Microscope system and method using immersion liquid

Abstract

A microscope device is provided that can be used to (i) move an immersion objective to a location where a liquid, such as oil or water, can be applied to the immersion objective, and (ii) move the immersion objective with the applied liquid to a sample holder and acquire an image of the sample. The liquid application process can be automated so that the liquid on the objective is replenished as needed. The device can be configured to automatically focus the lens to facilitate scanning and to replace the objective. Other embodiments are also described, including the use of such devices in high content / high throughput scanning.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims Paris Convention priority to and the U.S. benefit of U.S. Provisional Application Nos. 63 / 180693 and 63 / 180694, filed April 28, 2021, the contents of both of which are incorporated herein by reference.

[0002] The present invention relates to the field of precise optical scanning and imaging of samples, and more particularly to biological microscopy using a suitable immersion liquid, such as a stable oil, as a medium between the objective lens and the sample. [Background technology]

[0003] High content microscope systems used for observing biological samples are known in the art. Such microscope systems are usually based on a static and bulky microscope body, containing complex connections to the optical units such as objective turret, illumination unit, filter wheel, shutter, camera, internal optics, etc. To allow scanning of any sample, devices are added to the microscope body that allow the movement of the sample holder in X, Y and Z directions. In the imaging process of these microscope systems, the optical unit remains stationary, while the sample is moved to take images at different locations along the sample.

[0004] Various automated scanning systems have been developed in which the sample and objective lens are moved relative to one another, the objective lens is automatically focused, and scanning is performed without human intervention. Some such systems can also automatically change between different objective lenses; for example, one such system is described in U.S. Patent No. 9,170,412, the contents of which are incorporated herein by reference.

[0005] Although such automated scanning systems are useful for scanning biological materials with objectives operating in an air environment, they are not suitable for immersion microscopy. This is due in particular to the fact that the immersion liquid spreads along the sample plate as the objective and sample move relative to each other, and the immersion liquid needs to be replenished periodically. Although systems have been developed that attempt to replenish the liquid (see, for example, EP 1717628 B1, DE 202017000475 U1, US 7304793 B2, https: / / www.leica-microsystems.com / products / light-microscopes / p / leica-water-immersion-micro-dispenser / , and https: / / www.marzhauser.com / en / products / liquid-immersion.html), such systems have various drawbacks, such as, for example, the weight of the objective increases due to their design, the scanning area is reduced, and the throughput of the microscope system is reduced.

[0006] Therefore, there is a need in the art for a system and method for automated high-content microscopy using immersion liquid. [Brief description of the drawings]

[0007] Embodiments of the present invention will be better understood with reference to the following detailed description and drawings.

[0008] [Figure 1] FIG. 1 is a schematic diagram of one embodiment of a microscope system that can be adapted for use in accordance with an embodiment of the present invention.

[0009] [Diagram 2] FIG. 2 is an isometric view of an apparatus configured and operative for use in accordance with an embodiment of the present invention.

[0010] [Diagram 3]FIG. 3 is a cross-sectional view of an objective lens mounted in an objective lens holder and attached to a kinematic base that can be used in accordance with an embodiment of the present invention.

[0011] [Figure 4] FIG. 4 is an exploded top isometric view of the components of FIG.

[0012] [Diagram 5] FIG. 5 is an exploded isometric view from below of the components of FIG.

[0013] [Figure 6] FIG. 6 is an isometric view of the kinematics of FIGS.

[0014] [Figure 7] FIG. 7 shows how the lower portion of the objective lens unit of FIGS. 3-5 fits into the V-shaped groove of the kinematic base of FIG.

[0015] [Figure 8] FIG. 8 is an isometric view from above showing the kinematic base with the objective lens mounted in the objective lens holder and the magnet attached thereto. [Figure 9] FIG. 9 is an isometric view from below showing the kinematic base with the objective lens mounted in the objective lens holder and the magnet attached thereto.

[0016] [Figure 10-11] 10 and 11 show how objective lenses can be stored and replaced according to an embodiment of the present invention.

[0017] [Figure 12] FIG. 12 is a partially exploded perspective view of an oil-loaded subassembly having an immersion oil cartridge positioned above a lens exchange subassembly in accordance with an embodiment of the present invention.

[0018] [Figure 13] FIG. 13 is an exploded view of an oil loading subassembly according to an embodiment of the present invention.

[0019] [Figure 14A-14B] 14A and 14B are perspective and top elevational views, respectively, of the oil-loaded subassembly of FIG. 13 when assembled. [Fig. 14C-14D] 14C and 14D are plan side and cross-sectional views, respectively, of the oil-loaded subassembly of FIG. 13 when assembled.

[0020] [Figure 15A-15B] 15A and 15B are cross-sectional views illustrating the steps of inserting an immersion oil cartridge into the oil-loaded subassembly of FIGS. 13, 14A, 14B, 14C, and 14D. [Figure 15C] FIG. 15C is a cross-sectional view illustrating the step of inserting an immersion oil cartridge into the oil-loaded subassembly of FIGS. 13, 14A, 14B, 14C, and 14D.

[0021] [Figure 16A-16B] 16A and 16B are cross-sectional views showing oil flow paths within the oil loaded subassembly of FIGS. 13, 14A, 14B, 14C, 14D, 15A, 15B and 15C.

[0022] [Figure 17] FIG. 17 is a perspective view of the lens exchange assembly of FIGS. 1-11 and the oil loading subassembly of FIGS. 12, 13, 14A, 14B, 14C and 14D, including the objective lens in the immersion oil loading position of the lens exchange subassembly.

[0023] [Figure 18A-18B] 18A and 18B are front and side views, respectively, showing the loading of immersion oil into the objective lens in the structure of FIG.

[0024] [Figure 19A] FIG. 19A shows the spreading of immersion oil as the objective lens moves relative to the observation surface. [Figure 19B] FIG. 19B shows the spreading of the immersion oil as the objective lens moves relative to the observation surface. [Figure 19C] FIG. 19C shows the spreading of the immersion oil as the objective lens moves relative to the observation surface.

[0025] [Figure 20] FIG. 20 is a flow chart outlining a method for scanning a sample with an oil immersion lens and reloading the immersion oil on the oil immersion lens, according to an embodiment of the present invention.

[0026] [Figure 21A] FIG. 21A is a flow chart outlining a method for automatically focusing an oil-loaded oil immersion lens on a biological sample, according to an embodiment of the present invention. [Figure 21B] FIG. 21B is a flow chart outlining a method for automatically focusing an oil-loaded oil immersion lens on a biological sample, according to an embodiment of the present invention.

[0027] [Figure 22A] FIG. 22A is a flow chart outlining a method for scanning a sample, according to an embodiment of the present invention. [Figure 22B] FIG. 22B is a flow chart outlining a method for scanning a sample, according to an embodiment of the present invention. [Figure 23] FIG. 23 is a flow chart outlining a method for scanning a sample according to an embodiment of the present invention.

[0028] [Figure 24] FIG. 24 is a perspective view of an oil loading subassembly according to another embodiment of the present invention.

[0029] [Diagram 25]FIG. 25 is a cross-sectional view of an immersion oil cartridge according to another embodiment of the present invention.

[0030] [Figure 26A-26B] 26A and 26B are cross-sectional views of the step of inserting the immersion oil cartridge of FIG. 25 into the oil loading subassembly of FIG. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0031] Reference is now made to FIG. 1, which is a block diagram illustrating a schematic of an apparatus 10 adapted and operative for use in accordance with an embodiment of the present invention. The apparatus 10 includes a holder 12, which may be configured to hold a sample plate 13, e.g., a 6, 24, 96, 384, or 1536 well plate, typically used to hold biological samples, having a lower surface 13A and an upper surface 13B. The plate contains one or more samples to be observed, as is known in the art. The holder 12 may also be configured to hold a microscope slide, a Petri dish, or other substrate having a bottom transparent to electromagnetic radiation of one or more wavelengths of interest. For reference, the sample plate 13, which is not itself part of the apparatus, resides in an XY plane, and via its lower surface 13A, the samples contained therein face the components of the apparatus 10 described below.

[0032] The turretless objective lens, i.e. the single objective lens 14, which is part of the linear XYZ scanner 16, is arranged such that the lens faces the sample holder (and faces the underside 13A of the sample plate 13, if present), and the optical axis of the objective lens 14 is along the Z axis relative to the sample holder. By "linear XYZ scanner" we mean a mechanism that is configured and operative to move the objective lens 14 in three mutually orthogonal directions, the "Z" direction being used to indicate movement along the optical axis. Such scanners are known in the art, for example from Israel Patent No. 143836, entitled "Compact Linear Scanner System", filed on June 19, 2001, or U.S. Patent No. 6,850,362, the contents of both of which are incorporated herein by reference. For simplicity, only some of the components of the XYZ scanner 16 are shown in FIG. 1, it being understood that a more detailed description of said components is provided below. 1 include mirror 18 and mirror 20, which cooperate to redirect light along the optical axis of objective lens 14 and to reflect light from illumination unit 22 and light from autofocus unit 24 through objective lens 14, such as when the XYZ scanner is arranged to operate in an inverted microscope configuration. Mirror 18 is configured and operative to move in the X and Y directions with objective lens 14, and mirror 20 is configured and operative to move in the X direction with objective lens 14 and mirror 18 to allow light to move along the optical axis of objective lens 14. Mirror 18 and mirror 20 also reflect light received from the sample, including reflected incident light from illumination unit 22 or autofocus unit 24, or light resulting from fluorescence of the sample, away from the sample along the optical axis of objective lens 14.

[0033] As shown in Figure 1, the apparatus 10 also includes an autofocus unit 24. Autofocus units for focusing non-immersion objectives, i.e., objectives without oil or water, are known in the art as such. When used as shown in Figure 1, the autofocus unit is preferably an autofocus unit usable in high resolution, high throughput microscopy applications, such as the autofocus unit and method described in PCT Patent Publication WO 03 / 077008, filed March 13, 2003, entitled "Autofocus Method and Device," or U.S. Patent No. 7,109,459 of the same title, the contents of both of which are incorporated herein by reference.

[0034] In FIG. 1, the autofocus unit 24 emits a laser light beam at a wavelength, e.g., 635 nm, that is transmitted by the medium carrying one or more samples, which is reflected by a beam splitting device (dichroic filter 26) onto the optical axis of the objective lens 14, through the objective lens 14, off mirrors 18 and 20, and onto the medium in the sample holder. It is then reflected back along the same path, off the dichroic filter 26, and back to the autofocus unit, where it is sensed by a sensor (not shown) and a controller (not shown) programmed to adjust the focus of the objective lens along the Z axis as necessary. When the autofocus unit 24 is used with samples containing fluorescent labels, the wavelength of the autofocus light may be selected so as not to elicit a fluorescent response of the sample, although this is generally not critical, since the autofocus process is typically completed before the image capture process begins. When using, e.g., oil immersion lenses, a similar process can be employed, but with certain modifications to account for the use of oil, as described in more detail below.

[0035] Also shown in FIG. 1 is an illumination unit 22. The illumination unit 22 includes an illumination source (not shown), such as a mercury lamp, an LED lamp, a laser, or other suitable radiation source. Optionally, the illumination unit 22 includes collimating optics. If the sample includes one or more fluorescent probes or the like, a suitable beam splitting device is arranged to reflect the excitation light onto the optical axis of the objective lens 14. This beam splitting device may be a quad filter 28 that reflects light of the excitation wavelength generated by the illumination unit, but passes light of other wavelengths, in particular light generated by the fluorescence of the fluorescent probes in the sample. It will be appreciated that the illumination unit 22 may be configured to generate electromagnetic radiation of multiple wavelengths, or multiple illumination units may be employed to generate electromagnetic radiation of multiple wavelengths, if, for example, multiple fluorescent probes are employed in the sample being observed, then a suitable beam splitting device is also employed to ensure reflection of the excitation light onto the optical axis of the objective lens 14 and passage of light of wavelengths generated by the fluorescent probes in the sample, e.g. light of wavelengths of interest. Also, although FIG. 1 shows the autofocus unit 24 positioned between the illumination unit 22 and the objective lens 14, it will be appreciated that in principle the positions of the illumination unit 22 and the autofocus unit 24 could be reversed, provided that optics are provided to ensure that only light of the wavelengths of interest passes through the image capture device 30.

[0036] As shown in Figure 1, light reflected from or emitted by the sample (by fluorescence), or transmitted through the sample if the sample is illuminated from the top surface 13b, travels along the optical axis of the objective lens 14 and passes through a dichroic filter 26 and a quad filter 28 before being detected by one or more image capture devices 30. Figure 1 shows an arrangement in which there are three such image capture devices, namely three CCD cameras 30, 30' and 30", where after passing through the beam splitting devices 26 and 28, the light passes through a tube lens 32, reflects off a folding mirror 34, is split by an RGB prism 36, and then passes through emission filters 38, 38' and 38", which filter out all light except the emission bands of the fluorescent probes in the sample, before entering the CCD cameras. In FIG. 1, emission filter 38 passes red light, emission filter 38' passes green light, and emission filter 38" passes blue light. It will be appreciated that prism 36 may be other than an RGB prism and that filters 38, 38', 38" may result in filtering different wavelength ranges.

[0037] The operation of the system shown in Figure 1 is controlled by one or more controllers (not shown) collectively programmed to control the operation of the autofocus unit, the illumination unit, and the XYZ scanner. An analysis unit (not shown) for analyzing images obtained by the image capture device may also be provided, which may be part of the one or more controllers or may be a separate unit and may be configured to provide feedback to the one or more controllers. Furthermore, as will be appreciated by those skilled in the art, input and / or output devices such as keyboards, optical or magnetic storage readers and / or writers, printers, and display devices such as plasma displays or LCD displays, as well as storage devices may also be provided.

[0038] Those skilled in the art will appreciate that variations on the arrangement shown in Figure 1 may be employed in accordance with embodiments of the present invention. At least one such variation is described below for implementing an autofocus method when using an oil immersion lens.

[0039] An XYZ scanner such as that shown in Figure 1 can be incorporated into an apparatus according to an embodiment of the present invention. See, for example, Figure 2, which illustrates in isometric view a portion of an apparatus 810 configured and operative to be adapted for use according to an embodiment of the present invention. The apparatus 810 includes a sample holder 812 that holds a 96-well sample plate 813. The sample plate 813 lies in a plane perpendicular to the optical axis of an objective lens 814, which is part of a scanner 816 and is movable in three mutually orthogonal directions, namely the X, Y and Z directions. The scanner 816 includes a mirror 818 mounted on a mirror mount 819 movable in the X and Y directions, and a mirror 820 mounted on a mirror mount 821 movable in the X direction.

[0040] FIG. 2 also shows an autofocus unit 824 and a mirror 827 that directs light from the autofocus unit to a dichroic filter 826 and vice versa. The dichroic filter 826 directs light from the autofocus unit 824 to the optical axis of the objective lens 814. Also shown is a portion of the illumination unit 822, including a bundle of fiber optic cables 822a and collimation optics 822b, and a quad filter 828 positioned to reflect light from the illumination unit to the optical axis of the objective lens 814. Light reflected from the sample or caused by fluorescence in the sample plate (e.g., fluorescence of a fluorescent probe) that is not filtered by the quad filter 828 is focused by a tube lens 832 and reflected by a fold mirror 834 to the camera 830. A filter wheel 837 includes filters 838, 838′, and 838″ that can be selected to filter the light entering the camera 830.

[0041] According to some embodiments of the invention, the apparatus includes a coupling mechanism to facilitate the exchange of the objective lens, although it will be understood that the mechanism may be employed in other optical instruments. Reference is now made to Figures 3-9, which illustrate one embodiment of such a mechanism. As shown in Figures 3, 8 and 9, an objective lens 1010 is permanently attached to an objective lens base 1020. The objective lens 1010 and the objective lens base 1020 together form an objective lens unit 1060. The objective lens unit 1060 is attached to a kinematic base 1040. According to some embodiments of the invention, the kinematic base 1040 may be permanently attached to the top of a Z-axis component of an XYZ scanner, such that an objective unit 1060, including an objective lens such as lens 338, may rest thereon, as will now be described, or alternatively, the kinematic base may be formed as part of the top of the Z-axis component of an XYZ scanner.

[0042] As will be explained, the mounting between the objective lens base 1020 and the kinematic base 1040 shown in the figures uses a specific kinematic mount configuration that provides a positioning accuracy in the range of 50 nanometers or better. The objective lens base 1020 includes a number of coupling balls 1030 (three such balls are shown in Figures 3-9) that are set in position and then machined to ensure high precision in the spatial position of the coupling balls 1030 relative to the optical axis of the objective lens 1010. The coupling balls 1030 have high stiffness (e.g., hardness of 53-58 RC SS or better) and a suitable diameter, e.g., 3-3.5 mm. Each of the coupling balls 1030 is held in a predetermined position in one of the holes 1020a formed in the lower surface of the objective lens base 1020 such that 30-40% of the ball's diameter protrudes downward from the lower surface of the objective lens base 1020. The holes 1020a may extend only partially through the objective lens base 1020, thereby forming a cylinder closed at one end, or may extend completely through the bottom of the objective lens base 1020. Each of the holes 1020a has an interference diameter tolerance with a coupling ball 1030 disposed therein, thereby holding the ball securely in place. The objective lens base 1020 is made of ferromagnetic steel, such as 17-4PH.

[0043] As shown in the figure, the kinematic base 1040 is generally annular ring-shaped with several indentations in the form of V-shaped grooves 1040a on its upper side. The spacing between the coupling balls 1030 and the grooves 1040a is such that the coupling balls fit into three of the grooves, as shown in simplified form in FIG. 7. The kinematic base 1040 may be constructed of high performance ferromagnetic steel, such as 17-4PH, that has been heat treated to a surface hardness of 39RC or higher so that the kinematic base 1040 maintains its mounting accuracy during repeated movements of mounting and dismounting the lens unit 1060. To ensure that the kinematic base 1040 has the required surface hardness, the following manufacturing process is observed: (a) manufacture the part to the final dimensions leaving 50 microns for the grinding process of the final grooves 1040a, (b) perform a heat hardening process, and (c) grind the V-shaped grooves 1040a to the final dimensions.

[0044] As shown, the kinematic base 1040 is formed with three holes 1040b at approximately equal intervals around its circumference. A magnet 1050 is inserted and glued into each hole 1040b. The magnet 1050 creates a magnetic adhesion force between the objective lens unit 1060 when the parts are in close proximity. This adhesion force balances the adhesion force applied to the kinematic coupling to place the objective lens in a predetermined position and maintain the objective lens unit 1060 in a predetermined position while the optical system moves at high acceleration. It will be understood that the magnet does not necessarily have to contact the objective lens base 1060, or even to protrude through the holes 1040b, as seen, for example, in FIG. 9. Thus, when such a magnet is described herein as being "mounted" in a surface facing a ferromagnetic surface, the magnet may protrude from the surface "mounted", be embedded in the surface "mounted", or be below the surface "mounted", since the magnetic attraction acts without the need for direct physical contact.

[0045] Although a particular embodiment of the coupling mechanism is shown in Figures 3-9, it will be understood that variations on what is shown therein are possible. This is because the collective effect of the balls 1030 and grooves 1040a is both to accurately position the objective unit 1060 in the XY plane and to restrict the movement of the objective unit 1060 in the XY plane and in the negative Z direction, while the addition of the magnetic force restricts the movement of the objective unit in the positive Z direction, and these effects are in principle realizable with other arrangements. Thus, for example, more or fewer holes 1040b, and correspondingly more or fewer magnets, can be used, magnets can be located in the bottom of the lens objective unit and in the kinematic base, or magnets can be located only in the bottom of the lens objective unit. Similarly, the positions of the recess and ball can be reversed, with a recess such as a groove on the bottom surface of the objective unit 1060 and a ball protruding from it on the top surface of the kinematic base, or a recess and ball can be provided on both the objective unit and the kinematic base, or two grooves and a protrusion can be provided on the kinematic base and two corresponding protrusions and corresponding grooves can be provided on the bottom of the objective unit 1060.

[0046] Additionally, the recesses may be shaped other than V-shaped grooves; for example, one or more of the recesses may be shaped as a well that provides three contact points for the ball 1030 resting therein, rather than two as in the V-shaped groove. Such a well may be combined with a single V-shaped groove, a kinematic base surface, and a suitably positioned magnet of sufficient strength to achieve the same effect as three V-shaped grooves. Additionally, protrusions other than ball-shaped ones may be used to fit into the recesses. Thus, for example, one skilled in the art will appreciate that while the figures show multiple balls 1030 held in holes, other structural arrangements are possible, for example, round-headed nails may be used.

[0047] The relative positions of the recesses may also be different from those shown in Figures 3-9. The recesses may be arranged so that there is only one way for the protrusion from the opposing piece to fit therein, thus providing the only way for the objective unit to be set in place on the kinematic base. Alternatively, three protrusions may be used (to ensure that the piece with the protrusions fits on a flat surface), e.g. as described above with respect to the objective lens unit. However, instead of three or six V-shaped grooves on the opposing side (e.g. as described above with respect to the kinematic base), a larger number of equally spaced radially oriented V-shaped grooves (e.g. nine or twelve) may be used to facilitate positioning of the lens unit, e.g. when used in an objective lens exchanger, such as the lens exchanger described below.

[0048] Additionally, although Figures 3-9 show the objective unit 1060 formed from the objective lens 1010 and the objective lens base 1020, it will be understood that the objective lens 1010 may be formed in a manner that eliminates the need for the objective lens base 1020, for example, if the bottom of the objective lens 1010 is made from a ferromagnetic material and machined to have a coupling ball 1030 protruding therefrom. It will be understood that wherever in this specification or claims an objective lens is described as having a surface "associated with it," such a surface may be a surface of the objective lens assembly itself, or may be a surface of a lens holder or base to which the objective lens is attached, such as shown in Figures 3-9.

[0049] The coupling mechanism described above allows the objective lens to be repeatedly inserted and removed from the optical device with sufficient precision to perform high-precision observations with the objective lens. The mechanism described herein thus facilitates the use of multiple objective lenses in the optical device, since the lenses can be interchanged. In the case of the device shown in the figures, this can be achieved without burdening the XYZ scanner with the weight of the entire objective lens, facilitating higher accelerations and shorter settling times than could be achieved if the XYZ scanner were to bear the weight of a complete set of objective lenses. Alternatively, the objective lenses may be stored elsewhere in the device and replaced as required when a change in magnification is desired. Additionally, turrets carrying multiple objective lenses are known, but the objective lenses in such turrets are aligned with the focal path along the Z axis for the objective lenses that are used, while the objective lenses that are not used are positioned so that they are precisely aligned with the Z axis, so that replacing one oil immersion lens with another oil immersion lens will result in loss of oil as the turret rotates and moves the lenses to angles away from the vertical. Thus, as shown in Figs. 10 and 11, the lens exchange subassembly 1000 includes a magazine 1080 that holds a number of objective units 1060 in a number of stations 1070. Each objective unit 1060 in the magazine 1080 is substantially aligned with the Z-axis of the XYZ scanner 1090 and substantially perpendicular to the plane of the kinematic base 1040 that is attached to the top of the XYZ scanner 1090, and furthermore, a ball coupling 1030 protruding from the bottom of each objective unit 1060 is arranged to engage with a V-shaped groove 1040a of the kinematic base when the XYZ scanner is raised to contact the objective unit, as described below. Each station 1070 includes a pair of arms 1072, such that each pair of adjacent arms can hold an objective unit 1060. As shown in Figs. 10 and 11, the magazine 1080 holds three objective units 1060, but in principle the optical device may be designed to hold many more such objective units.

[0050] To explain how the coupling mechanism can be used to exchange objective lenses, assume that the scanner 1090 is initially empty of objective units and that at least one objective unit 1060 is loaded in one station 1070 of the magazine 1080, as shown in FIG. 10. The scanner 1090 is first moved to a Z position low enough to move under the selected objective unit 1060. The XYZ stage is then moved in the XY plane to a position where the optical axis of the selected objective lens resting in the station 1070 is approximately aligned with the required optical axis of the objective lens placed for use on the XYZ scanner 1090. The Z stage is then moved upwards to contact the bottom of the selected objective lens unit 1060. As a result, the coupling ball 1030 comes to rest in the groove 1040a. The magnet 1050 comes close to the bottom of the objective unit 1060, which is made of a ferromagnetic material and is therefore attracted to the magnet, so that the coupling ball 1030 settles in the groove and is held there. As shown in Figures 3-11, if the coupling balls and V-shaped grooves are machined with sufficient precision (e.g., within a tolerance of 50 nm), with three coupling balls spaced approximately 120° apart and oriented radially from the center of the kinematic base and the V-shaped grooves into which the coupling balls fit, the optical axis of the objective lens is set with sufficient precision to allow the objective lens to be used without further calibration. The Z stage is moved further upwards, lifting the objective unit 1060 sufficiently off the arm 1072. The XY stage may then be moved out of station 1070. The optical system is now ready to operate using the selected objective lens.

[0051] A similar process is repeated when changing from the first objective lens to the second objective lens. The scanner is moved to the open station 1070 and operates in reverse order to place the first objective lens unit 1060 at station 1070. The XYZ stage then moves to another station in the magazine 1080 and loads the second objective lens in a manner similar to that described above for the first objective lens.

[0052] It will be appreciated that the movement of the XYZ scanner may be automated, by control of suitable motors and a microprocessor programmed for such purpose.

[0053] Also, while Figures 10 and 11 show an XYZ optical scanner 1090 as a device for facilitating movement of objective lenses in and out of the optical system, it will be understood that the methods described are not limited to use with an XYZ optical scanner, but may be used in any system having moving optical elements controlled by a combination of motors, encoders, sensors, servo controllers or other automated elements. Additionally, in the event that the moving optics cannot reach all of the objective units, a secondary motion system (not shown) may be used to move the magazine 1080 such that the particular lens in question is in a position reachable by the optics.

[0054] The lens exchange subassembly 1000 described herein with respect to Figures 3-11 is particularly advantageous for performing automated high content or high throughput screening of biological samples using an immersion objective lens. However, it will be appreciated that in some embodiments of the invention, an apparatus or device does not have a lens exchange subassembly, uses only a single objective lens, and the apparatus or device is sufficiently large in the X and / or Y directions. (a) the objective lens can be moved away from the sample holder along the Z axis while maintaining its 3D orientation (in terms of pitch, roll, yaw, etc., 3D orientation), can be moved in the XY plane to an oil refill position, and then after refilling the oil, can be moved back to the same location in the XY plane so that scanning can continue from where it was interrupted after refocusing in the Z direction; (b) while maintaining its orientation in the XYZ coordinate space, the sample holder can be moved from its rest position for scanning to facilitate refilling the oil, and then moved back to the same location where the scanning was interrupted so that scanning can continue from where it was interrupted after refocusing the objective lens in the Z direction; or (c) both (a) and (b) can be combined.

[0055] One of the challenges of immersion scanning of biological samples is the need to periodically replenish the liquid on the objective. This is particularly important when scanning in an environment where the objective moves relative to the sample while in close proximity to or contact with the surface of the sample holder (e.g., multi-well plate, petri dish, specimen-bearing slide), as relative motion between the objective and the sample holder can cause the immersion liquid to spread to a point where the layer of immersion liquid is insufficient to properly observe the sample. Scanning is therefore limited to the distance the objective can travel before the immersion liquid absolutely needs to be replenished.

[0056] As noted in the background discussion above, prior art systems for refilling oil suffer from various drawbacks. For example, they require additional items to be placed on the objective lens itself, which increases the weight and effective size of the objective lens assembly. This reduces the area of ​​the sample that can be scanned and can limit how close the lens itself can be to the sample holder. Furthermore, if this is done manually, a human user must stop operation of the system and manually refill the objective lens with immersion liquid. Both are done manually or with the assistance of specially designed devices. This significantly slows down operation of the system and requires regular attention from a human operator. As such, it is also error-prone. Furthermore, the need to manually refill immersion liquid limits or prevents the implementation of large screening sessions that scan many locations located at long distances from each other.

[0057] Another challenge with manually applying immersion oil to the objective lens is that the human operator may apply too little oil, resulting in frequent shutdowns of the microscope system. Alternatively, the human operator may apply too much oil, causing the oil to spill out of the objective unit during the movement of the objective lens, for example, where the human operator applies immersion fluid to the scanning location. A further problem is that in order to avoid moving the lens from its focused position, it was previously necessary to refill the oil while the objective lens was placed adjacent to the surface of the sample holder (e.g., close to the bottom of a multi-well plate), leaving little room to maneuver in order to refill the lens with oil.

[0058] According to an embodiment of the invention, as described below with respect to Figures 12-19B, a lens exchange subassembly forming part of a microscope system can automatically move the objective lens and ensures that there is a fixed position where the objective lens is not located under the sample. The existence of such a fixed position facilitates the construction of the inventive immersion liquid loading subassembly described below in this specification directly above a predetermined position in the magazine of the lens exchange assembly. As described below, according to an embodiment of the invention, in use, the immersion objective lens is periodically moved to a predetermined position in the magazine of the lens exchange assembly and the objective lens is automatically returned to the scanning position by the system to continue the scanning process after oil is automatically applied to the objective lens from the liquid loading subassembly.

[0059] It will be understood that the immersion liquid subassembly and process of its use described below are suitable for use with any suitable immersion liquid, such as synthetic hydrocarbon-based oil, water, glycerol, silicon oil, etc., and the scope of the disclosure herein should be interpreted to include all types of immersion liquid suitable for microscopy, and in particular for microscopy of biological samples. However, for the sake of brevity and clarity, the following disclosure relates exclusively to synthetic hydrocarbon-based immersion oils, i.e., oil objective lenses, immersion oil loading, and the like.

[0060] Reference is now made to FIG. 12, which is a partially exploded view of an oil loading subassembly 200 having an immersion oil cartridge 300 disposed above a lens exchange subassembly 400, in accordance with an embodiment of the present invention. The lens exchange subassembly 400 is substantially similar in structure and operation to the lens exchange subassembly 1000 described herein above. As shown, the lens exchange subassembly 400 includes a lens holding magazine 410 having a plurality of positions, shown here as three positions, for holding objective lenses. The leftmost position 412 of the magazine 410 is shown as temporarily housing an oil immersion objective lens unit 500 similar to the objective lens units described herein with respect to FIGS. 3-9. The leftmost position 412 is predefined to be an oil loading position such that an oil outlet of the oil loading subassembly 200 is disposed above the oil loading position 412, in a position to drip oil onto the objective lens of the objective unit 500. An immersion oil cartridge 300, described in more detail herein, is adapted to be positioned above and inserted into a corresponding well of the oil loading subassembly 200, as described herein. It will be appreciated that since position 412 is reserved for oil loading, the lens exchange subassembly 400 as shown may perform an exchange between two lenses that may be stored in two other positions to the right of position 412. It will also be appreciated that the lens exchange subassembly 400 may be formed with additional positions to facilitate an exchange between more lenses. Alternatively, as mentioned above, in principle, there may be no lens exchange subassembly, for example if there is sufficient space in the device to provide an oil loading position far enough away from the sample so as not to limit the scanning to only a portion of the sample.

[0061] The oil loading position 412 is selected to always be outside the scan path of the lens that scans the sample.

[0062] In some embodiments, the oil loading position 412 of the lens exchange subassembly 400 may include a weighing mechanism to determine the weight of the objective unit 500 when the objective unit 500 is placed in that position. For example, the weighing mechanism can obtain a baseline weight or perform a tare when the objective unit 500 is placed in the oil loading position 412. In this manner, the weighing mechanism can determine, for example, a change in weight of the objective unit after application of immersion oil. A controller associated with the weighing mechanism can determine, based on the characteristics of the immersion oil, how much immersion oil has been applied to the objective unit 500 and whether additional application of oil is required.

[0063] The weighing mechanism may be any suitable mechanism for weighing the objective unit 500. In some embodiments, the weighing mechanism may include a flexible leaf 420 attached to the frame structure 402 of the lens exchange subassembly 400. A pair of flexible arms 422 extend from the leaf 420 and are arranged around the objective lens unit 500 such that the objective lens unit 500 rests on the arms 422 when the objective lens unit is in position 412. Attached to one or more surfaces of each arm 422, for example in the free area of ​​the arm, but in some embodiments near the leaf 420 and / or near the frame structure 402, are thin, flat strain gauges (not explicitly shown). Each of the strain gauges is electrically connected (e.g., by thin wires not shown) to a thin, flat electronic card (not explicitly shown) located below the arm 422. The electronic card(s) may be arranged to minimize the length of the connection between the strain gauges and the card(s). The electronic card(s) are electrically coupled to a processor (not shown). It will be appreciated that the use of the strain gauges and the electronic card allows correlation of the deflection of the arm 422 with the change in electrical resistance of a circuit, measured for example using a Wheatstone bridge, also arranged on the electronic card, to calculate the change in the weight of the objective unit 500, i.e. the mass of immersion oil applied to the objective lens. Thus, the arm 422 together with the strain gauges forms a signal provider for providing a signal indicative of the amount of immersion oil applied to the objective lens unit. If the density of the immersion oil is known, it is easier to calculate the amount of immersion oil to be added. For example, the amount of immersion oil applied to the objective lens can be calculated repeatedly each time immersion oil is applied to the objective lens, and thus it is possible to identify in real time whether a sufficient amount of immersion oil has been applied. Thus, a controller associated with the weighing mechanism may be functionally associated with the oil pump of the oil loading subassembly 200 described later in this specification, such that the operation of the pump for applying immersion oil is started and stopped based on information related to the weight of the objective unit.

[0064] In some embodiments, an overflow tray (not explicitly shown) may be located below the magazine 410, or at least below the oil loading position 412, to collect any overflow of immersion oil that may have dripped from the oil loading subassembly 200 onto the objective lens unit 500, or that may have spilled out while the objective lens is not placed in the oil loading position. It will also be appreciated that the presence of a holder for the lens, such as the objective lens base 1020 of Figure 5, may provide a trap for oil if it spills out of the objective lens.

[0065] Alternatively, the amount of oil added can be calculated by calibrating the average amount of oil per drop released from the oil loading subassembly, and the number of drops applied to the lens can be counted to calculate the amount of oil added, for example, by a light beam connected to a sensor that provides a signal each time a drop breaks the beam, or by a camera coupled to an automated image analysis program that detects and counts the falling drops of oil. Alternatively, each time the oil on the objective lens is replenished, the amount of oil removed from the oil reservoir can be calculated, and the amount of oil applied to the lens calculated, for example, by measuring the change in weight in the reservoir.

[0066] Whether the amount of oil added is determined by direct weight measurement or by counting the number of drops, the total amount of oil added, and therefore the total amount of oil remaining in the assembly for further application to the lens, can be calculated and optionally displayed on a screen or other output device.

[0067] Reference is now made to FIG. 13, which is an exploded view of an oil-loaded subassembly 200 according to an embodiment of the present invention, and to FIGS. 14A, 14B, 14C, and 14D, which are a perspective view, a plan front view, a plan side view, and a cross-sectional view, respectively, of the oil-loaded assembly 200 when constructed and having an immersion oil cartridge 300 disposed therein. Such a cartridge may be made of any suitable material that does not significantly interact with or lead to deterioration of the oil or other liquid contained therein. In the embodiment shown in FIGS. 13-18B, the cartridge is preferably made of a metal, such as aluminum. In the embodiment shown in FIGS. 24-26B, the cartridge is preferably made of plastic.

[0068] As shown, the oil loading subassembly 200 includes a body portion 210 having a central oil sump 212 in fluid communication with an oil viewing opening 214, and a pump seat hole 216 terminating in a pump inlet conduit 217 (see FIG. 14D). As seen in FIG. 14D, an oil flow conduit 218 extends from the base of the oil sump 212 and adjacent the pump inlet conduit 217 and terminates at an end of the body portion 210, the oil flow conduit being sealed by a stopper 220. An observation window 222 is disposed within and seals the oil viewing opening 214. As can be clearly seen in FIG. 14B, the observation window 222 includes a generally circular transparent portion 224, allowing an operator to observe the oil level in the oil loading subassembly 200 and to determine when the immersion oil cartridge 300 should be replaced, as described in further detail herein. In a variation of this, the window may have a cover to prevent light from entering through the window when the user is not directly viewing the oil level, or the oil loading subassembly 200 may have neither the viewing opening 214 nor the window 222.

[0069] The cartridge piercing element 230 is placed in the central oil sump 212 and attached thereto by fasteners 232, such as screws or bolts. However, the fasteners 232 may be replaced by any other suitable attachment mechanism, such as soldering, adhesives, etc. The cartridge piercing element 230 includes a base 234 and a piercing pin 236, having a hollow channel 238 extending longitudinally therethrough, the channel including a hole 239 (see FIGS. 15B-15C) and terminating in a sharp tip. As shown in FIGS. 14A-14D, when the immersion oil cartridge 300 is placed in the central oil sump 212, the hollow channel 238 is in fluid communication with the central oil sump 212 and also in fluid communication with the oil flow conduit 218 (see FIGS. 14D and 16A).

[0070] A diaphragm pump 250 is positioned within the pump seat hole 216 such that its inlet 252 fits within the pump inlet conduit 217 and its outlet conduit 254 extends downwardly from the body portion 210. The diaphragm pump 250 is adapted to draw oil from the oil flow conduit 218, through the outlet conduit 254 and onto an article (in use, an immersion oil objective) positioned below the outlet conduit.

[0071] The oil-loading subassembly 200 further includes a base mount 260 attached to the bottom side of the body portion 210 above the lens exchange subassembly 400 and adapted for mounting the oil-loading subassembly 200 to other components of the microscope system. A back mount 265 is attached to the body portion 210 behind the central oil sump 212. Both the base mount 260 and the back mount 265 may be connected to the body portion 210 by fasteners 268, such as screws, bolts, etc. However, any other suitable connection method, such as gluing, soldering, etc., is considered to be within the scope of the present invention.

[0072] Reference is now made to Figures 15A, 15B and 15C, which are cross-sectional views of an insertion step of the immersion oil cartridge 300 into the oil-loading subassembly 200. As seen in Figure 15A, the immersion oil cartridge 300 includes a housing 301 formed of a generally cylindrical wall 302 defining a hollow 304 having a first inner diameter and terminating in a lip 306. At one end thereof, distal to the lip 306, the wall 302 extends to a generally transverse shoulder 310 which narrows the inner diameter of the wall to form a generally cylindrical hollow neck 312 having a second inner diameter. The second inner diameter of the neck 312 may be smaller than the first inner diameter of the wall 302. At the end of the neck 312, distal to the shoulder 310, the wall of the neck 312 reduces in thickness to define a first chamber having a third inner diameter greater than the second inner diameter. The first chamber includes an annular shoulder 316 and terminates in a lip 318. On its outer surface, the neck includes snap-fit ​​engagement projections and / or grooves 319 adapted for snap-fit ​​engagement with corresponding grooves and / or projections 219 on the inner circumference of the central sump 212.

[0073] A generally annular lateral wall 320 is disposed between the lip 306 and the neck 312 and extends radially inward from the wall 302 at a location substantially closer to the lip 306 than the neck 312. The annular lateral wall 320 terminates radially inward at a cowl portion 322 that is substantially concentric with the wall 302. The volume between the annular lateral wall 320 and the lip 306 defines a second chamber. A fluid flow path exists between the first and second chambers through the hollow defined by the neck 312, the hollow defined by the wall 302, and the hollow of the cowl portion 322.

[0074] A puncture piston 330 is disposed within the housing 301. The puncture piston 330 includes a generally circular base 332 from which extends a central shaft 334 that terminates in a pointed edge 336 that may include one or more points. At an upper portion of the shaft 334 near the edge 336, the shaft 334 defines a hollow 338 and includes one or more holes 340 that connect the hollow 338 with the environment surrounding the shaft 334. The remainder of the shaft 334 is solid.

[0075] In an initial closed operating orientation of the immersion oil cartridge 300, illustrated in FIG. 15A, a first seal 350, which may be a material inert to oil, such as nylon or aluminum foil, is disposed within the first chamber and engages the annular shoulder 316. A base 332 of the piercing piston 330 is disposed within the hollow of the neck 312 and engages the first seal 350, such that a shaft 334 extends through the hollow defined by the neck 312, the wall 302, and the cowl 322. A sharp edge 336 may not extend beyond the end of the annular lateral wall 320, but may be flush therewith. A second seal 352, which may be a material inert to oil, such as nylon or aluminum foil, is disposed within the second chamber and engages the lateral wall 320. Thus, in the closed operating orientation of the immersion oil cartridge 300, the cartridge is sealed and oil is disposed therein (for clarity, the oil is not shown in FIG. 15A).

[0076] To use the immersion oil in the cartridge 300, a user moves the cartridge in the direction of arrow 360 so that the first seal 350 faces the central oil reservoir 212 and the piercing element 230 disposed therein.

[0077] With reference to FIG. 15B, it can be seen that when the user presses the oil immersion cartridge 300 into the central oil sump 212, its piercing element 230, in particular the piercing pin 236, penetrates and pierces the first seal 350, pushing the base 332 of the piercing piston 330 towards the second seal 352. As a result, the entire piercing piston 330 moves in the direction of the arrow 362 towards the wall 320, and the sharp edge 336 of the shaft 334 penetrates and pierces the second seal 352. The snap-fit ​​engagement projections and / or grooves 319 of the oil immersion cartridge 300 engage with corresponding grooves and / or projections 219 on the inner circumference of the central oil sump 212, ensuring that the cartridge is snap-fitted into the oil well. In this orientation, as shown in FIG. 15C, the seals 350 and 352 are pierced, allowing air to flow into the oil immersion cartridge 300 and oil to flow out of the cartridge. Thus, oil flows out of the cartridge, around the base 332 of the piercing piston 330 , through the channels 238 and holes 239 in the piercing element 230 and into the central oil sump 212 .

[0078] Reference is now further made to Figures 16A and 16B, which are cross-sectional views showing the oil flow paths within the oil loading subassembly 200. As shown, after unsealing the immersion oil cartridge 300 shown in Figures 15A-15C, oil flows from the central oil sump 212, via the oil flow conduit 218, to the pump inlet conduit 217. To reduce the possibility of dirt or other material contaminating the oil, it will be appreciated that the oil cartridge 300 may be positioned to release oil into the oil sump 212 and left in place after being punctured, or alternatively, the apparatus may include a cover (not shown) which covers the oil sump 212 when the oil cartridge 300 is removed.

[0079] Operation of the diaphragm pump 250, for example in response to an input or signal received from a controller, causes the pump to draw oil into the pump via an inlet 252 located in the pump inlet conduit 217, as shown in Figure 16A. The oil drawn into the diaphragm pump 250 then drips out of the pump via the pump's outlet conduit 254, as shown in Figure 16B. In operation, an oil immersion objective lens unit is positioned below the outlet conduit 254 such that oil drips onto the objective lens, as described in more detail herein below.

[0080] 17, which is a perspective view of the lens exchange subassembly 400 and the oil loading subassembly 200, in which the objective lens unit 500 is disposed at the immersion oil loading position 412. Further, reference is made to FIGS. 18A and 18B, which are a front view and a side view, respectively, of the loading of immersion oil into the objective lens in the configuration of FIG. 17.

[0081] As shown, the immersion oil cartridge 300 is mounted on the oil loading subassembly 200, which is mounted above the lens exchange subassembly 400. The objective lens unit 500, which includes an oil immersion lens 502, is positioned in the oil loading position 412 of the lens exchange subassembly such that the outlet conduit 254 of the diaphragm pump is directly above the oil immersion lens 502. As seen in FIG. 18A, upon receiving an appropriate control signal to trigger operation of the diaphragm pump 250, a droplet 504 of immersion oil is expelled from the outlet conduit 254 onto the immersion lens 502. In some embodiments, the droplet of oil includes 20-30 μl of oil. The droplet remains as a mound 506 on the lens due to the surface tension of the immersion oil. In some embodiments, the lens unit 500 may be raised, for example by movement of the lens exchange subassembly 200, such that the lens 502 is in close proximity to the outlet opening of the outlet conduit 254. This may ensure that the expelled oil is dripped directly onto the lens and not wasted.

[0082] In some embodiments, multiple drops of oil may be applied to the lens unit 500 during each oil loading. Once a sufficient amount of oil has been applied to the lens unit 500, the lens unit may be returned to its scanning location, for example, as described herein above with respect to Figures 10 and 11. As described in more detail herein below, the amount of oil to be dropped onto the lens may be estimated, for example, based on the expected spread of oil given the movements already performed by the lens, or may be estimated by a weighing mechanism, for example, as described herein above.

[0083] Reference is now made to Figures 19A, 19B and 19C, which show the spread of immersion oil as the objective lens unit moves relative to the observation surface during use of the microscope system.

[0084] As seen in Figures 19A and 19B, a multiwell plate 600 is placed in a plate holder 602. The multiwell plate has a lower surface 604 and a number of wells 606, each containing a biological sample. An oil immersion lens unit 500 is placed under the lower surface 604 and is initially positioned to scan a first sample in well 606a. As seen in the enlarged portion of the figure, at this stage, which is an initial stage after loading the lens unit 500 with immersion oil, a drop of oil placed on the lens of the lens unit 500 spreads due to the lens unit's proximity to the lower surface 604, forming an oil layer 610. Excess oil may flow to the beveled edge 510 of the objective lens unit and get trapped in the circumferential groove 512 on the top of the objective lens unit. The surface tension of the immersion oil ensures that the oil and excess oil remain engaged with the objective unit on one side and the plate on the other side. As will be described later, the oil is typically formulated to have the same refractive index as the bottom of the multiwell plate or other sample-holding container. In most cases the base is made of glass.

[0085] FIG. 19B shows the lens unit 500 after moving along the plate 600, scanning wells 606b and 606c, and reaching well 606d. At this stage, the thickness of the oil layer 610 has decreased relative to the thickness illustrated in FIG. 19A. This is because the movement of the objective lens has caused the immersion oil to spread along the lower surface 604 of the plate. As described in more detail later herein, a controller associated with the microscope system or the objective unit, such as a controller for the XYZ motion described above herein, may be configured to determine that the movement distance of the objective unit reaches a threshold value at which point the immersion oil is expected to spread too thin, and to control the operation of the objective unit to return the objective unit to the oil loading position and apply additional oil thereto.

[0086] 19C shows five different states of a droplet of oil placed on top of a lens unit 500. As shown, in the initial stage (i), which typically occurs immediately after applying the oil to the lens unit, the droplet of oil forms a dome based on the surface tension of the oil. In fact, at this time, the droplet of oil is actually in the shape of a droplet.

[0087] In (ii), the lens unit approaches the plate surface 604 but has not yet spread along the surface. The drop now extends vertically between the lower surface of the plate and the upper surface of the lens unit. This configuration can occur when approaching a plate or when the lens unit is lowered relative to a plate during removal of a drop of oil from the plate.

[0088] In (iii), the lens unit approaches surface 604 and the oil droplet spreads along a portion of the plate surrounding the area of ​​the lens unit, but the oil is still contained only in the area of ​​the lens unit. In some embodiments, the oil droplet configuration as shown in (iii) may be at a height that corresponds to the initial scan height, as described below with respect to Figures 21A and 21B.

[0089] Image (iv) shows the configuration of the oil during scanning of an initial area or well of the plate after focusing of the objective lens, as described in detail later in this specification. As shown, the oil is more spread out relative to (iii), but is still located only above the objective lens and is substantially concentric with the objective lens. After the lens unit is moved, for example to scan another field of wells, or another well, some oil remains in the part of the plate previously scanned by the objective lens, and some oil moves with the objective lens to a new area, resulting in the oil spreading to areas that are not located directly above the objective lens, as shown in (v), where the objective lens is moved to the right relative to the plate and the oil spreads above and to the left of the objective lens.

[0090] 20 is a flow chart outlining a method for scanning a sample using an oil immersion lens and reloading immersion oil on the oil immersion lens, according to an embodiment of the present invention. The following description is provided with respect to the plate 600 shown in FIGs. 19A and 19B having a lower surface 604 and each well having an upper surface on which a biological sample is placed. However, the disclosure is equally relevant to any other sample carrying structure having a lower surface and an upper surface.

[0091] As seen in Figure 20, in a first set-up step 700, the Z-axis location of the underside 604 of the plate 600 is approximated. The approximate value is saved and used as the plate height approximation for the entire plate scan. Such an approximation step may be performed for each individual plate checked, or may be performed once for multiple plates or sample carriers.

[0092] In some embodiments, approximation of the Z-axis location of the underside of the plate may be performed using rapid laser scanning to identify the underside of the plate at several locations of the planned scan. In some other embodiments, multiple foci, for example three or four foci, may be determined for the plate or regions of the plate using laser scanning, and an approximation plane of the underside of the plate is determined based on the multiple foci.

[0093] As described below, the approximate height of the lower surface of the plate is used as the basis for subsequent focusing operations of the plate, which are performed using a search for a maximum signal of the laser beam reflection, as described herein. There are several ways in which this initial approximation can be performed. One option is to use an air objective to determine the approximate height of the lower surface of the plate, and then switch to an oil immersion objective for the actual focusing and scanning. Another option is to determine the height based on the structure of the plate and the device, knowing that the average distance of the bottom of a particular model number of a particular manufacturer is a predetermined distance above the bottom edge of the plate, and the dimensions of the device. In some embodiments, an initial approximation of the height of the lower surface of the plate may be performed with an oil immersion objective having a lower magnification than the oil immersion objective used to scan the sample, and then switching to a higher magnification oil immersion objective for the actual scanning.

[0094] In a second setup step 702, one or more scan areas of a plate or slide are defined, for example using a user interface, and provided to a controller that controls the XYZ motion of the objective lens, as described herein. In a third setup step 704, a maximum threshold amount of oil that can be lost by the oil immersion objective lens before replenishing oil thereon is determined. This amount can be tracked according to the total movement of the objective lens, by weight loss, or by oil thickness, as described below. In some embodiments, the system can be programmed to dynamically adjust this threshold after each instance of oil replenishing according to the amount of oil applied to the lens.

[0095] Although the setup steps 700, 702 and 704 are described in a particular order in FIG. 20, they may be performed in a different order, provided they are performed before scanning of the biological sample begins.

[0096] In step 706, which is also performed before the start of the scan, the objective unit is placed in an oil loading position of the lens exchange assembly and an initial amount of oil is dripped onto the objective lens from the oil loading subassembly as described herein, for example, a controller associated with a diaphragm pump provides a signal to the pump to drip immersion oil onto the objective lens when the objective lens is in the correct position.

[0097] In some embodiments, the initial amount of oil is greater than the subsequent amount of oil that is loaded at a later stage as described herein.

[0098] In some embodiments, the exact amount of oil to be loaded onto the objective lens is determined using a weighing system as described herein, hi some other embodiments, the amount of oil to be loaded onto the objective lens is approximated based on the number of drops of oil applied to the objective lens, the volume of each drop of immersion oil aspirated by the pump, which is a known value, and the desired volume of oil to be aspirated.

[0099] In step 708, the controller controls the XYZ motion of the oil-loaded objective lens to move the lens to the appropriate XY position for scanning, as determined in step 702. When starting the scan, the XY position is the position where the scan should begin. After a later iteration of replenishing oil on the objective lens, as described later in this specification, the XY position is the XY position where the scan stopped to replenishing the immersion oil, or the next XY position. In step 710, the controller raises the objective lens to a predetermined initial scan height, typically a few microns from the approximate Z-axis location of the underside of the plate established in step 700. The predetermined height is selected to be close enough to the underside of the plate so that the drop of oil on the objective lens spreads to the bottom surface of the plate, as shown in FIG. 19A.

[0100] The distance from the plate at which the objective lens will focus on the sample is automatically calculated in step 712, for example using the method described below with respect to Figure 21, and the objective lens is moved to the focal length. Once the objective lens reaches the established focal length, scanning of the sample begins in step 714.

[0101] During scanning, the objective lens is moved relative to the sample or plate 600 between different positions as described herein above with respect to Figures 19A and 19B. The distance traversed by the objective lens is tracked, for example by a controller associated therewith, including both movement when the oiled objective lens is in contact with the surface of the plate and when the objective lens is not in contact with the plate. The controller can be configured to calculate the amount of oil lost from the objective lens as a result of such movement, and the amount of oil lost when the objective lens is lowered along the Z-axis so that it is no longer in contact with the plate. In some embodiments, the controller also considers the effect of differences in plate materials on the amount of oil lost when the objective lens is moved along the plate or removed from contact with the plate. Alternatively or additionally, the amount of oil lost can be tracked by monitoring the weight change of the oiled objective lens from when it was oiled and / or by inspecting the thickness of the oil on the objective lens, for example by measuring with a camera or with a laser.

[0102] In step 716, which may occur continuously or periodically, the controller checks whether the amount of oil lost as a result of the objective lens movement is greater than the maximum threshold amount established in step 704. If the distance traversed is less than the maximum threshold oil loss, the scan continues in step 714.

[0103] Otherwise, if the calculated amount of lost oil is equal to or greater than the maximum threshold amount, in step 718, the objective lens is lowered relative to the plate 600 and moved in the XY plane to an oil-loaded position. A replenishment amount of oil is then loaded onto the objective lens in step 720, substantially as described above with respect to step 706, except that the replenishment amount of oil may be less than the initial amount of oil. As described herein above, the amount of oil required to replenish the drop of oil on the objective lens may be calculated, for example, based on a weighing mechanism, may be approximated based on the distance traversed by the objective lens, or based on any other relevant parameters. Flow then returns to step 708, where the objective lens is returned to the scanning position to continue scanning the sample.

[0104] Reference is now made to Figures 21A, 21B and 21C, which are flow charts outlining an embodiment of a method for automatically focusing an oil immersion lens on a biological sample, according to an embodiment of the present invention. The method of Figures 21A-21C assumes that an initial scan height, typically a few microns, is known from the approximate Z-axis location of the lower surface of the plate. The initial scan height is typically based on the height of the lower surface of the plate, for example, established in step 700 of Figure 20. This automatic focusing is performed every time the lens is repositioned to a different XY position, including after an oil refill.

[0105] The initial scan height is the height at which a drop of oil placed on the oil objective is known to engage and spread between the oil objective and the lower surface of the plate. As mentioned above, typically the oil is formulated to have the same refractive index as the material of which the lower surface of the plate is made, which is often glass. In this way, the immersion oil and the initial scan height are selected so that there is no unwanted reflection or refraction as the light passes between the oil and the material of the multiwell plate, i.e., the oil contacts both the lens and the bottom of the plate. Thus, as the light beam passes from the objective through the oil and the plate material to the sample, the light is refracted only once at the transition between the plate material and the material of the sample. This occurs at the top surface of the well.

[0106] Turning to Figure 21A, it can be seen that in an initial step 750 of the focusing process, the objective lens is brought to a known and predetermined initial height so that the oil contacts the underside of the plate. This corresponds to step 710 of Figure 20. To allow the oil-surface interaction to stabilize, a delay of a predetermined time is waited, for example 500 ms to 2000 ms, before proceeding to the next step of the method. In step 752, the reflection of the emitted laser signal from the objective lens is measured.

[0107] In step 754, the objective lens is moved along the Z axis to a different height closer to the bottom surface of the plate, and in step 756 the reflection of the laser signal at the new position is measured. The reflection value at the new Z axis height is recorded. This process is essentially repeated continuously. The measurement is expected to increase until the focal length is reached, and then begin to decrease.

[0108] In step 758, the controller evaluates the collected reflectance data to assess whether there has been a decrease in the measured signal reflectance over the past few iterations. If not, flow returns to step 754 to measure the signal at a new Z-axis position. However, if the measured reflectance has decreased, in step 760, the objective lens is moved to the Z-axis position where the signal was maximum. This position along the Z-axis is considered the focal length of the objective lens relative to the sample.

[0109] In some embodiments, a continuous decay of 0.1V of the laser signal is considered to be significant enough.

[0110] The method of FIG. 21 is used to determine the focal length of a sample every time a new sample or plate is scanned and after refilling the immersion oil.

[0111] In some embodiments, a similar process is used to ensure that the objective lens is focused, for example, when moving to a new field of the plate, or when the objective lens remains stationary in a single position and images at the same position for a long time (e.g., to avoid issues of drift from a predetermined position). However, the previous focal length is known when moving to a new field, or when recalculating when the objective lens remains stationary in a single position for a long time. Thus, in this situation, in step 752, the objective lens is moved between heights within the range of "known focal length ± predetermined search range". Again, the focal length is determined to be the point at which the maximum signal is obtained.

[0112] Turning to FIG. 21B, it can be seen that in an initial step 770 of the focusing process, the objective lens is brought to a known and predetermined initial height so that the oil contacts the underside of the plate. This corresponds to step 710 of FIG. 20. A delay of a predetermined time, for example 500 ms to 2000 ms, is waited for before proceeding to the next step of the method to allow the oil-surface interaction to stabilize. In step 772, the objective lens is moved a predetermined distance along the Z axis while repeatedly measuring the reflection of the emitted laser signal through the objective lens. The predetermined distance along the Z axis is selected such that the measured reflection of the signal is expected to peak and then decrease during the Z axis movement of the objective lens.

[0113] In step 774, a best fitting Gaussian (or polynomial) curve of the signal peaks collected over the entire measurement obtained in step 772 is calculated to determine the peak maximum, and the objective lens is moved to the Z-axis position where the Gaussian (or polynomial) curve was maximum. This position along the Z-axis is taken as the focal length of the objective lens relative to the sample.

[0114] In step 776, the objective lens is moved to the focal distance determined in step 774 and scanning begins. As part of the scan, images of the sample are taken at the focal position until the area has been scanned or the maximum scan time has elapsed.

[0115] In step 778, the controller evaluates whether scanning of the area is complete. If scanning of the area is complete, the objective lens is lowered and moved to a new XY position in step 780. Flow then returns to step 770 for refocusing of the objective lens at the new location.

[0116] Otherwise, if the scan of the area is not complete, then in step 782 the controller evaluates whether the maximum scan time has elapsed. If not, flow returns to step 776 and scanning continues. If the maximum scan time has elapsed, flow returns to step 772 to refocus the objective lens and ensure that there has been no drift of the objective lens or sample during the scanning process. However, when moving to a new field or remaining in the same position for an extended period of time, the previous focal length is known. Thus, in this situation, in step 772 the objective lens is moved between heights within the "known focal length ± predetermined search range". Again, the focal length is determined to be the point at which the maximum signal is obtained.

[0117] In some embodiments, a decay of 0.1V continuous decay in the laser signal is considered to be significant enough.

[0118] The method of FIG. 21B is used to determine the focal length of the sample each time a new sample or plate is scanned and after refilling the immersion oil.

[0119] Reference is now made to Figs. 22A, 22B and 23, which are flow charts outlining a scanning method and image processing and analysis according to embodiments of the present invention. The flow chart in Fig. 22A gives an overview of a scanning process according to some embodiments of the present invention. First, a first scanning operation of an object is performed according to user-defined parameters such as size (e.g., ignoring objects larger and / or smaller than a certain size), shape (ignoring non-circular or non-semicircular objects), intensity, etc. The scanned image thus obtained is processed in order to identify objects of interest and their features. This processing may be performed using image processing algorithms currently known in the art or that may be developed in the future. Next, the image processing results are analyzed according to predefined rules in order to determine optimal parameters for performing further scanning operations of the same area. Based on the analysis results, new parameters are defined for a scan to obtain new images of the same object. At least one second scanning operation is then performed using the new scanning parameters. Thus, for example, the system may scan a biological sample plate having 96 wells, each with a diameter of 6 mm. During the scan, the image processing algorithm recognizes each living cell present in the plate. If two or more cells are attached together or if there are individual cells larger than a certain size, the system may record this as an abnormal event to be further observed using higher magnification optics. With an objective lens exchanger as described above, the lenses can be exchanged to facilitate such observation. The system then determines parameters that define the appropriate image quality of the high magnification scan. In some embodiments, the image processing is performed while the first scanning operation is still being performed, in which case the analysis results can affect the operation of the scan in real time according to the newly defined parameters. In some embodiments, at least one second scanning operation is performed at a higher magnification than the first scanning operation.In some embodiments in which the method is utilized in conjunction with an apparatus having an objective lens changer as described above, the second scanning operation at a higher magnification is performed by changing to a higher magnification objective lens using the objective lens changer and then scanning the area of ​​interest at the higher magnification.

[0120] FIG. 22B is a flow chart of a scanning process according to some embodiments of the present invention. In the first stage of scanning, a low magnification image of the defined location is acquired. This process is repeated until images of multiple defined locations are acquired. Immediately after acquisition, each of the scanned images is transferred to an image processing and analysis module, which starts processing and analyzing the images upon receiving the first image. This module may be incorporated into the software that controls the overall operation of the optical device, or may be located in a separate software application or computer. The processing and analysis module uses the results of the processing and analysis to start generating a location matrix containing information about the areas of interest according to the objects and their characteristics identified by the initial scan. In this way, the location matrix may be completed immediately after the completion of the low magnification scan. Alternatively, the location matrix may be generated after all processing and analysis are completed. Based on this location matrix, a second stage scan is performed during which high magnification images of the specific locations of interest are acquired.

[0121] FIG. 23 is a flow chart of an image processing and analysis process according to some embodiments of the present invention. In the initial stage of image processing, the following steps are performed: acquire low magnification images, apply a low pass filter to the obtained data, apply a high pass filter to the obtained data, and perform a watershed transformation. Based on these image processing steps, objects and object attributes / characteristics are detected and extracted. Then, related objects are selected using user parameters / attributes / characteristics, and object centers (L[Cx,Cy]) are extracted. Then, high magnification images of the selected objects are obtained, from which a 3D transformation matrix (M) between objects is created and utilized.

[0122] 24 is a perspective view of an oil-loaded subassembly 1200 according to another embodiment of the present invention. The oil-loaded subassembly 1200 is substantially similar to the oil-loaded subassembly 200 of FIGS. 13-14D, and like numbers represent like elements. For simplicity, the following description focuses on the distinction between the oil-loaded subassembly 1200 and the oil-loaded subassembly 200.

[0123] As shown, the oil loading subassembly 1200 includes a body portion 1210 including a central oil sump 1212 that may be in fluid communication with an oil observation opening, as described herein above with respect to FIG. 14D, and a pump seat hole that terminates in a pump inlet conduit. Similar to that shown in FIG. 14D, an oil flow conduit extends from a base of the oil sump 1212 adjacent to the pump inlet conduit and terminates at an edge of the body portion 1210 where the oil flow conduit is sealed by a stopper. An observation window, as described herein above with respect to FIG. 14B, may be disposed within and may seal the oil observation opening, although as described above such a window is not required.

[0124] The cartridge piercing element 1230 is seated within the central oil sump 1212 and may be attached thereto by any suitable attachment mechanism, such as fasteners, soldering, adhesives, etc. The cartridge piercing element 1230 includes a base 1234 and a central piercing pin 1236 having a hollow channel 1238 extending longitudinally therethrough that terminates in a sharp tip and is in fluid communication with a hole 1239 formed in a side of the cartridge piercing element 1230. A pair of peripheral piercing pins 1240 may extend from an edge of the base 1234 and be substantially parallel to one another. Each piercing pin 1240 is substantially planar and has a first thickness for a majority of its longitudinal length. An end of each piercing pin 1240 distal from the base 1234 has a second thickness less than the first thickness such that a shoulder 1242 is formed near the end of the piercing pin. Each puncture pin 1240 terminates in a sharp tip 1244 .

[0125] As described below with respect to Figures 26A and 26B, when the immersion oil cartridge 1300 is installed in the central oil reservoir 1212, the hollow channel 1238 of the central puncture pin 1236 is fluidly connected to the central oil reservoir 1212, as is the oil flow conduit, in a manner similar to that shown in Figures 14D and 16A.

[0126] The diaphragm pump 1250 is positioned within the pump seat hole such that the inlet of the pump 1250 seats within the pump inlet conduit, and the pump outlet conduit 1254 (see Figures 26A and 26B) extends downwardly from the body portion 1210 substantially similar to that shown in Figures 13-14D. As described herein above with reference to the diaphragm pump 250, the diaphragm pump 1250 is adapted to draw oil from an oil flow conduit, through a pump outlet conduit, and into an article positioned below the pump outlet conduit, which, in use, is an immersion oil objective lens.

[0127] The oil loading subassembly 1200 further includes a base mount 1260 attached to the bottom side of the body portion 1210 and adapted for mounting the oil loading subassembly 1200 to other components of a microscope system above the lens exchange subassembly 400. A back mount 1265 is attached to the body portion 1210 behind the central oil sump 1212. Both the base mount 1260 and the back mount 1265 may be connected to the body portion 1210 by any suitable connection method, such as fasteners, adhesives, soldering, etc.

[0128] Reference is now made additionally to FIG. 25, which is a cross-sectional view of an immersion oil cartridge 1300 according to another embodiment of the present invention.

[0129] As seen in FIG. 25, the immersion oil cartridge 1300 includes a generally cylindrical housing 1301. The housing 1301 includes a base 1302 from which extends a first cylindrical wall portion 1304 having a first width and defining a first chamber 1305 having a first inner diameter d1. A second cylindrical wall portion 1306 extends from the first cylindrical wall portion 1304. The second wall portion 1306 has a second width less than the first width. An outer surface of the second wall portion 1306 is flush with an outer surface of the first wall portion 1304, and an internal shoulder 1308 is formed between the inner surfaces of the walls 1304 and 1306. A third cylindrical wall portion 1310 extends from the second cylindrical wall portion 1306. The third wall portion 1310 has a third width less than the second width and forms a neck of the cartridge 1300. An interior surface of the third wall 1310 is flush with an interior surface of the second wall 1306, and an exterior shoulder 1312 is formed between the exterior surfaces of the walls 1310 and 1306. The third cylindrical wall 1310 terminates at a lip 1314 distal from the base 1302. The second wall 1306 and the third wall 1310 together define a second chamber 1315 having a second inner diameter d2. The second chamber 1315 is in fluid communication with the first chamber 1305.

[0130] In some embodiments, the outer surface of the third wall portion 1310 distal to the external shoulder 1312 may include snap-fit ​​engagement protrusions and / or grooves adapted for snap-fit ​​engagement with corresponding grooves and / or protrusions on the inner circumference of the central oil reservoir 1212.

[0131] A generally cylindrical oil reservoir 1320 is disposed within the housing 1301 and defines a hollow 1321. A first end 1322 of the oil reservoir 1320 is disposed within the first chamber 1305 and engages an inner surface of the base 1302. A second end 1324 of the oil reservoir 1320 is substantially flush with the lip 1314 such that a majority of the oil reservoir 1320 is disposed within the second chamber 1315. The oil reservoir 1320 includes a plurality of holes 1326 that are closer to the first end 1322 than the second end 1324 while in fluid communication with the second chamber 1315. A cylindrical gap 1330 is formed between the housing 1301 and the oil reservoir 1320 within the second chamber 1315.

[0132] A generally cylindrical push element 1340 is disposed in the oil container 1320 within the gap 1330 and is longitudinally movable relative to the oil container. A first end 1342 of the push element 1340 is adapted to be disposed closer to the first end 1322 of the oil container 1320 and a second end 1344 of the push element is adapted to be disposed closer to the second end 1324 of the oil container. The cylindrical push element 1340 includes a plurality of holes 1346 adapted to be radially aligned with the holes 1326 of the oil container 1320. The longitudinal alignment of the holes 1346 with the holes 1326 depends on the position of the push element 1340 relative to the oil container 1320, as described in further detail herein below.

[0133] A first end 1342 of the pressing element 1340 is adapted to press against an annular O-ring 1350 disposed within the gap 1330. As will be described in further detail with respect to Figures 26A and 26B, when the O-ring 1350 is disposed under the hole 1326, it seals the gap 1330 such that there is no fluid path between the hollow 1321 and the gap 1330. In contrast, as shown in Figure 25, when the O-ring 1350 is pressed to lie against the shoulder 1308 and the holes 1326 and 1346 are aligned with one another, a fluid path is formed between the gap 1330 and the hollow 1321 through the holes 1326 and 1346.

[0134] In the storage orientation of cartridge 1300, a sealing foil 1360 (see FIG. 26A ), or other seal, is positioned against lip 1314 of housing 1301 and second end 1324 of oil reservoir 1320 to seal the oil within the cartridge. In the storage orientation, O-ring 1350 is positioned under hole 1326 to seal gap 1330 such that there is no fluid path between hollow 1321 and gap 1330.

[0135] Reference is now made additionally to FIGS. 26A and 26B, which are cross-sectional views of a step of inserting the immersion oil cartridge 1300 of FIG. 25 into the oil loading subassembly 1200 of FIG.

[0136] 26A, the cartridge 1300 is in a storage orientation, with the lip 1314 of the housing 1301, the second end 1324 of the oil container 1320, and the second end 1344 of the pusher element 1340 all flush with one another and positioned against the sealing foil 1360. In the storage orientation, the O-ring 1350 is positioned below the hole 1326 and against the first end 1342 of the pusher element 1340. The O-ring seals the gap 1330 such that there is no fluid path between the hollow 1321 and the gap 1330. Oil 1365 is positioned within the hollow 1321 such that the oil level does not extend above the hole 1326.

[0137] To load the cartridge 1300 into the oil loading subassembly 1200, the cartridge 1300 is moved in the direction of arrow 1370 toward the central oil sump 1212 until the central pin 1236 pierces the sealing foil 1360 and enters the hollow 1321. In this initial installation step, the O-ring 1350 remains below the hole 1326, blocking the fluid path between the hollow 1321 and the gap 1330.

[0138] As the cartridge 1300 continues to be moved in the direction of arrow 1370, the tips 1244 of the peripheral piercing pins 1240 pierce the periphery of the foil 1360 near the gap 1330. The shoulder 1242 of the peripheral piercing pins 1240 engages the second end 1344 of the pusher element 1340 and as the cartridge continues to be lowered, the pressure exerted by the shoulder 1242 on the second end 1344 moves the pusher element 1340 towards the base 1302 of the housing 1301 which in turn pushes the O-ring 1350 towards the shoulder 1308.

[0139] 26B, in the installed operating orientation of the cartridge 1300, the peripheral piercing pins 1240 and the shoulder 1242 of the pressing element 1340 press against the O-ring 1350 and rest against the shoulder 1308. In this arrangement, the hole 1346 of the pressing element 1340 is aligned with the hole 1326 of the oil reservoir 1320 such that a fluid path exists between the central oil sump 1212, the gap 1330, the hollow 1321 of the oil reservoir 1320, and the channel 1238 of the central piercing element 1236. In this arrangement, oil can flow from the reservoir 1320 into the central oil sump 1212 and operation of the system continues substantially as described herein above.

[0140] It will be understood that the oil or other liquid ejection device as described herein can also be used in combination with a confocal microscope. Furthermore, the illumination source does not necessarily have to be a conventional laser, but can be a light-emitting diode (LED) or a combination or array of LEDs. As a result, a microscope or scanner equipped with the oil or other liquid ejection device as described herein can be used in high content imaging (HCI, also called high content screening) to obtain images that can be processed according to known techniques such as photoactivated localization microscopy (PALM) (see, for example, Betzig, E. et al., "Imaging intracellular fluorescent proteins at nanometer resolution", Science 313, 1642-1645 (2006)) or stochastic optical reconstruction microscopy (STORM) (see, for example, Rust, MJ, Bates, M. & Zhuang, X., "Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)", Nature Methods 3, 793-795 (2006)). In one embodiment, images obtained using an acquisition device comprising an oil ejection apparatus as described herein can be processed according to super-resolution radial fluctuations (SRRF) as described in Gustafsson et al., "Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-resolution radial fluctuations," Nature Communications 7:12471 (published August 12, 2016). Such processing can be performed using the ImageJ software plugin freely available at https: / / henriqueslab.github.io / resources / NanoJ-SRRF / .The fact that the apparatus and methods described herein facilitate automatic dispensing of oil (or other liquid) onto the objective lens means that the apparatus can be combined in a fully automated image acquisition: automatic scanning, automatic focusing, automatic objective lens changing, automatic dispensing of lens immersion medium, and automatic detection of objects of interest.

[0141] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods are described herein.

[0142] All publications, patent applications, patents, and other documents mentioned herein are incorporated by reference in their entirety. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0143] Those skilled in the art will understand that the present invention is not limited to what has been specifically shown and described hereinabove, but rather, the scope of the present invention is defined by the general combination of parts performing the same function as illustrated in the embodiments, and includes both combinations and subcombinations of the various features described hereinabove, as well as variations and modifications thereof that may occur to those skilled in the art after reading the foregoing description.

Claims

1. 1. A microscope device for observing or imaging a sample placed on a sample holder, comprising: a first immersion objective lens having a three-dimensional orientation; a first liquid application location where the sample holder cannot be placed, and a second location different from the first location where the sample holder can be placed; an apparatus separated from the first immersion objective lens and disposed at the first liquid application location; a mechanism configured to move the first immersion objective lens in an XYZ coordinate space from a first liquid application position to a second scanning position having different X and Y coordinates from the first liquid application position without changing the three-dimensional orientation of the first immersion objective lens; the apparatus is configured to apply liquid to the first immersion objective when the first immersion objective is disposed at the first liquid application position beneath the apparatus; A microscope device wherein when the sample holder is disposed at the second location, liquid at the second scanning position on the first objective lens contacts the sample holder.

2. 10. The microscope device of claim 1, further comprising a controller, A microscope device, wherein the controller loads the first immersion objective lens with liquid, places it in the second scanning position, and then moves it along the Z axis towards the sample holder, enabling determination of a focused Z axis position.

3. A microscope device according to claim 1 or 2, the device comprises a second immersion objective lens different from the first immersion objective lens; The microscope device, wherein the mechanism is further configured to place the first immersion objective lens in a first storage location, remove the second immersion objective lens from a second storage location, and move the second immersion objective lens to at least one of the first liquid application position and the second scanning position.

4. Providing a microscope device according to any one of claims 1 to 3; using the apparatus to apply liquid to the first immersion objective lens disposed at the first liquid application position; moving the first immersion objective lens with the liquid disposed thereon to the second scanning position, A method according to claim 1, wherein the microscope device is arranged at the second scanning location such that a sample holder containing a sample is positioned within the microscope device.

5. 5. The method of claim 4, A method wherein at the second scanning position, a distance between the first immersion objective lens with the liquid disposed thereon and a surface of the sample holder is a predetermined distance from the surface of the sample holder.

6. 6. The method of claim 5, A method comprising determining a Z coordinate of the surface using an air objective lens that is replaced with the first immersion objective lens before using the apparatus to apply the liquid, before moving the first immersion objective lens on which the liquid is disposed to the second scanning position.

7. 6. The method of claim 5, prior to moving a first immersion objective lens with the liquid disposed thereon to the second scanning position, a Z coordinate of the surface is determined using a second immersion objective lens having a lower magnification than the first immersion objective lens used to scan the sample; A method wherein the second immersion objective lens is replaced with the first immersion objective lens prior to the step of applying liquid to the first immersion objective lens using the apparatus.

8. 6. The method of claim 5, the sample holder is a commercially available sample plate of a particular model number; A method in which the Z coordinate of the surface is determined based on the average distance of the bottom of the particular model plate from the bottom edge of the plate in combination with the dimensions of a microscope device before moving the first immersion objective lens to the second scanning position.

9. The method according to any one of claims 4 to 8, The method further comprises determining a focus position of the first immersion objective lens after moving the first immersion objective lens with the liquid disposed thereon to the second scanning position.

10. 10. The method of claim 9, The method further comprises, after determining the focus position of the first immersion objective, returning the first immersion objective to the first liquid application position and applying liquid thereto.

11. 11. The method of claim 10, After determining the focal position of the first immersion objective lens, scanning the sample; The method of claim 1, wherein the returning step is performed in response to the scan reaching a threshold that includes one or more of the following: (a) the liquid lost from the first immersion objective lens as a function of the distance traveled by the first immersion objective lens in the XY plane during scanning; (b) the amount of liquid lost from the first immersion objective lens as a result of moving the first immersion objective lens along the Z axis away from the sample holder; (c) the measured weight loss of the first immersion objective lens holding the liquid. (d) a measured thickness of liquid on the first immersion objective lens.

12. 12. The method of claim 10 or 11, returning the first immersion objective lens to the first liquid application position and applying liquid thereto, and then moving the first immersion objective lens to either (a) the second scanning position or (b) a third position having X and Y coordinates different from the first liquid application position and having at least one X or Y coordinate different from the second scanning position; In this step, liquid at the third position on the first immersion objective lens can be brought into contact with a sample holder disposed at the second location.

13. The method according to any one of claims 4 to 12, further comprising the step of acquiring a plurality of images of the sample through the first immersion objective.

14. The method of claim 13 further comprising the step of processing the plurality of images.

15. The method according to any one of claims 4 to 14, wherein the liquid is an oil.

16. 16. The method of claim 15, wherein the oil is a hydrocarbon-based oil.

17. 16. The method of claim 15, wherein the liquid is a silicone oil.

18. The method according to any one of claims 4 to 14, wherein the liquid is water.

19. 4. The microscope device according to claim 1, wherein the liquid is oil.

20. 20. The microscope device of claim 19, wherein the oil is a hydrocarbon-based oil.

21. 20. The microscope device of claim 19, wherein the liquid is silicone oil.

22. 4. A microscope device according to claim 1, wherein the liquid is water.

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