CN117581145A - Microscope system and method using immersion liquid - Google Patents

Abstract

There is provided a microscope apparatus capable of (i) moving a liquid immersion objective to a position where a liquid such as oil or water can be applied to the liquid immersion objective, and (ii) moving the liquid immersion objective with the liquid applied thereto to a sample holder for acquiring an image of a sample. The liquid application process can be automated to replenish the liquid on the objective lens as needed. The device is capable of automatically focusing the lens to facilitate scanning and is configurable to replace the objective lens. Other embodiments are also described, including the use of such devices in high content/high throughput scanning.

Description

Microscope system and method using immersion liquid

Cross Reference to Related Applications

The present application claims Paris convention priority and U.S. rights and interests of U.S. provisional application Nos. 63/180693 and 63/180694 filed concurrently on month 28 of 2021. The contents of the two provisional applications are incorporated herein by reference.

Technical Field

The present invention relates to the field of accurate optical scanning and imaging of samples, and more particularly to biological microscopes that use a suitable immersion liquid (e.g., a stable oil) as the medium between the objective lens and the sample.

Background

High content microscope systems for viewing biological samples are known in the art. Such microscope systems are typically based on a static large microscope body and include complex connections to optical units (e.g. objective turret, illumination unit, filter wheel, shutter, camera, internal optics and other units). To be able to scan a given sample, a device is added to the microscope body that enables movement of the sample holder in the X, Y and Z directions. During imaging of these microscopy systems, the sample is moved to capture images at different locations along the sample while the optical unit is stationary.

Various automatic scanning systems have been developed in which the sample and the objective lens are moved relative to each other, the objective lens is auto-focused, and scanning is performed without manual intervention. Some such systems are also capable of automatically changing between a plurality of different objectives, for example, one such system is described in U.S. patent No.9170412, the contents of which are incorporated herein by reference.

While such automatic scanning systems may be used to scan biological materials using an objective lens operating in an air environment, these systems are not suitable for use with liquid immersion microscopes. This is due in particular to the fact that: as the objective lens and the sample move relative to each other, the immersion liquid diffuses along the sample plate, so that periodic replenishment of the immersion liquid is required. While systems have been developed to try to replenish the liquid (see for example EP 1717628B1, DE 202017000475 U1, U.S. Pat. No. 3,7304793 B2, https:// www.leica-microsystems. Com/products/light-microscope/p/leica-water-immersion-micro-dis-penser/and https:// www.marzhauser.com/en/products/liquid-immersion. Html), such systems have various drawbacks, such as their design increases the weight of the objective lens, reduces the scanning area and reduces the throughput of the microscope system.

Accordingly, there is a need in the art for a system and method for automated high content microscopy using immersion liquid.

Detailed Description

Embodiments of the invention will be better understood from the following detailed description and by reference to the drawings, in which:

FIG. 1 is a schematic diagram of an embodiment of a microscope system that can be adapted to embodiments in accordance with the invention;

FIG. 2 is an isometric view constructed and operative to adapt a device according to an embodiment of the present invention;

FIG. 3 is a cross-sectional view of an objective lens mounted on an objective lens holder and attached to a motion base, which may be used in an embodiment in accordance with the present invention;

FIG. 4 is a top exploded isometric view of the component of FIG. 3;

FIG. 5 is a bottom exploded isometric view of the components of FIG. 3;

FIG. 6 is an isometric view of the mover of FIGS. 4 and 5;

fig. 7 shows how the lower part of the objective lens unit of fig. 3-5 fits into the V-shaped groove of the motion base of fig. 6;

fig. 8 and 9 are top and bottom isometric views, respectively, showing an objective lens mounted in an objective lens holder and a motion base with a magnet attached;

FIGS. 10 and 11 illustrate how an objective lens is stored and changed according to an embodiment of the present invention;

FIG. 12 is a partially exploded view of an oil loading subassembly having an oil immersion cartridge disposed above a lens replacement subassembly according to an embodiment of the present invention;

FIG. 13 is an exploded view of an oil loading subassembly according to an embodiment of the present invention;

14A, 14B, 14C and 14D are perspective, front plan, side plan and cross-sectional views, respectively, of the oil loading subassembly of FIG. 13 in its configuration;

15A, 15B and 15C are cross-sectional views of a step of inserting an oil dip tube into the oil loading subassembly of FIGS. 13, 14A, 14B, 14C and 14D;

FIGS. 16A and 16B are cross-sectional views illustrating oil flow paths within the oil loading sub-assemblies of FIGS. 13, 14A, 14B, 14C, 14D, 15A, 15B and 15C;

FIG. 17 is a perspective view showing the lens exchange sub-assembly of FIGS. 1-11 and the oil loading sub-assembly of FIGS. 12, 13, 14A, 14B, 14C and 14D, including the objective lens in the oil-immersed loading position of the lens exchange sub-assembly;

fig. 18A and 18B are front and side views, respectively, of the immersion oil loaded onto the objective lens in the configuration of fig. 17.

FIGS. 19A, 19B and 19C illustrate the diffusion of immersion oil as the objective lens moves relative to the viewing surface;

FIG. 20 is a flow chart summarizing a method of scanning a sample using an oil immersed lens and reloading immersion oil onto the oil immersed lens according to an embodiment of the invention;

FIGS. 21A, 21B and 21C are flowcharts outlining methods for auto-focusing an oil-borne oil saturation mirror on a biological sample in accordance with embodiments of the present invention;

FIGS. 22A, 22B and 23 are flowcharts outlining methods for scanning a sample according to embodiments of the present invention;

FIG. 24 is a perspective view of an oil loading subassembly according to another embodiment of the present invention;

FIG. 25 is a cross-sectional view of an oil immersion cartridge according to another embodiment of the present invention; and

fig. 26A and 26B are cross-sectional views of a step of inserting the oil impregnated cartridge of fig. 25 into the oil loading subassembly of fig. 24.

It should be appreciated that although color drawings and photographs are popular in the scientific literature and are readily presented in electronic format, PCT regulations remain a dilemma in the 19 th century and still do not allow for submission of color drawings or photographs. While the united states patent and trademark office only allows the use of color drawings or photographs as requested. Accordingly, fig. 13, 14A, 14B, 14C, 14D, 15A, 15B, 15C, 16A, 16B, 17, 18A, 18B, 19C, 24, 25, 26A and 26B are filed in the form of black and white drawings with the PCT application. However, the original picture is colored, which has been uploaded to the public photosharing service Shutteofly for disclosure, and can be accessed by anyone on the Internet using the link https:// papercompptordrandraws. This link is first shared at the time of filing this patent application and the color drawings are incorporated herein by reference.

Reference is now made to fig. 1, which is a block diagram schematically illustrating an apparatus 10, the apparatus 10 being constructed and operative to be adapted for use in accordance with an embodiment of the present invention. The device 10 comprises a holder 12, which holder 12 may be configured to hold a sample plate 13, such as a 6-, 24-, 96-, 384-, or 1536-well plate commonly used for holding biological samples and having a lower surface 13A and an upper surface 13B, the lower surface 13A and the upper surface 13B respectively comprising the sample or samples to be observed, as is known in the art. The holder 12 may also be configured to hold a microscope slide, a culture dish, or another substrate having a bottom that is transparent to electromagnetic radiation of one or more wavelengths of interest. For reference, the sample plate 13, which is not itself part of the device, is located in the XY plane, so that the sample contained in the sample plate will be opposed by its lower surface 13A to the components of the device 10 described below.

The turret-free objective, i.e. the single objective 14 as part of the linear XYZ scanner 16, is arranged such that the lens is directed towards the sample holder (and towards the lower surface 13A of the sample plate 13 when the sample plate 13 is present), the optical axis of the objective 14 being along the Z-axis with respect to the sample holder. "Linear XYZ scanner" refers to a mechanism constructed and operative to move the objective lens 14 in three mutually perpendicular directions, where the "Z" direction is used to denote movement along the optical axis. Such scanners are known per se in the art, for example from israel patent No.143836 or us patent No.6850362, filed on even date 19 at 6 of 2001, the contents of both of which are incorporated herein by reference, entitled "Compact Linear Scanner System". It should be understood that for simplicity, only some of the components of the XYZ scanner 16 are shown in fig. 1; this component will be described in more detail below. Among the components of the XYZ scanner shown in fig. 1 are mirrors 18 and 20 that work together to redirect light along the optical axis of the objective lens 14, for example, when the XYZ scanner is arranged to operate in an inverted microscope configuration, to reflect light from the illumination unit 22 and from the autofocus unit 24 through the objective lens 14. The mirror 18 is configured and operable to move with the objective lens 14 in the X and Y directions, and the mirror 20 is configured and operable to move with the objective lens 14 and the mirror 18 in the X direction to ensure that light can travel along the optical axis of the objective lens 14. The mirrors 18 and 20 also reflect light received from the sample (including light from the illumination unit 22 or the autofocus unit 24 that reflects incident light or light generated by fluorescence of the sample) away from the sample along the optical axis of the objective lens 14.

As shown in fig. 1, the apparatus 10 further comprises an autofocus unit 24. The autofocus unit itself is used to focus a non-fluid immersion objective, i.e. an objective known in the art that does not use oil or water. The use as shown in fig. 1, autofocus is preferably an autofocus unit useful for high resolution, high throughput microscopy applications, such as that described in PCT patent publication WO 03/077008 entitled "Auto-focusing method and device" filed on 13/3/2003 or in U.S. patent No.7109459 of the same title, both of which are incorporated herein by reference.

In fig. 1, the autofocus unit 24 emits a laser beam having a wavelength that is transparent to the carrier carrying the sample, e.g. 635nm, which is then reflected by the beam splitter device (dichroic filter 26) onto the optical axis of the objective lens 14, from the mirrors 18 and 20 through the objective lens 14 onto the carrier in the sample holder. It is then reflected back along the same path and reflected back to the autofocus unit by the dichroic filter 26 where it is sensed by a sensor (not shown) and a controller (not shown) programmed to adjust the focal point of the objective lens along the Z-axis as necessary. When the autofocus unit 24 is used with a sample containing fluorescent markers, the wavelength of the autofocus light may be selected so as not to induce a fluorescent response in the sample, although this is typically not important, as the autofocus process will typically be completed before the image capture process begins. A similar procedure can be employed when using, for example, an oil immersed lens, but with certain changes due to the use of oil, as will be described in more detail below.

Also shown in fig. 1 is a lighting unit 22. The illumination unit 22 comprises an illumination source (not shown), such as a mercury lamp, LED lamp, laser or other suitable radiation source. The illumination unit 22 includes collimating optics, if desired. Where the sample comprises one or more fluorescent probes or the like, a suitable beam splitting device is arranged to reflect excitation light onto the optical axis of the objective lens 14. The beam splitter device may be a four-way filter 28 that reflects light of the excitation wavelength generated by the illumination unit, but allows light of other wavelengths to pass, particularly light generated by fluorescence of a fluorescent probe in the sample. It should be appreciated that illumination unit 22 may be configured to produce electromagnetic radiation of more than one wavelength, or more than one illumination unit may be employed to produce electromagnetic radiation of more than one wavelength, for example if multiple fluorescent probes are employed in the sample being observed, assuming appropriate beam splitting devices are also employed to ensure that excitation light is reflected onto the optical axis of objective lens 14 and that light of the wavelengths of interest (e.g., fluorescence produced by fluorescent probes in the sample) is passed. In addition, it should be appreciated that while fig. 1 shows the autofocus unit 24 positioned between the illumination unit 24 and the objective lens 14, in principle the positions of the illumination unit 22 and the autofocus unit 24 may be interchanged, as long as appropriate optics are provided to ensure that only light of the wavelength of interest passes through to the image capturing device 30.

As shown in fig. 1, light reflected by the sample or generated by the sample (by fluorescence), or if the sample is illuminated from the side of the upper surface 13b, light transmitted through the sample travels along the optical axis of the objective lens 14 and passes through the dichroic filter 26 and the four-way filter 28 before being detected by the one or more image capturing devices 30. FIG. 1 depicts an apparatus in which there are three such image capturing devices, namely three CCD cameras 30, 30' and 30", and in which after passing through beam splitter devices 26 and 28 but before impinging on the CCD cameras, light passes through tube lens 32, reflects off fold mirror 34 and is split by RGB prism 36, and then passes through emission filters 38, 38' and 38", which emission filters 38, 38' and 38 "filter out all light outside the emission band of the fluorescent probe in the sample. In fig. 1, emission filter 38 allows red light to pass, emission filter 38' allows green light to pass and emission filter 38 "allows blue light to pass. It should be appreciated that prism 36 may not be an RGB prism, and filters 38, 38' and 38 "may therefore filter different ranges of wavelengths.

The operation of the system shown in fig. 1 is controlled by one or more controllers (not shown) that are programmed together to control the operation of the autofocus unit, the illumination unit, and the movement of the XYZ scanner. An analysis unit (not shown) for analysing the image obtained by the image capturing device may also be provided, which may be part of one or more controllers or may be a separate unit, and may be configured to provide feedback to the one or more controllers. Furthermore, input and/or output devices (e.g., keyboard, optical or magnetic storage reader and/or writer, printer), display devices (e.g., plasma or LCD display), and storage devices may also be provided, as will be appreciated by those skilled in the art.

Those skilled in the art will appreciate that variations of the apparatus shown in fig. 1 may be employed in accordance with embodiments of the present invention; at least one such variant is described below for implementing an auto-focusing method when an oil immersed lens is used.

The XYZ scanner as shown in fig. 1 can be incorporated into an apparatus according to an embodiment of the present invention. For example, referring to fig. 2, a portion of a device 810 is shown in an isometric view, the device 810 being constructed and operative such that it can be adapted for use in accordance with an embodiment of the present invention. The device 810 includes a sample holder 812, the sample holder 812 holding a 96-well sample plate 813. The sample plate 813 is located in a plane perpendicular to the optical axis of the objective 814, and the objective 814 is part of the scanner 816 and is movable in three mutually orthogonal directions (i.e., X, Y and Z directions). The scanner 816 includes a mirror 818 and a mirror 820, the mirror 818 being mounted in a mirror mount 819 movable in the X and Y directions, the mirror 820 being mounted in a mirror mount 821 movable in the X direction.

Fig. 2 also shows an autofocus unit 824 and a mirror 827, the mirror 827 directing light from the autofocus unit to a dichroic filter 826 or vice versa; the dichroic filter 826 directs light from the autofocus unit 824 to the optical axis of the objective 814. Also shown is a portion of the illumination unit 822 (which includes an optical cable bundle 822a and collimating optics 822 b) and a four-way filter 828 arranged to reflect light from the illumination unit to the optical axis of the objective 814. Light reflected from the sample or generated by fluorescence in the sample plate (e.g., fluorescence of a fluorescent probe) and not filtered by the four-way filter 828 is then focused by the tube lens 832 and reflected by the folding mirror 834 to the camera 830; filter wheel 837 contains filters 838, 838', 838 "that can be selected to filter light entering camera 830.

According to some embodiments of the invention, the device is equipped with a coupling mechanism to facilitate replacement of the objective lens, but it should be understood that the mechanism can be used in other optical instruments. Referring now to fig. 3-9, one embodiment of such a mechanism is shown. As shown in fig. 3, 8 and 9, the objective 1010 is permanently mounted to an objective mount 1020. The objective 1010 and the objective mount 1020 together form an objective unit 1060. An 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 mounted on top of the Z-axis component of the XYZ scanner such that an objective lens unit 1060 containing an objective lens (e.g., lens 338) may rest thereon, as now described; alternatively, the motion base may be formed as part of the top of the Z axis component of the XYZ scanner.

As now described, the attachment between the objective lens mount 1020 and the kinematic mount 1040 uses a particular kinematic mount that provides positioning accuracy in the 50 nm range or better, as shown. The objective mount 1020 contains a plurality of coupling balls 1030 (three such balls are depicted in fig. 3-9) and is machined such that, after being set in place, the spatial position of the coupling balls 1030 relative to the optical axis of the objective 1010 has a high accuracy. The coupling ball 1030 has a high rigidity (e.g., a hardness of 53-58RCSS or higher) and has a suitable diameter, e.g., 3-3.5 mm. Each coupling ball 1030 is held in place in one of the holes 1020a formed in the lower surface of the objective lens base 1020 such that 30-40% of the ball diameter protrudes downwardly beyond the lower surface of the objective lens base 1020. The apertures 1020a may only partially penetrate into the objective base 1020, forming a closed-end cylinder, or they may pass completely through the bottom of the objective base 1020. Each aperture 1020a has an interference diameter tolerance with a coupling ball 1030 placed therein, thereby firmly securing the ball in place. The objective lens mount 1020 is made of ferromagnetic steel such as 17-4 PH.

As shown, the kinematic base 1040 is generally annular in shape and has a plurality of notches formed in its upper side in the form of V-shaped grooves 1040 a. The spacing of the coupling balls 1030 and the grooves 1040a is such that the coupling balls fit into three grooves, as shown in simplified form in fig. 7. Kinematic base 1040 may also be made of high performance ferromagnetic steel (e.g., 17-4 PH) that has been heat treated to a surface hardness of 39RC or higher such that kinematic base 1040 maintains attachment accuracy during periodic loading and unloading operations of lens unit 1060. To ensure that the kinematic base 1040 has the desired surface hardness, the following manufacturing process is performed: (a) Manufacturing the part to final dimensions, leaving 50 microns for the final groove 1040a grinding process; (b) performing a thermal hardening process; (c) grinding the V-shaped grooves 1040a to a final size.

As shown, the kinematic base 1040 is formed with three apertures 1040b therethrough, with the three apertures 1040b being substantially evenly spaced about the base. A magnet 1050 is inserted into each hole 1040b and glued in place. When the components are very close together, the magnets 1050 together cause a magnetic attachment force with the objective lens unit 1060. The attachment force positions the objective lens in place by balancing the attachment force applied to the kinematic coupling and holds the objective lens unit 1060 in place when the optical system is moving with high acceleration. It should be appreciated that the magnet does not have to be in contact with the objective lens base 1060 nor does it have to protrude through the aperture 1040b as shown, for example, in fig. 9. Thus, in this application, when describing such magnets as being "mounted within" a surface "opposite to a ferromagnetic surface, the magnets may protrude from, or be embedded within, or even beneath the surface in which they are" mounted "because the magnetic attraction forces may function without direct physical contact.

Although fig. 3-9 illustrate a particular embodiment of a coupling mechanism, it should be understood that variations of the illustrated content are possible. This is because the combined action of the ball 1030 and the recess 1040a is to not only precisely position the objective unit 1060 in the XY plane but also to limit the movement of the objective unit 1060 in the XY plane and in the negative Z direction, and the addition of magnetic force limits the movement of the objective unit in the positive Z direction, and these effects can in principle be achieved by other means. Thus, for example, more or fewer apertures 1040b and correspondingly more or fewer magnets may be employed, and the magnets may be mounted in the bottom of the lens objective unit as well as in the motion base, or the magnets may be mounted exclusively in the bottom of the lens objective unit. Similarly, the positions of the recess and the ball may be interchanged such that the bottom of the objective unit 1060 contains the recess (e.g., the groove) and the upper surface of the motion base has the ball protruding therefrom, or both the objective unit and the motion base may have the recess and the ball, or the motion base may have the protrusion and two grooves and the bottom of the objective unit 1060 may have the corresponding groove and two corresponding protrusions.

Further, the notches may be other than V-shaped grooves, for example, one or more of the notches may be well-shaped, providing three points of contact for the ball 1030 resting therein instead of two points of contact as in a V-shaped groove. One such well, in combination with a single V-groove and surface of the motion base and a suitably positioned magnet of sufficient strength, may have the same effect as three V-grooves. In addition, protrusions other than spheres that fit into these recesses may also be used. Thus, for example, those skilled in the art will appreciate that while a plurality of balls 1030 are shown in the figures as being held in holes, other construction means are possible, such as a round head nail may be used.

The relative positions of the notches may also be different from those shown in fig. 3-9: the recess may be arranged such that the protrusion from the counter piece fits therein in only one way, thereby providing only one way to set the objective unit in place on the motion base. Alternatively, three protrusions (to ensure that the part with protrusions is located on a plane) may be used, for example as described above in relation to the objective lens unit, but there are three or six V-shaped grooves in the counter piece (e.g. as shown above in relation to the motion base), a larger number (e.g. 9 or 12) of evenly spaced, radially oriented V-shaped grooves may be employed in order to more easily place the lens unit, for example when used in an objective lens changer like the one described below.

Furthermore, while fig. 3-9 illustrate an objective lens unit 1060 formed from the objective lens 1010 and the objective lens base 1020, it should be appreciated that the objective lens 1010 may be formed in a manner that does not require the objective lens base 1020, for example if the bottom of the objective lens 1010 is made of ferromagnetic material and is machined to have a coupling ball 1030 protruding therefrom. It should be understood that when it is stated in the description or claims that the objective lens has a "surface associated with it", this surface may be the surface of the objective lens assembly itself, or it may be the surface of the lens holder or the surface of the base on which the objective lens is mounted, as shown in fig. 3-9.

The above-described coupling mechanism enables the objective lens to be repeatedly inserted into and removed from the optical device with sufficient accuracy to allow high-accuracy observation using the objective lens. Thus, the mechanisms described herein facilitate the use of multiple objectives in an optical device, as lenses can be swapped in and out; in the case of the apparatus shown in the figures, this can be achieved without adding a weight load to the whole objective lens to the XYZ scanner, which is therefore advantageous in achieving a higher acceleration and a faster settling time than is achieved by the XYZ scanner bearing the weight of the whole objective lens. Instead, the objective lens may be stored elsewhere in the device and replaced as needed when it is desired to change magnification. In addition, although turrets carrying multiple objectives are known, the objectives in such turrets are arranged such that the objective in use is aligned with the focal path along the Z-axis, while the objective not in use is at an angle to the Z-axis, so switching one oil immersed lens to another causes oil loss as the turrets rotate and the lenses move to angles away from the vertical. Thus, as shown in fig. 10 and 11, the lens changing subassembly 1000 includes a cassette 1080, the cassette 1080 holding a plurality of objective lens units 1060 in a plurality of stations 1070. Each of the objective units 1060 in the cassette 1080 is substantially aligned with the Z-axis of the XYZ scanner 1090 and substantially perpendicular to the plane of the motion base 1040 connected to the upper portion of the XYZ scanner 1090, and further, as described below, the coupling balls 1030 protruding from the bottom of each of the objective units 1060 are positioned such that they will engage with the V-shaped grooves 1040a in the motion base when the XYZ scanner is raised to contact the objective units. Each station 1070 includes a pair of arms 1072, such that each pair of adjacent arms is capable of holding an objective lens unit 1060. As shown in fig. 10 and 11, the cassette 1080 holds three objective lens units 1060, and in principle the optical device may be designed to hold more of these objective lens units.

To illustrate how the coupling mechanism is used to replace the objective lens, it is assumed that the scanner 1090 has no objective lens unit attached initially, as shown in fig. 10, and that at least one objective lens unit 1060 is loaded onto one station 1070 of the cassette 1080. Scanner 1090 first moves to a Z position that is low enough to enable it to move under the selected objective lens unit 1060. The XYZ stage is then moved in the XY plane to a position in which the optical axis of the selected objective lens placed in station 1070 is approximately aligned with the optical axis required for the objective lens once positioned for use on XYZ scanner 1090. The Z stage is then moved upward to contact the lower portion of the selected objective lens unit 1060. This causes the coupling ball 1030 to rest in the recess 1040 a. The magnet 1050 is close to the bottom of the objective unit 1060, which is made of ferromagnetic material and is thus attracted by the magnet, causing the coupling ball 1030 to settle in the groove and be held there. As shown in fig. 3-11, when the coupling balls and V-grooves are machined to be sufficiently precise (e.g., within a 50nm tolerance), three coupling balls spaced about 120 ° apart and V-grooves oriented radially from the center of the motion base are used, and the coupling balls fit in the V-grooves, which allow the optical axis of the objective lens to be set with sufficient precision so that the objective lens can be used without further calibration. The Z stage moves further upward enough to lift the objective unit 1060 off of the arm 1072. The XY stage may then be moved out of station 1070. The optical system is now ready to be operated with the selected objective lens.

When changing from the first objective lens to the second objective lens, a similar process is repeated. The scanner is moved to the open station 1070 and operated in reverse order of the above order, placing the first objective lens unit 1060 in the station 1070. The XYZ stage is then moved to a different station of the cassette 1080 and loads the second objective lens in a similar manner as described above for the first objective lens.

It will be appreciated that the movement of the XYZ scanner may be automated, by means of a suitable motor and controlled by a microprocessor programmed for this purpose.

It should also be appreciated that although in fig. 10 and 11, XYZ optical scanner 1090 is shown as a device for facilitating transfer of an objective lens onto and off of an optical system, the methods described are not limited to use with XYZ optical scanners and can be used in any system having moving optical elements controlled by motors, encoders, sensors, servo controllers, or other automated element combinations. Furthermore, in the event that the moving optical system is not able to reach all of the objective lens units, an auxiliary motion system (not shown) may be employed to move the cassette 1080 so that the particular lens to be used is in a position accessible to the optical system.

The lens exchange subassembly 1000 described herein with reference to fig. 3-11 is particularly advantageous for automated high content or high throughput screening of biological samples using a liquid immersion objective. However, it should be understood that in some embodiments of the present invention, the apparatus or device does not have a lens replacement subassembly, an apparatus or device using only a single objective lens is large enough in the X and/or Y directions that (a): the objective lens is capable of moving away from the sample holder along the Z-axis while maintaining its three-dimensional orientation (e.g., pitch, roll, and yaw), and moving to an oil-filled position in the XY-plane, then returning to the same position in the XY-plane after oil filling, and continuing to scan from the neutral position after refocusing in the Z-direction; (b): the sample frame can move from a fixed position to scan while maintaining the orientation of the sample frame in an XYZ coordinate space so as to facilitate oil supplement, and then move back to the same position where the scanning is interrupted, so that the scanning can be continued from the position where the scanning is interrupted after the objective lens is refocused in the Z direction; or (c): a combination of (a) and (b).

One of the challenges of immersion scanning of biological samples is the need to periodically replenish the liquid on the objective lens. This is particularly important when scanning is performed with the objective lens moving relative to the sample and approaching or contacting the surface of a sample holder (e.g. a multiwell plate, petri dish or sample carrier slide), as the relative movement between the objective lens and the sample holder causes diffusion of the immersion liquid until the immersion liquid layer is insufficient to properly view the sample. Thus, the scanning is limited to the distance the objective can move before the immersion liquid has to be replenished.

As noted in the background discussion above, prior art systems for oil replenishment have various drawbacks, such as that they require additional items to be placed on the objective itself, thereby increasing the weight of the objective assembly and its effective size, which reduces the area of the sample being scanned and limits the proximity of the lens itself to the sample holder. In addition, when this is done manually, the human user must stop the operation of the system and replenish the immersion liquid onto the objective either manually or with the aid of specially designed devices. This greatly delays the operation of the system and requires periodic attention from the operator. It is therefore also more prone to error. In addition, the need to manually replenish the immersion liquid limits or prevents large screening processes that scan many locations that are far apart from each other.

Another challenge with manually applying immersion oil to the objective lens is that the operator may apply too little oil, resulting in frequent stops of the microscope system. Alternatively, the operator may apply too much oil, resulting in oil spillage during movement of the objective unit, e.g. from the point where the operator applies immersion liquid to the scanning position. Another problem is that in order to avoid the lens moving from its focus position, it has been necessary to date to perform oil replenishment while keeping the objective lens in a position close to the surface of the sample holder (e.g. close to the bottom of the perforated plate), which leaves little operating space for adding oil to the lens.

According to an embodiment of the present invention, as described below with respect to fig. 12-19B, the lens changing subassembly forming part of the microscope system ensures that there is a fixed position to which the objective lens can be automatically moved and in which the objective lens is not arranged below the sample. The presence of such a fixed position facilitates the construction of the innovative immersion liquid loading sub-assembly discussed below directly above the predetermined position of the cassette of the lens exchange assembly. As described below and in accordance with an embodiment of the invention, in use, the immersion objective is periodically moved to a predetermined position of the cassette of the lens changing assembly and oil is automatically applied to the objective from the liquid loading subassembly, after which the system automatically returns the objective to the scanning position in order to continue the scanning process.

It should be appreciated that the immersion liquid subassemblies described below and methods of use thereof are suitable for use with any suitable immersion liquid, such as synthetic hydrocarbon-based oils, water, glycerol, silicone oils, and the like, and the scope of the disclosure herein should be construed to include all types of immersion liquids suitable for microscopy, particularly for microscopy of biological samples. However, for the sake of brevity and clarity, the following discussion relates to synthetic hydrocarbon-based oil impregnation, i.e., to oil targets, loading of the oil impregnation, etc.

Reference is now made to fig. 12, which is a partially exploded view of an oil loading subassembly 200 according to an embodiment of the present invention, the oil loading subassembly 200 having an oil immersion cartridge 300 disposed above a lens changing subassembly 400. The lens changing subassembly 400 is substantially similar in structure and operation to the lens changing subassembly 1000 described above. As shown, the lens changing subassembly 400 includes a lens holding cassette 410, the lens holding cassette 410 having a plurality of positions, here shown as three positions, for holding the objective lens. The leftmost position 412 of the cassette 410 is shown temporarily accommodating an oil immersion objective unit 500, similar to the objective units described above with respect to fig. 3-9. The leftmost position 412 is predetermined as an oil loading position such that the oil outlet of the oil loading subassembly 200 is disposed above the oil loading position 412 in a position to drop oil onto the objective lens of the objective lens unit 500. The oil immersion cartridge 300, which will be described in further detail below, is positioned above a corresponding well in the oil loading subassembly 200 and is adapted for insertion therein, as explained herein. It should be appreciated that because location 412 is reserved for oil loading, lens exchange subassembly 400 as shown may enable exchange between two lenses that may be stored in the other two locations to the right of location 412. It should also be appreciated that the lens exchange subassembly 400 may be formed with additional locations to facilitate exchange between a greater number of lenses. Alternatively, as previously mentioned, there is in principle no need for a lens exchange sub-assembly, for example if there is enough space in the device to provide an oil loading location far enough from the sample so that scanning is not limited to scanning only a portion of the sample.

The oil loading position 412 is selected such that it is always outside the scan path of the lens that scans the sample.

In some embodiments, the oil loading position 412 of the lens changing subassembly 400 may include a weighing mechanism for determining the weight of the objective lens unit 500 when in that position. For example, the weighing mechanism can obtain a reference weight or a peeling weight when the objective unit 500 is placed in the oil loading position 412. The weighing mechanism is thus able to recognize a change in weight of the objective unit, for example after immersion oil has been applied thereto. Based on the nature of the immersion oil, a controller associated with the weighing mechanism can determine how much immersion oil is applied to the objective unit 500 and whether additional oil application is required.

The weighing mechanism may be any suitable mechanism for weighing the objective lens unit 500. In some embodiments, the weighing mechanism may include a flexible blade 420 attached to the frame structure 402 of the lens replacement subassembly 400. A pair of flexible arms 422 extend from the blade 420, the arms being arranged around the objective unit 500 when the objective unit is in position 412, such that the objective unit 500 rests on the arms 422. For example, in the free regions of the arms, attached to one or more surfaces of each arm 422, but in some embodiments there are thin, flat strain gauges (not explicitly shown) near the blade 420 and/or near the frame structure 402. Each strain gauge is electrically connected (e.g., by a thin wire, not shown) to a thin, flat electronic card (not explicitly shown) located below the arm 422. The electronic card may be positioned such that the length of the connection between the strain gauge and the card is minimized. The electronic card is electrically coupled to a processor (not shown). It will be appreciated that the use of a strain gauge and electronic card allows the deflection of the arm 422 to be correlated to the change in resistance in the circuit, for example using a wheatstone bridge measurement also located on the electronic card, allowing the weight change of the objective unit 500 to be calculated, and thus the mass of immersion oil applied to the objective. Thus, the arm 422 together with the strain gauge forms a signal provider for providing a signal indicative of the amount of immersion oil applied to the objective unit. If the density of the immersion oil is known, it helps calculate the volume of immersion oil added. For example, the amount of immersion oil applied to the objective lens may be iteratively calculated after each application of immersion oil, and thus it is recognized in real time whether a sufficient amount of immersion oil has been applied. As such, a controller associated with the weighing mechanism may be functionally associated with the oil pump of the oil loading subassembly 200 such that operation of the pump applying the immersion oil is started and stopped based on information related to the weight of the objective lens unit, as described below.

In some embodiments, an overflow tray (not explicitly shown) may be provided below the cassette 410, or at least below the oil loading position 412, to collect any overflow immersion oil that drips from the oil loading subassembly 200 onto the objective lens unit 500 or immersion oil that may overflow when no objective lens is placed in the oil loading position. It should also be appreciated that the presence of a holder for a lens (e.g., the objective lens mount 1020 in fig. 5) may provide an oil catcher as the oil flows out of the objective lens.

Alternatively, the amount of oil added can be calculated by calibrating the average amount of a drop of oil 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 beam of light connected to a sensor that provides a signal each time the drop of oil interrupts the beam of light; or by a camera coupled to an automated image analysis program to detect and count dropped oil droplets. Alternatively, each time the oil on the objective lens is replenished, the amount of oil removed from the oil reservoir can be calculated (e.g. by measuring the weight change in the reservoir) to calculate the amount of oil applied to the lens.

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

Reference is now made to fig. 13, which is an exploded view of an oil loading subassembly 200 in accordance with an embodiment of the present invention, and to fig. 14A, 14B, 14C and 14D, which are perspective, front plan, side plan and cross-sectional views, respectively, of the oil loading subassembly 200 as constructed and with an oil immersion cartridge 300 disposed therein. Such cartridges may be made of any suitable material that does not significantly interact with or cause degradation of the oil or other liquid contained therein. In the embodiment shown in fig. 13-18B, the cassette is preferably made of metal, such as aluminum. In the embodiment shown in fig. 24-26B, the cassette is preferably made of plastic.

As shown, the oil loading subassembly 200 includes a body portion 210 and a pump seat bore 216, the body portion 210 having a central oil basin 212 in fluid communication with an oil viewing opening 214, the pump seat bore 216 terminating in a pump inlet conduit 217 (see fig. 14D). As shown in fig. 14D, an oil flow conduit 218 extends from the base of the oil basin 212 and adjacent the pump inlet conduit 217 and terminates at the edge of the body portion 210, with the oil flow conduit being sealed by a barrier 220. A viewing window 222 is disposed within the oil viewing opening 214 and seals the oil viewing opening 214. As best seen in fig. 14B, the viewing window 222 includes a generally circular transparent portion 224 that allows an operator to view the oil level in the oil loading subassembly 200 and determine when the oil immersion cartridge 300 must be replaced, as explained in further detail below. In a variant thereof, the window may be provided with a cover to block light out of the window when the user does not observe the oil level directly; alternatively, the oil loading subassembly 200 can be provided with neither the viewing opening 214 nor the window 222.

A cartridge piercing element 230 is located within the central oil basin 212 and is attached thereto by fasteners 232 (e.g., screws or bolts). However, the fasteners 232 may be replaced by any other suitable attachment mechanism, such as welding, adhesive, or the like. Cartridge piercing element 230 includes a base 234 and a piercing needle 236, with piercing needle 236 having a hollow passageway 238 extending longitudinally therethrough, which includes an aperture 239 (see fig. 15B-15C) and terminates in a sharp point. When the dip tank 300 is installed in the center oil pan 212, as shown in fig. 14A-14D, the hollow passage 238 is in fluid communication with the center oil pan 212 and the oil flow conduit 218 (see fig. 14D and 16A).

The diaphragm pump 250 is disposed within the pump seat bore 216 such that its inlet 252 is located within the pump inlet conduit 217 and its outlet conduit 254 extends downwardly out of the body portion 210. The diaphragm pump 250 is adapted to draw oil from the oil flow conduit 218 through the outlet conduit 254 onto an item arranged below the outlet conduit, which item in use is an oil immersion objective.

The oil loading subassembly 200 also includes a base mount 260, the base mount 260 being mounted to the underside of the body portion 210 and adapted to attach the oil loading subassembly 200 to other components of the microscope system, above the lens replacement subassembly 400. A rear mount 265 is mounted to the main body portion 210 rearward of the center oil basin 212. Both the base mount 260 and the rear mount 265 may be connected to the body portion 210 by fasteners 268 (e.g., screws, bolts, etc.). However, any other suitable attachment means, such as bonding, welding, etc., are considered to be within the scope of the present invention.

Referring now to fig. 15A, 15B and 15C, there are cross-sectional views illustrating the steps of inserting the dip tank 300 into the oil loading subassembly 200. As shown in fig. 15A, the oil immersion cartridge 300 includes a housing 301 formed of a generally cylindrical wall portion 302, the wall portion 302 defining a cavity 304 having a first inner diameter and terminating in a lip 306. At its end remote from the lip 306, the wall portion 302 extends to a generally transverse shoulder 310 which narrows the inner diameter of the wall portion to form a generally cylindrical hollow neck portion 312 having a second inner diameter. The second inner diameter of the neck portion 312 may be smaller than the first inner diameter of the wall portion 302. At an end of the neck portion 312 distal from the shoulder 310, the wall of the neck portion 312 is reduced in thickness, thereby defining a first chamber having a third inner diameter that is 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 portion comprises snap-fit protrusions and/or recesses 319 adapted to snap-fit with corresponding recesses and/or protrusions 219 in the inner periphery of the central oil basin 212.

A generally annular transverse wall 320 extends radially inward from the wall portion 302 at a location disposed between the lip 306 and the neck portion 312, with a distance to the lip 306 being substantially less than to the neck portion 312. The annular transverse wall 320 terminates radially inward in a shroud portion 322, the annular transverse wall 320 being generally concentric with the wall portion 302. The volume between the annular transverse wall 320 and the lip 306 defines a second chamber. A fluid flow path exists between the first chamber and the second chamber via the hollow defined by the neck portion 312, the hollow defined by the wall portion 302, and the hollow of the cap portion 322.

A puncture piston 330 is disposed within the housing 301. The piercing piston 330 includes a generally circular base 332 from which a central shaft 334 extends and terminates in a sharp edge 336, which sharp edge 336 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 connecting the hollow 338 with an environment surrounding the shaft 334. The remainder of the shaft 334 is non-hollow.

In the initial, closed, operational orientation of the oil dip canister 300 as shown in fig. 15A, the first seal 350 may be a material inert to oil (e.g., nylon or aluminum foil) disposed in the first chamber and engaging the annular shoulder 316. The seat 332 of the piercing piston 330 is disposed within the hollow of the neck portion 312 and engages the first seal 350 such that the shaft 334 extends through the hollow defined by the neck portion 312, the wall portion 302, and the cap portion 322. The sharp edge 336 does not extend beyond the end of the annular transverse wall 320 and may be flush therewith. The second seal 352 may be a material inert to oil (e.g., nylon or aluminum foil) disposed in the second chamber and engaging the transverse wall 320. Thus, in the closed operational orientation of the oil impregnated canister 300, the canister is sealed with oil disposed therein (oil not shown in fig. 15A for clarity).

To use the immersion oil in the cartridge 300, the user moves the cartridge in the direction of arrow 360 such that the first seal 350 faces the center oil basin 212 and the piercing element 230 disposed therein.

Turning to fig. 15B, it can be seen that when a user pushes the dip tank 300 into the center oil basin 212, the piercing element 230 of the center oil basin 212, and in particular the piercing pin 236, pierces the first seal 350 and pushes the base 332 of the piercing piston 330 toward the second seal 352. Thus, the entire piercing piston 330 is moved in the direction of arrow 362 toward the wall portion 320 and the sharp edge 336 of the shaft 334 pierces the second seal 352. The snap-fit protrusions and/or recesses 319 of the dip tank 300 engage with corresponding recesses and/or protrusions 219 in the inner periphery of the central oil basin 212, ensuring a snap-fit of the tank within the well. In this orientation, seals 350 and 352 are pierced, air can flow into the oil impregnated cartridge 300, and oil can flow out of the cartridge, as shown in fig. 15C. Thus, the oil flows out of the cartridge, around the base 332 of the piercing piston 330, and through the passageway 238 and bore 239 of the piercing element 230 into the central oil basin 212.

Reference is now additionally made to fig. 16A and 16B, which are cross-sectional views illustrating the oil flow path within the oil loading subassembly 200. As shown, after unsealing the dip tank 300 as shown in fig. 15A to 15C, oil flows from the center oil pan 212 to the pump inlet pipe 217 via the oil flow pipe 218. It should be appreciated that the cartridge 300 may remain in place after it has been placed and pierced to release oil into the basin 212 in order to reduce the likelihood of dirt or other material contaminating the oil, or the device may be provided with a cover (not shown) that covers the basin 212 when the cartridge 300 is removed.

Operation of the diaphragm pump 250, for example in response to a signal or input received from a controller, causes the pump to draw oil therein via an inlet 252 located in the pump inlet conduit 217, as shown in fig. 16A. The oil drawn into the diaphragm pump 250 is then dropped from the pump via the pump outlet conduit 254, as shown in fig. 16B. In operation, an oil-filled objective unit is disposed below outlet conduit 254 such that oil droplets fall onto the objective, as described in further detail below.

Reference is now made to fig. 17, which is a perspective illustration of the lens changing subassembly 400 and the oil loading subassembly 200, wherein the objective lens unit 500 is disposed in the oil immersion loading position 412. Referring additionally to fig. 18A and 18B, there are front and side views, respectively, of the immersion oil loaded onto the objective lens in the configuration of fig. 17.

As shown, the dip tank 300 is mounted in the oil loading subassembly 200, and the oil loading subassembly 200 is mounted above the lens changing subassembly 400. The objective unit 500 containing the oil immersed lens 502 is located in the oil loading position 412 of the lens changing subassembly such that the outlet tube 254 of the diaphragm pump is located directly above the oil immersed lens 502. As shown in fig. 18A, upon receipt of a suitable control signal triggering operation of the diaphragm pump 250, immersion oil droplets 504 are discharged from the outlet conduit 254 onto the immersion lens 502. In some embodiments, the oil droplets comprise 20 to 30 μl of oil. The oil droplets remain as hills 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 changing subassembly 200, such that the lens 502 is proximate to the outlet opening of the outlet conduit 254. This ensures that the drained oil drips directly onto the lens and is not wasted.

In some embodiments, multiple drops of oil may be applied to the lens unit 500 each time an oil load is applied. Once a sufficient amount of oil is applied to the lens unit 500, the lens unit may be returned to its scanning position, for example as described above with respect to fig. 10 and 11. As explained in further detail below, the amount of oil that is dropped onto the lens may be estimated, for example, based on an expected oil spread taking into account the motion that the lens has performed, or may be calculated, for example, by a weighing mechanism as described above.

Reference is now made to fig. 19A, 19B and 19C, which illustrate the diffusion of immersion oil as the objective unit moves relative to the viewing surface during use of the microscope system.

As shown in fig. 19A and 19B, a porous plate 600 is provided in a plate holder 602. The multiwell plate has a lower surface 604 and a plurality of wells 606, each well comprising a biological sample. Oil immersion lens unit 500 is disposed below lower surface 604 and is initially positioned to scan a first sample in well 606 a. As shown in the enlarged portion of the figure, at this stage, i.e., at an initial stage after the immersion oil is loaded onto the lens unit 500, oil droplets disposed on the lenses of the lens unit 500 are spread to the lower surface 604 by close proximity to the lens unit 500, forming an oil layer 610. Any excess oil may flow to the inclined edge 510 of the objective unit and may be caught in the circumferential groove 512 at the top portion of the objective unit. The surface tension of the immersion oil ensures that the oil and excess oil will remain engaged with the objective lens unit on one side and the plate on the other side. As will be discussed in detail below, the oil is typically formulated to have a refractive index that is the same as the refractive index of the bottom of a multiwell plate or other sample-holding container; typically the base is made of glass.

Fig. 19B shows lens unit 500 after lens unit 500 has moved along plate 600, scanned wells 606B and 606c, and now has reached well 606 d. At this stage, the thickness of the oil layer 610 is reduced relative to that shown in fig. 19A. This is because immersion oil has spread along the lower surface 604 of the plate by the movement of the objective lens unit. As explained in further detail below, a controller associated with the objective unit (e.g., the microscope system discussed above or the controller of XYZ movement) may be configured to determine that the distance of movement of the objective unit has reached a threshold value, at which point immersion oil is expected to spread too thin, and to control operation of the objective unit to return the objective unit to the oil loading position to apply additional oil thereto.

Fig. 19C shows five different states of oil droplets disposed on top of the lens unit 500. As shown, in the initial stage (i), which typically occurs immediately after the oil is applied to the lens unit, the oil droplets form domes according to the surface tension of the oil. In fact, the oil droplets at this time are actually in the shape of droplets.

At (ii), the lens unit is proximate to, but not yet diffusing along, the surface 604 of the plate. At this time, the droplet is vertically stretched between the lower surface of the plate and the upper surface of the lens unit. This configuration may occur when approaching the plate or when the lens unit is lowered relative to the plate during separation of the oil droplets from the plate.

At (iii), the lens unit has been brought closer to the surface 604 such that the oil droplets spread along a portion of the plate surrounding the lens unit area. However, the oil is still contained only in the lens unit region. In some embodiments, the configuration of oil droplets as shown in (iii) may occur when the height is equal to the initial scan height, as described below with respect to fig. 21A and 21B.

Image (iv) shows the configuration of the oil after focusing of the objective lens and during scanning of the initial area or well of the plate, as described in detail below. As shown, the oil is more dispersed with respect to (iii) but still only above the objective lens and substantially concentric therewith. After the lens unit is moved to scan another area of the well or another well, for example, some oil remains on the previously scanned plate portion of the objective lens, and some oil moves with the objective lens to the new area, causing the oil to also spread to an area not directly above the objective lens, as shown in (v), wherein the objective lens moves to the right with respect to the plate, and the oil now spreads over the objective lens and to the left of the objective lens.

Fig. 20 is a flowchart outlining a method for scanning a sample using an oil immersed lens and reloading immersion oil onto the oil immersed lens in accordance with an embodiment of the present invention. The following description is made with respect to the plate 600 shown in fig. 19A and 19B, the plate 600 having a lower surface 604, and each well having an upper surface on which a biological sample is disposed. However, the present disclosure is equally applicable to any other sample carrying structure having a lower surface and an upper surface.

As shown in fig. 20, in a first setting step 700, the Z-axis position of the lower surface 604 of the board 600 is roughly estimated. This approximation is stored and will be used as an approximation of the plate height for scanning the entire plate. Such a rough estimation step may be performed for each individual plate examined, or may be performed once for a plurality of plates or sample carriers.

In some embodiments, a rapid laser scan may be used to perform a rough estimate of the Z-axis position of the lower surface of the plate, identifying the bottom surface of the plate at several locations for which the scan is planned. In some other embodiments, a plurality of focal points, e.g., 3 or 4 focal points, may be determined for a plate or region of a plate using laser scanning, and an approximate plane of the lower surface of the plate is determined based on the plurality of focal points.

As explained below, the approximate height of the lower surface of the plate is used as a basis for a subsequent focusing operation of the plate, which is performed using a search of the maximum signal reflected by the laser beam, as explained herein. There are various ways in which this initial rough estimate may be performed. One option is to determine the approximate height of the lower surface of the plate using an air objective, and then switch to an oil immersion objective for 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 of plate from a particular manufacturer will be a given distance above the bottom edge of the plate, and knowing the size of the device. In some embodiments, an initial rough estimate of the height of the lower surface of the plate may be performed by 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 used for the actual scan.

In a second setting step 702, one or more scan areas of the plate or slide are defined, for example using a user interface, and provided to a controller that controls XYZ movement of the objective lens as described herein. In a third setting step 704, before the oil-immersed objective is replenished with oil, a maximum threshold amount of oil that it can lose is determined; this amount can be tracked based on total movement of the objective lens, weight loss, or oil thickness, as described below. In some embodiments, the system may be programmed to dynamically adjust the threshold after each oil refill based on the amount of oil applied to the lens.

Although the setting steps 700, 702 and 704 are depicted in a particular order in fig. 20, they may be performed in a different order as long as they occur before the scanning of the biological sample is initiated.

In step 706 (which also occurs before starting scanning), the objective lens unit is placed in the oil loading position of the lens changing assembly, and an initial amount of oil is dripped from the oil loading subassembly onto the objective lens, as described herein. For example, when the objective lens is in the correct position, a controller associated with the diaphragm pump provides a signal to the pump to drop the immersion oil onto the objective lens.

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

In some embodiments, the exact amount of oil loaded onto the objective lens is determined using a weighing system as described above. In some other embodiments, the amount of oil loaded onto the objective lens is roughly estimated based on the number of oil droplets applied to the objective lens, the volume of immersion oil per droplet pumped by the pump (which is a known value), and the amount of oil required to be aspirated.

In step 708, the controller controls the XYZ motion of the oil loaded objective lens to bring the lens to the appropriate XY position for scanning, as determined in step 702. When scanning is started, the XY position is a position where scanning should be started. After a later iteration of replenishing the objective lens with oil, the XY position is the XY position where the scanning is stopped to replenish the immersion oil, or the next XY position, as described below. In step 710, the controller raises the objective lens to a predetermined initial scan height, typically a few microns from the approximate Z-axis position of the lower surface of the plate, as established in step 700. The predetermined height is selected to be close enough to the lower surface of the plate so that oil droplets on the objective lens spread onto the bottom surface of the plate, as shown in fig. 19A.

In step 712, the distance of the plate relative to the sample while the sample is in focus is automatically calculated, for example, using the method described below with reference to fig. 21, and the objective lens is moved to focus. Once the objective lens is at the established focal length, scanning of the sample begins in step 714.

During scanning, the objective lens is moved between different positions relative to the sample or plate 600, as described above with respect to fig. 19A and 19B. The distance travelled by the objective lens is tracked, for example by a controller associated with the objective lens, including movement when the oiled objective lens is in contact with the surface of the plate and movement when the objective lens is not in contact with the plate. The controller can be configured to calculate the amount of oil lost by the objective lens due to such movement, as well as the amount of oil lost when the objective lens is lowered along the Z axis out of contact with the plate. In some embodiments, the controller also considers the effect of differences in plate material on the amount of oil lost as the objective lens moves along or out of contact with the plate. Alternatively or additionally, the amount of oil loss may be tracked by monitoring the change in weight of the oiling objective lens since it was oiled, and/or by checking the thickness of the oil on the objective lens (e.g., measured with a camera or by a laser).

In step 716 (which may occur continuously or periodically), the controller checks whether the amount of oil lost due to 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, then the scan continues in step 714.

Otherwise, if the calculated oil loss amount is equal to or greater than the maximum threshold amount, the objective lens is lowered relative to the plate 600 in step 718 and moved in the XY plane to the oil loading position. A supplemental 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 supplemental oil amount may be less than the initial oil amount. As mentioned above, the amount of oil drop on the objective lens that needs to be replenished may be calculated, for example based on a weighing mechanism, or may be coarsely estimated based on the distance travelled by the objective lens or based on any other relevant parameter. The flow then returns to step 708 for moving the objective lens back to the scanning position to continue scanning the sample.

Reference is now made to fig. 21A, 21B, and 21C, which are flowcharts summarizing methods for automatically focusing an oil-borne oil saturation mirror on a biological sample, in accordance with embodiments of the present invention. The method of fig. 21A-21C assumes that an initial scan height is known, typically a few microns from the approximate Z-axis position of the lower surface of the plate. The initial scan height is typically based on the height of the lower surface of the plate, such as the initial scan height established in step 700 of fig. 20. This autofocus is performed each time the lens is repositioned at a different XY position, including after oil replenishment.

The initial scan height is the height at which the oil droplets provided on the oil objective engage and spread between the lower surfaces of the oil objective and the plate, as is known. As mentioned above, the oil is typically formulated to have the same refractive index as the material (typically glass) that makes up the bottom of the plate. Thus, the immersion oil and initial scan height are selected to ensure that no undesired reflections or refractions occur as light passes between the oil and the porous plate material, i.e., to ensure that the oil is in contact with both the lens and the plate bottom. Thus, when a light beam enters the sample from the objective lens via the oil and the plate material, the light will only be refracted once at the transition between the plate material and the sample material, which occurs at the upper surface of the well.

Turning to fig. 21A, it can be seen that in an initial step 750 of the focusing process, the objective lens is brought to a known predetermined initial height such that the oil is in contact with the lower surface of the plate. This corresponds to step 710 of fig. 20. A delay of a predetermined duration (e.g., 500 to 2000 milliseconds) is waited before proceeding to the next step of the method in order to allow the oil-surface interaction to stabilize. In step 752, the reflection of the laser signal emitted via the objective lens is measured.

In step 754, the objective lens is moved along the Z-axis to another height closer to the lower surface of the plate, and the reflection of the laser signal at the new location is measured in step 756. The reflection value at the new Z-axis height is recorded. The process is then repeated substantially continuously. The expected measurement increases until the focal length is reached, and then begins to decrease.

In step 758, the controller evaluates the collected reflection data and evaluates whether there is a decrease in the measured signal reflection in the last few iterations. If not, flow returns to step 754 to measure the signal at the new Z-axis position. However, if the measured reflection is reduced, the objective lens is moved to the Z-axis position where the signal is maximum in step 760. This position along the Z-axis is considered to be the focal length of the objective lens relative to the sample.

In some embodiments, the attenuation is considered sufficiently significant if the attenuation in the laser signal is a sustained attenuation of 0.1V.

The method of fig. 21 is used to find the focal length of the sample each time a new sample or plate is scanned and after each oil dip replenishment.

In some embodiments, a similar procedure is used to ensure that the objective lens is focused, for example, when moving to a new area of the plate or when the objective lens has been in a single position, imaging is performed for a long period of time in the same position (e.g., to avoid the problem of deviating from a predetermined position). However, when moving to a new area, or when the object lens has been in a single position for a long time to recalculate, the previous focal length is known. Therefore, in this case, in step 752, the objective lens is moved between the known focal length ±heights within the predetermined search range. Here, the focal length is also determined as the point at which the maximum signal is obtained.

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 predetermined initial height such that the oil is in contact with the general lower surface. This corresponds to step 710 of fig. 20. A delay of a predetermined duration (e.g., 500 to 2000 milliseconds) is waited before proceeding to the next step of the method in order to allow the oil-surface interaction to stabilize. In step 772, the reflection of the laser signal emitted via the objective lens is repeatedly measured as the objective lens is moved a predetermined distance along the Z-axis. The predetermined distance along the Z-axis is selected such that the measured signal reflection is expected to peak and then decrease during Z-axis movement of the objective lens.

In step 774, a best fit gaussian (or polynomial) curve of the signal peaks collected in all measurements made in step 772 is calculated to determine peak maximum and the objective lens is moved to the Z-axis position where the gaussian (or polynomial) is maximum. This position along the Z-axis is considered to be the focal length of the objective lens relative to the sample.

In step 776, the objective lens is moved to the focal length determined in step 774 and scanning is initiated. As part of the scan, an image of the sample is taken at the focal position until the scan of the area is completed, or until a maximum scan duration has elapsed.

In step 778, the controller evaluates whether the scanning of the region is complete. If the scanning of the area is completed, the objective lens is lowered and moved to a new XY position in step 780. The flow then returns to step 770 to refocus the objective lens at the new location.

Otherwise, if the scanning of the region is not complete, the controller evaluates whether the maximum scan duration has elapsed in step 782. If the maximum scan duration has not elapsed, flow returns to step 776 and the scan continues. If the maximum scan duration has elapsed, flow returns to step 772 to refocus the objective lens and ensure that the objective lens or sample does not drift during the scan. However, when moving to a new area or remaining in the same position for a long time, the previous focal length is known. In this case, therefore, the objective lens is moved between the known focal length and the height within the predetermined search range in step 772. Here, the focal length is also determined as the point at which the maximum signal is obtained.

In some embodiments, the attenuation is considered sufficiently significant if the attenuation in the laser signal is a continuous attenuation of 0.1V.

The method of fig. 21B is used to find the focal length of the sample each time a new sample or plate is scanned and after each oil dip replenishment.

Reference is now made to fig. 22A, 22B, and 23, which illustrate a flowchart outlining a scanning and image processing and analysis method according to an embodiment of the present invention. The flowchart in fig. 22A outlines a scanning process according to some embodiments of the invention. Initially, a first scanning operation is performed on the target according to user-defined parameters, such as size (e.g., ignoring objects that are larger and/or smaller than a particular size), shape (ignoring non-circular or non-semicircular objects), intensity, and the like. The scanned image thus obtained is then processed to identify the object of interest and its features; the processing may be implemented using image processing algorithms currently known in the art or that may be developed in the future. The image processing results are then analyzed according to predefined rules to determine optimal parameters for performing further scanning operations on the same region. And defining new scanning parameters according to the analysis result to acquire new images of the same target. At least one second scanning operation is then performed using the new scanning parameters. For example, the system may thus scan a biological sample plate having 96 wells (each well having a diameter of 6 mm). During the scan, the image processing algorithm will identify each living cell present in the plate; if two or more cells are attached together, or if a single cell is larger than a certain size, the system may treat this as an unusual event, which should be further observed using higher magnification optics; if an objective changer such as described above is present, the lens can be changed to facilitate such viewing. The system then determines parameters defining the appropriate image quality for the high magnification scan. In some embodiments, image processing is performed while the first scanning operation is still ongoing, in which case the result of the analysis may affect the scanning operation in real time, according to the newly defined parameters. In some embodiments, the at least one second scanning operation is performed at a higher magnification than the first scanning operation; in some embodiments of the method used in combination with a device equipped with an objective changer as described above, a second scanning operation of higher magnification is performed by using the objective changer to change to a higher magnification objective, and then the region of interest is scanned at the higher magnification.

Fig. 22B is a flow chart of a scanning process according to some embodiments of the invention. In a first stage of scanning, acquiring a low-magnification image at a designated position; this process is repeated until images at a plurality of specified locations are acquired. After acquisition, transmitting each scanned image to an image processing and analyzing module, which starts image processing and analysis after receiving the first image; the module may be incorporated in software that controls the overall operation of the optical device, or may be located at a different software application or computer. The processing and analysis module uses the results of the processing and analysis to begin generating a location matrix containing information about the region of interest of the initially scanned object and its features. Thus, the position matrix can be completed soon after the low magnification scanning is completed. Alternatively, the location matrix may be generated after all processing and analysis is completed. From the position matrix, a second stage of scanning is performed during which a high magnification image at the specified position of interest is acquired.

FIG. 23 is a flow chart of an image processing and analysis process according to some embodiments of the invention. In a first stage of image processing, the following steps are performed: acquiring a low-magnification image; applying a low pass filter to the obtained data; applying a high pass filter to the obtained data; and performing a watershed transformation. Based on these image processing steps, objects and object properties/features are detected and extracted. Next, the relevant object is selected using the user parameters/attributes/features, and the object center (L [ Cx, cy ]). A high magnification image of the selected object is then acquired, from which a 3D transformation matrix (M) between the objects is created, and the 3D transformation matrix is then used.

Fig. 24 is a perspective view of an oil loading subassembly 1200 according to another embodiment of the present invention. The oil loading subassembly 1200 is substantially similar to the oil loading subassembly 200 of fig. 13-14D, like reference numerals representing like elements. For brevity, the following description focuses on the differences between the oil loading subassembly 1200 and the oil loading subassembly 200.

As shown, the oil loading subassembly 1200 includes a body portion 1210, the body portion 1210 including a center oil basin 1212 and a pump seat bore, the center oil basin 1212 being in fluid communication with an oil viewing opening, the pump seat bore terminating in a pump inlet conduit, as described above with respect to fig. 14D. Similar to that shown in fig. 14D, the oil flow conduit extends from the base of the oil basin 1212 adjacent to the pump inlet conduit and terminates at the edge of the main body portion 1210, wherein the oil flow conduit is sealed by a barrier. The viewing window as described above with respect to fig. 14B may be disposed within and seal the oil viewing opening, but as explained above, such a window is not required.

The cartridge piercing element 1230 is located within the central oil basin 1212 and may be attached thereto by any suitable attachment mechanism (e.g., fasteners, welding, adhesive, etc.). The cartridge piercing element 1230 includes a base 1234 and a center piercing pin 1236, the center piercing pin 1236 having a hollow passageway 1238 extending longitudinally therethrough that terminates in a sharp tip and is in fluid communication with an aperture 1239 formed in the side of the cartridge piercing element 1230. A pair of peripheral spike pins 1240 extend from the edge of base 1234 and may be substantially parallel to one another. Each of the penetration pins 1240 is substantially planar and has a first thickness over a majority of its longitudinal length. The end of each piercing pin 1240 distal from base 1234 has a second thickness that is less than the first thickness such that a shoulder 1242 is formed near the end of the piercing pin. Each penetration pin 1240 terminates in a sharp tip 1244.

When the dip tank 1300 is installed in the center oil basin 1212, as shown below with respect to fig. 26A and 26B, the hollow passage 1238 of the center piercing pin 1236 is in fluid communication with the center oil basin 1212 and the oil flow conduit in a manner similar to that shown in fig. 14D and 16A.

The diaphragm pump 1250 is positioned in the pump seat bore such that the inlet of the pump 1250 is positioned in the pump inlet conduit and the outlet conduit 1254 (see fig. 26A and 26B) of the pump extends downwardly out of the body portion 1210, substantially similar to that shown in fig. 13-14D. As described above with reference to diaphragm pump 250, diaphragm pump 1250 is adapted to draw oil from the oil flow conduit through the pump outlet conduit onto an article disposed below the pump outlet conduit, which in use is an oil immersion objective.

The oil loading subassembly 1200 also includes a base mount 1260, the base mount 1260 being mounted to the underside of the body portion 1210 and adapted to attach the oil loading subassembly 1200 to other components of the microscope system above the lens replacement subassembly 400. A rear mount 1265 is mounted to the main body portion 1210 rearward of the center oil basin 1212. Both the base mount 1260 and the rear mount 1265 may be connected to the body portion 1210 by any suitable connection means (e.g., fasteners, adhesion, welding, etc.).

Reference is now additionally made to fig. 25, which is a cross-sectional view of an oil impregnated cartridge 1300 according to another embodiment of the invention.

As shown in fig. 25, the oil immersion cylinder 1300 includes a generally cylindrical housing 1301. The housing 1301 includes a base 1302, a first cylindrical wall portion 1304 extending from the base 1302, the first cylindrical wall portion 1304 having a first width and defining a first chamber 1305 having a first inner diameter d 1. A second cylindrical wall portion 1306 extends from the first cylindrical wall portion 1304. The second wall portion 1306 has a second width that is less than the first width. The outer surface of the second wall portion 1306 is flush with the outer surface of the first wall portion 1304, and an inner shoulder 1308 is formed between the inner surfaces of the wall portions 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 that is less than the second width and forms a neck portion of the barrel 1300. The inner surface of the third wall portion 1310 is flush with the inner surface of the second wall portion 1306, and an outer shoulder 1312 is formed between the outer surfaces of the wall portions 1310 and 1306. The third cylindrical wall portion 1310 terminates at a lip 1314 remote from the base 1302. The second wall portion 1306 and the third wall portion 1310 together define a second chamber 1315, the second chamber 1315 having a second inner diameter d2. The second chamber 1315 is in fluid communication with the first chamber 1305.

In some embodiments, the outer surface of the third wall portion 1310 remote from the outer shoulder 1312 may include snap-fit protrusions and/or grooves adapted to snap-fit with corresponding grooves and/or protrusions in the inner periphery of the center oil basin 1212.

A substantially cylindrical oil container 1320 is disposed within the housing 1301 and defines a hollow 1321. A first end 1322 of the oil container 1320 is disposed within the first chamber 1305 and engages an inner surface of the base 1302. The second end 1324 of the oil container 1320 is substantially flush with the lip 1314 such that a majority of the oil container 1320 is disposed within the second chamber 1315. The oil reservoir 1320 includes a plurality of holes 1326 that are in fluid communication with the second chamber 1315 at a smaller distance from the first end 1322 than the second end 1324. Within the second chamber 1315, a cylindrical gap 1330 is formed between the housing 1301 and the oil container 1320.

A generally cylindrical push element 1340 is disposed within gap 1330 about oil reservoir 1320 and is longitudinally movable relative to the oil reservoir. The 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 the 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 member 1340 includes a plurality of apertures 1346, which apertures 1346 are adapted for radial alignment with the apertures 1326 of the oil reservoir 1320. The longitudinal alignment of the apertures 1346 and 1326 depends on the position of the push element 1340 relative to the oil reservoir 1320, as will be explained in further detail below.

The first end 1342 of the pusher element 1340 is adapted to push an annular O-ring 1350 disposed within the gap 1330. As further explained with respect to fig. 26A and 26B, when the O-ring 1350 is disposed below the bore 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 fig. 25, when the O-ring 1350 is pushed against the shoulder 1308 and the holes 1326 and 1346 are aligned with each other, a fluid path is formed between the gap 1330 and the hollow 1321 through the holes 1326 and 1346.

In the storage operational orientation of the cartridge 1300, a sealing foil 1360 (see fig. 26A) or any other seal abuts the lip 1314 of the housing 1301 and the second end 1324 of the oil container 1320 to seal the oil within the cartridge. In the storage orientation, O-ring 1350 is disposed below bore 1326 and seals gap 1330 such that no fluid path exists between hollow 1321 and gap 1330.

Reference is now additionally made to fig. 26A and 26B, which are sectional views of a step of inserting the oil impregnated cartridge 1300 of fig. 25 into the oil loading subassembly 1200 of fig. 24.

As shown in fig. 26A, the cartridge 1300 is in the storage orientation, the lip 1314 of the housing 1301, the second end 1324 of the oil container 1320, and the second end 1344 of the push element 1340 are all flush with one another, and are disposed against the sealing foil 1360. In the storage orientation, the O-ring 1350 is disposed under the bore 1326 against the first end 1342 of the push element 1340. The O-ring seals the gap 1330 such that there is no fluid path between the hollow 1321 and the gap 1330. The oil 1365 is disposed within the hollow 1321 such that the height of the oil does not extend above the bore 1326.

To load the cartridge 1300 into the oil loading subassembly 1200, the cartridge 1300 is moved toward the center oil basin 1212 in the direction of arrow 1370 until the center pin 1236 pierces the sealing foil 1360 and enters the hollow 1321. During this initial installation step, O-ring 1350 remains under bore 1326 and the fluid path between hollow 1321 and gap 1330 remains blocked.

As cartridge 1300 continues to move in the direction of arrow 1370, tips 1244 of peripheral piercing pins 1240 pierce the periphery of foil 1360 adjacent gap 1330. The shoulder 1242 of the peripheral spike 1240 engages the second end 1344 of the push element 1340 and as the cartridge continues to descend, the pressure applied by the shoulder 1242 to the second end 1344 will move the push element 1340 toward the base 1302 of the housing 1301, which in turn pushes the O-ring 1350 toward the shoulder 1308.

As shown in fig. 26B, in the installed operational orientation of cartridge 1300, shoulder 1242 of peripheral piercing pin 1240 and pushing element 1340 push O-ring 1350 against shoulder 1308. In such a device, the aperture 1346 of the push element 1340 is aligned with the aperture 1326 of the oil reservoir 1320 such that a fluid path exists between the center oil basin 1212, the gap 1330, the hollow 1321 of the oil reservoir 1320, and the passageway 1238 of the center piercing element 1236. In this arrangement, oil will flow out of the container 1320 into the center oil basin 1212 and operation of the system continues substantially as described above.

It should be understood that an oil dispensing device or other liquid dispensing device such as described herein may also be used in conjunction with a confocal microscope. In addition, the illumination source need not be a conventional laser, but may be a Light Emitting Diode (LED) or a combination or array of LEDs. Thus, a microscope or scanner equipped with an oil dispensing device or other liquid dispensing device such as described herein may be used for high content imaging (HCI, sometimes also referred to as high content screening) to obtain images, which can then be processed according to known techniques, such as according to a light activated positioning microscope (PALM) (see, e.g., betzig, e.g., "Imaging intracellular fluorescent proteins at nanometer resolution", science 313,1642-1645 (2006)) or a random optical reconstruction microscope (stop) (see Rust, M.L, bates, m. And Zhuang, x. Sub diffraction-limit imaging by stochastic optical reconstruction microscopy "(stop), nature Methods 3,793-795 (2006)). In one embodiment, images obtained using an acquisition device equipped with an oil distribution apparatus such 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 (release 8, 12 of 2016). Such processing may be performed using ImageJ software plug-ins, which are available free of charge at https:// henrimiquslab. The device and method described herein facilitate automatic dispensing of oil (or other liquid) to the objective lens, which means that the device can be combined with fully automated image acquisition: auto-scan, auto-focus, auto-objective lens replacement, auto-dispense lens immersion medium, and auto-detect an object of interest.

Unless defined otherwise, 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, suitable methods are described herein.

All publications, patent applications, patents, and other references 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 not intended to be limiting.

It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described hereinabove. Rather, the scope of the present invention is defined by the generic combination of components that perform the same function as the embodiments described above and includes the combinations and sub-combinations of the various features described hereinabove as well as variations and modifications thereof which would occur to persons skilled in the art upon reading the foregoing description.

Claims (22)

1. A microscope device for viewing or imaging a sample disposed on a sample holder, comprising:

A first liquid immersion objective having a three-dimensional orientation;

a first liquid application position at which the sample holder cannot be placed, and a second position different from the first position at which the sample holder can be placed;

means separate from the first liquid immersion objective and arranged at the first liquid application position, the means being configured to apply liquid to the first liquid immersion objective when the first liquid immersion objective is arranged at the first liquid application position below the means; and

a mechanism configured to move the first liquid immersion objective from the first liquid application position to a second scanning position in XYZ coordinate space without changing the three-dimensional orientation of the first liquid immersion objective, the second scanning position having different X and Y coordinates from the first liquid application position, and in which the liquid on the first objective is in contact with the sample holder when the sample holder is placed in the second position.

2. The microscope device according to claim 1, further comprising a controller capable of determining a focus Z-axis position when the first immersion objective is loaded with liquid and placed at the second scanning position and then moved along the Z-axis towards the sample holder.

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

4. A method, comprising:

providing a microscope device according to any one of claims 1 to 3, in which a sample holder containing a sample is placed at the second scanning position;

applying liquid with the device to a first immersion objective located at the first liquid application location; and

the first immersion objective with the liquid disposed thereon is moved to the second scanning position.

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

6. The method of claim 5, wherein an air objective is used to determine the Z coordinate of the surface prior to moving a first immersion objective with the liquid disposed thereon to the second scanning position, the air objective being replaced with the first immersion objective prior to applying the liquid with the device.

7. The method of claim 5, wherein a second liquid immersion objective having a lower magnification than the first liquid immersion objective used to scan a sample is used to determine the Z coordinate of the surface prior to moving the first liquid immersion objective with the liquid disposed thereon to the second scanning position, the second liquid immersion objective being replaced with the first liquid immersion objective prior to applying liquid to the first liquid immersion objective with the device.

8. The method of claim 5, wherein the sample holder is a commercial sample plate of a particular model, and the Z coordinate of the surface is determined based on a distance of a bottom of the particular model plate from a bottom edge of the plate in combination with a size of the microscope device before moving the first immersion objective to the second scanning position.

9. The method of any of claims 4 to 8, further comprising: after moving a first immersion objective with the liquid disposed thereon to the second scanning position, a focus position of the first immersion objective is determined.

10. The method of claim 9, further comprising: after determining the focal position of the first liquid immersion objective, the first liquid immersion objective is returned to the first liquid application position and liquid is applied thereto.

11. The method of claim 10, further comprising: scanning the sample after the determining the focal position of the first immersion objective, and wherein the returning is performed in response to the scanning reaching a threshold, the threshold comprising one or more of: (a) A liquid loss of the first liquid immersion objective as a function of a distance traveled by the first liquid immersion objective in an XY plane during scanning; (b) Liquid loss of the first liquid immersion objective due to movement of the first liquid immersion objective along the Z axis away from the sample holder; (c) A measured reduction in weight of the liquid-loaded first immersion objective; and (d) the measured thickness of the liquid on the first immersion objective.

12. The method of claim 10 or 11, further comprising: after returning the first liquid immersion objective to the first liquid application position and applying liquid to the first liquid immersion objective, the first liquid immersion objective is moved to either (a) the second scanning position or (b) a third position having different X-and Y-coordinates from the first liquid application position and at least one of the X-or Y-coordinates is different from the second scanning position, in which third position liquid on the first liquid immersion objective is able to contact a sample holder placed in the second position.

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

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

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

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

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

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

19. A microscope device according to any one of claims 1 to 3 wherein the liquid is an oil.

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

21. The microscope device according to claim 19, wherein the liquid is silicone oil.

22. A microscope device according to any one of claims 1 to 3 wherein the liquid is water.