining biological and non-biological samples independent of ambient lighting.Another advantage is that the microscope device has transparent sample surface, whichprovides an open and large examination area for simultaneous examination of several containersplaced adjacently or in any desired spatial arrangement in x and y dimensions. The invention is notlimited to sample / sample container size due to the large open examination surface. In certain cases,holders can be mounted on the examination surface to fix containers in predefined positions. Theapplication of holders can be useful when a robotic manipulator is used to manipulate samples in thecontainers. In a specific case, the transparent sample surface may be removed completely andsubstituted with a holder designed to hold a specific number and type of holders, whereinlarge magnification digital image captures suitable to observe more clinically relevant details andstructures of the sample.An advantage of the invention that an integrated battery can power the microscope deviceand / or the microscope system for multiple days or even weeks in a completely wireless manner.On the one hand, the microscope device and, on the other hand, the microscope system canbe placed in an incubator, preferably CO2 incubator. In this case, the examination performed by themicroscope device and / or microscope system can take place in a closed, stable environment insidethe incubator. The incubator applied to the microscope device and / or the microscope system can bea fully wireless incubator, in which no wires are required for power supply, data transmission orcontrol. The methods according to the invention can be controlled by a user-friendly software UI,which was designed to have every functionality easily reachable and understandable. This is achievedby a design in which steps and tasks are organised and ordered such that barely minimal userinteraction is needed to select which kind of operation, step or task the user desires to execute, andparameters or settings are inferred automatically and intrinsically, so that their understanding ortuning is not needed either. Previous configurations are stored to enable repeating workflows easily.The UI works well with desktop, tablet or mobile devices, keeping interactions easy and convenient.The software includes many functionalities for convenience (for example scheduling, notifications)and analysis (for example counting, detection, segmentation, classification, statistics, plots).Another advantage is that no external device is needed at the installation site, only a singleindependent remote system or server connected through a network wirelessly.Furthermore, it is also an advantage that no storage device needs to be connected in order towork, data is safely stored in the cloud data storage.The control and processing unit and the external processing and monitoring system can alsooperate the microscope device and / or the microscope system via wireless communication.Other advantages of the invention that it can support custom applications e.g., WSI(whole-slide imaging), live cell monitoring, patient sample observation, bacteria or fungi growth,virus infection, standard tabletop microscopy, automated ART, etc.The disclosed embodiments of above solutions have practical applications in many fieldsincluding, but not limited to, material inspections, medicine-related applications, biology application(e.g., biological research, microbiology, biochemistry, botany), biotechnology, medical diagnosis (e.g.pathology, haematology, microbiology, cytology), forensic science, environmental science,environment protection (e.g. water quality control, pollutant detection), nanotechnology, geology(e.g., minerals, rocks), agricultural science, food industry (e.g. quality control of yeast or mold),industrial applications, education application (e.g. plant leaf, onion cell, insect, dust particles,microorganisms in dirty water, printed paper, microelectronics, pollen etc.), jewellery industry,beauty industry (e.g., cosmetics), archaeology, energy industry (e.g., semiconductors, solar panelcells etc.) and entertainment applications.Other embodiments of the invention will be apparent to those skilled in the art fromconsideration of the specification and practice of the invention disclosed herein. It is intended thatthe specification and examples be considered as exemplary only, with a true scope and spirit of theinvention being indicated by the following claims.

Claims

AMENDED CLAIMS received by the International Bureau on 27 April 2025 (27.04.2025)

1. A microscope device (100) for examining samples (201) illuminated by visible light, comprising• a microscope housing (110) having a frame (111),• a horizontal, optically transparent examination surface (125) arranged on the top of the frame (111) of the microscope housing (110) for holding at least one sample (201) visible in natural light,• a bottom-up brightfield microscope module (130) arranged under the examination surface (125), includes o an illumination unit (131) parallel to the examination surface (125) for emitting visible light upward from below the sample (201), wherein the illumination unit (131) is a single or multiple LED(s), preferably the colour of the LED(s) is white, o a microscope objective (132), which is positioned vertically with respect the examination surface (125) and directly under said examination surface (125) for magnifying the sample (201), o a beam splitter (133), which is essentially in line with the illumination unit (131) and positioned between the illumination device (131) and the microscope objective (132) for controlling and directing light along an optical path, o a detector unit (134) for detecting light reflected from the sample (201) and capturing digital image(s) and / or video(s) of the sample (201), wherein the bottom-up brightfield microscope module (130) is movable along x -y-z axes,• a moving unit (140) connected to the frame (111) and configured to position the bottom-up brightfield microscope module (130) in any of the x-y-z directions;• a control and processing unit (150) for controlling operation of the microscope device (100) and receiving, storing and processing digital image(s) and / or video(s) from the detector unit (134); characterized in that the bottom-up brightfield microscope module (130) is enclosed by a light-excluding house (135) having an opening around the microscope objective (132), wherein the light-excluding house (135) is configured to essentially exclude external light, i.e., non-illuminated light from the illumination unit (131), wherein essentially the optical path between the sample (201) and the detector unit (134) is not affected by external light; wherein the beam splitter (133) is arranged to create the optical path by directing visible light from the illumination unit (131) through the microscope objective (132) and then transmitting light essentially directly reflected from the sample (201) to the detector unit (134), wherein the light is emitted and reflected along the same optical path between the sample (201) and the detector unit (134);that the examination surface (125) is configured as a flat surface, so that the at least one sample (201) visible in natural light can be placed on the examination surface (125) in any non-predefined arrangement; that the microscope device (100) further comprising• an integrated battery (160) arranged in the microscope housing (110), which can power the microscope device (100) in a completely wireless manner;• an external processing and monitoring system (500) wirelessly connected to the control and processing unit (150), wherein the external processing and monitoring system (500) is configured to remotely control the control and processing unit (150), as well as store and process digital image(s) and / or video(s) transmitted from the control and processing unit (150).

2. Microscope device (100) according to claim 1, wherein the microscope device (100) uses visible light with wavelengths between 380 and 700 nanometres to examine samples (201).

3. Microscope device (100) according to any one of claims 1 to 2, wherein the examination surface is adapted to be able to receive one or more optically transparent sample containers (200) each comprising a sample (201) visible in natural light and said sample containers (200) can be placed on the examination surface (125), wherein each sample (201) stored in the optically transparent containers (200) and illuminated by visible light is a biological sample and selected from the group essentially comprising live-cell cultures, bacteria, fungi, blood or pap smears, histopathology tissues or similar entities, and subcellular structures such as nucleoli or similar entities, unlabelled living or fixed samples, or samples labelled with such markers that are visible in natural light.

4. Microscope device (100) according to any one of claims 1 to 3, wherein the microscope device (100) is adapted to be able to receive such samples (201) visible in natural light that are non-biological samples such as organic solid materials, inorganic solid materials, crystalline materials, amorphous solid materials, composite materials, or nanostructured materials.

5. Microscope device (100) according to any one of claims 1 to 4, wherein the detector unit (134) is a digital camera, wherein the detector unit (134) is configured to be adapted to a focus capability for imaging structures of the samples (201) visible in natural light.

6. Microscope device (100) according to any one of claims 3 to 5, wherein the microscope device (100) is configured to be able to receive the one or more optically transparent containers (200) which is / are selected from the group essentially comprising optically transparent multi-well micro-titer plates, flasks, dishes, slides, chambers, and / or any other optically transparent sample containers or vessels.

7. Microscope device (100) according to any one of claims 3 to 6, wherein the examination surface (125) is provided at least one additional fixed optically transparent holder and / or a bottom-opened holder arranged at a desired position of the examination surface (125) adapted to be able to secure the one or more optically transparent containers (200) on the examination surface (125).

8. Microscope device (100) according to any one of claims 1 to 7, wherein the bottom-up brightfield microscope module (130) further including at least one of the following elements: lens (136), tube (138), dichroic mirrors (137).

9. Microscope device (100) according to claim 8, wherein the bottom-up brightfield microscope module (130) is provided with at least one lens (136) along the optical path for focusing visible light emitted from the illumination unit (131) or controlling the amount of visible light along the optical path.

10. Microscope device (100) according to any one of claims 8 to 9, wherein the bottom-up brightfield microscope module (130) is provided with at least one tube (138) arranged along the optical path for encapsulating the visible light beam from the illumination unit (131) to the detector unit (134).

11. Microscope device (100) according to any one of claims 8 to 10, wherein the bottom-up brightfield microscope module (130) is provided with at least one pair of dichroic mirrors (137) for reflecting light between the beam splitter (133) and the detector unit (134).

12. Microscope device (100) according to any one of claims 1 to 11, wherein the inside of the microscope housing (110) is coated a cooling surface on at least connecting surfaces of the microscope device (100) and / or the microscope housing (110) is provided with a cooling unit.

13. Microscope device (100) according to any one of claims 1 to 12, wherein the integrated battery (160) is configured to have such capacity that it can power the microscope device (100) for several days, but at least approximately 72 hours, preferably about 1-7 days, i.e. at least 1 Ah, preferably about 5-35 Ah, more preferably about 35-250 Ah capacity.

14. Microscope device (100) according to any one of claims 1 to 13, comprising at least one additional integrated battery (160), each configured to have such capacity that it can power the microscope device (100) for several days such as about 1-7 days.

15. Microscope device (100) according to any one of claims 1 to 14, wherein the microscope device (100) further comprising a non-transparent lid to enclose the examination surface (125) for protecting the samples (201) and excluding external light.

16. Microscope device (100) according to any one of claims 1 to 15, wherein the microscope objective (132) is configured to allow the observation of the samples (201) withsuitable examination magnification such as lx,2x,4x,10x,20x,40x,60x,63x,100x or 150x magnification.

17. Microscope device (100) according to any one of claims 1 to 16, wherein the microscope device (100) is adapted to be placed in an incubator, preferably in a CO2incubator, wherein the incubator allows the placement of a device having at least 15x10x5 cm external dimensions (width x length x height) inside the incubator, preferably the placement of a device having larger external dimensions in any of the three aforementioned dimensions.

18. A microscope system (10), comprising• a microscope device (100) according to any one of claims 1 to 17;• at least one robotic manipulator (300) is mounted on an external surface of an upper part of a microscope housing (110), wherein the at least one robotic manipulator (300) can be controlled wirelessly by a control and management unit (150) and / or an external processing and monitoring system (500).

19. Microscope system (10) according to claim 18, wherein the at least one robotic manipulator (300) is a robotic arm having tilting parts and is configured to allow movement in a direction of x-y-z axes and rotation around the x axis, wherein the at least one robotic manipulator (300) is configured to connect a respective experimental tool such as a pipette, needle, syringe.

20. Microscope system (10) according to any one of claims 18 to 19, wherein the robotic manipulator (300) has an integrated rechargeable battery for power supply.

21. Microscope system (10) according to any one of claims 18 to 19, wherein the robotic manipulator (300) is configured to power by the integrated battery (160) of the microscope device (100).

22. Microscope system (10) according to any one of claim 18 to 21, wherein the two robotic manipulator (300) are mounted on the external surface of the upper part of the microscope housing (110), wherein one of the robotic manipulators (300) is a pipetting injector unit for injecting sperms, and an other of the robotic manipulators (300) is a pipetting needle unit for holding an oocyte.

23. Microscope system (10) according to claim 22 is used to implement Assisted Reproductive Technology (ART) automatically, without human intervention, controlled by the method, performed using deep learning algorithms, machine learning and / or image-processing.

24. Microscope system (10) according to any one of claim 18 to claim 23, wherein the microscope system (10) is adapted to be placed in an incubator, preferably in a CO2incubator, wherein the incubator allows the placement of a device having at least 15x10x5 cm externaldimensions (width x length x height) inside the incubator, preferably the placement of a device having larger external dimensions in any of the three aforementioned dimensions.

25. A method for using a microscope device (100) according to any one of claims 1 to 17 for examining samples (201) illuminated by visible light, wherein said method comprising:• an emitting step of emitting visible light from an illumination unit (131) (S10);• a transmitting step of transmitting emitted light through a microscope objective (132) to a given sample (201) by a beam splitter (133) (S20);• a reflecting step of reflecting transmitted emitted light directly off the given sample (201) (S30); and• a detecting step of detecting light reflected essentially directly from the given sample (201) through the microscope objective (132) and the beam splitter (133) by a detector unit (134) (S40).

26. The method according to claim 25 for using a microscope device (100) according to any one of claims 8 to 17, wherein, after step S10, the method comprising:• at least one focusing step of focusing emitted visible light from the illumination unit (131) along an optical path by at least one lens (136) in connection to any of steps S20-S40, or a combination thereof, and / or• at least one controlling step of controlling the amount of visible light along the optical path by the at least one lens (136) in connection to any of steps S20-S40, or a combination there-of, and / or• at least one guiding step of guiding light along the optical path by at least one tube (138) in connection to any of steps S20-S40, or a combination thereof.

27. The method according to any one of claims 25 to 26 for using a microscope device (100) according to any one of claims 8 to 17, wherein, after step 530, the method comprising:• an additional reflecting step of reflecting visible light between the beam splitter (133) and the detector unit (134) by at least one pair of dichroic mirrors (137).

28. The method according to any one of claims 25 to 27 for using a microscope device(100) according to any one of claims 1 to 17, wherein, after step S40, the method comprising:• an image-capturing step of capturing at least one digital image, preferably a plurality of digital images and / or video, of the detected light reflected directly from the given sample (201) by the detector unit (134) (S50).

29. The method according to any one of claims 25 to 28 for using a microscope device (100) according to any one of claims 1 to 17, wherein:• repeating at least steps S10-S40, preferably steps S10-S50 for each sample (201) to be examined.

30. The method according to any one of claims 19 to 30 for using a microscope device(100) according to any one of claims 1 to 17, wherein, after step S50, the method comprising:• a processing step of processing the captured at least one digital image, preferably a plurality of digital images and / or video (S60); and / or• a storing step of storing the captured and / or the processed at least one digital image, preferably a plurality of digital images and / or video (S70).

31. The method according to claim 30 for using a microscope device (100) according to any one of claims 1 to 17, wherein:• the processing step (S60) comprising at least one of the following: quality improvement of the at least one digital image of the samples (201); confluency calculation of the at least one digital image of biological samples (201); segmentation, detection, counting of given objects of the samples (201) based on at least one digital image of the samples (201); classification of given objects and / or samples (201) based on at least one digital image of the samples (201); statistical analysis of quantitative features of the at least one digital image of the samples (201); or a combination thereof.

32. The method according to any one of claims 30 to 31 for using a microscope device (100) according to any one of claims 1 to 17, wherein:• the processing step (S60) is performed automatically using deep learning algorithms, machine learning and / or image-processing.

33. The method according to any one of claims 30 to 32 for using a microscope device (100) according to any one of claims 1 to 17, wherein:• the storing step (S70) is performed by local storing and / or cloud-based storing.

34. Use of a microscope system (10) according to any one of claims 18 to 24 for remotely controlled examination of each sample (201) illuminated by visible light upward from below using automatic methods for examination.

35. A method for using a microscope system (10) according to any one of claims 22 to 24 for automated implementation of Assisted Reproductive Technology (ART), wherein said method comprising:• providing a first optically transparent container (200) comprising living sperms (S100);• providing a second optically transparent container (200) comprising living oocyte (S200);• automatically detecting and imaging moving sperms in the first container (200) in real time (S300);• automatically analysing digital image(s) and / or video(s) of the detected moving sperms based on predetermined features to select at least one desired sperm for injection (S400);• tracking the at least one selected sperm in real time and collecting the at least one selected sperm using a negative pressure by a pipetting injector unit (S500);• holding the oocyte by a pipetting needle unit (S600);• penetrating the oocyte by the pipetting injector unit to enter the cytoplasm of the oocyte (S700);• collecting a part of the cytoplasm of the oocyte by the pipetting injector unit using a negative pressure (S800);• changing the negative pressure of the pipetting injector unit to positive pressure in order to insert the collected sperm and the collected part of the cytoplasm of the oocyte into the oocyte (S900), and• removing the pipetting injector unit and the pipetting needle unit from the ooctyte (S1000).

36. The method according to claim 35, wherein the method comprising:• before step S600 and / or S1000, confirming step S500 and / or S900 based on digital imaging performed by the microscope system (10) (S510, S910).

37. The method according to any one of claims 35 to 36, wherein the method comprising:• after step S1000, real-time monitoring and qualifying selected features of the fertilized oocyte continuously or at predetermined time based on digital imaging performed by the microscope system (10) (S1100).

38. The method according to any one of claims 35 to 37, wherein the method comprising:• after step S1100, determined the quality of the fertilized oocyte by comparing qualified features of step S1100 with predetermined features (S1200).

39. The method according to any one of claims 35 to 38, wherein the steps of the method are performed using deep learning algorithms, machine learning and / or image-processing.STATEMENT UNDER ARTICLE 19 (1 )Amendment to claim 1 : The light-excluding house is characterized according to the description.The light-excluding house is not obvious for the person skilled in the arts, since the devices available on the market apply a different solution to this problem. This relates to the standard problem in microscopy where the illumination from a lamp in the laboratory and the external ambient natural light (e.g. coming from the window) influences imaging. Our invention offers a solution to this problem with the light-excluding housing which excludes external light from the light path, therefore makes imaging independent of external light, thus a desirable feature / improvement in microscopy, since usually a lid is applied to enclose the microscope in order to exclude light, however, in our solution such a lid is not a necessary element of the microscope but an auxiliary optional element. See also the next point.Amendment to claim 15: The non-transparent lid is characterized according to the description.The non-transparent lid is a typically applied element in microscopy to protect the samples and exclude external light, however, in our proposed solution this element is only optional as an addition to the microscope device, since the light-excluding house performs the exclusion of external light, to which purpose the non-transparent lid contributes and additionally serves a protective purpose.Amendment to claim 23: The phrasing of the claim is clarified according to the description.The automatic manner in which ART is performed is defined according to the description, furthermore, we specify that the automatic implementation of the process is performed by machine learning, deep learning and / or image-processing algorithms.Amendment to claim 29: The missing word “according” added to the expression “according to”.Amendment to claim 30: The missing word “according” added to the expression “according to”.

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