Note: Descriptions are shown in the official language in which they were submitted.
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MICROSCOPY SYSTEM AND METHOD USING AN IMMERSION LIQUID
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims Paris Convention Priority from, and the US
benefit of, US
provisional applications nos. 63/180693 and 63/180694, both of which were
filed on April 28,
2021. The contents of both of said provisional applications are incorporated
herein by reference.
FIELD
[0002] The present invention relates to the field of accurate optical scanning
and imaging of
samples and more particularly, to biological microscopy using a suitable
immersion liquid, such
as a stable oil, as a medium between the objective and the sample.
BACKGROUND
[0003] High-content microscopy systems, used for observing biological samples,
are known in
the art. Such microscopy systems are typically based on a static and massive
microscope body,
and include complex connections to optical units such as an objective turret,
an illumination
unit, filter wheels, shutters, a camera, internal optics and other units. To
enable the scanning of a
given sample, devices to enable motion of the sample holder in the X, Y and Z
directions are
added to the microscope body. During the imaging process of these microscopic
systems, the
sample is moved to capture images at different locations along the sample,
while the optics units
are static.
[0004] Various automated scanning systems have been developed, in which the
sample and the
objective are moved relative to one another, the objective is automatically
focused, and scanning
occurs without human intervention. Some such systems also enable automatically
changing
between multiple different objective lenses, for example U.S. Patent No.
9170412, the contents
of which are incorporated herein by reference, describes one such system.
[0005] While such automated scanning systems are useful for scanning of
biological material
using an objective lens working in an air environment, these systems are
unsuitable for liquid
immersion microscopy. This is due, among other things, to the fact that, as
the objective and
sample move relative to one another, the immersion liquid is spread along the
sample plate, such
that the immersion liquid needs to be periodically replenished. While systems
have been
developed to try to replenish the liquid, see, e.g., EP 1717628 B 1, DE
202017000475 Ul, US
7304793 B2, 'Taps //www. I ei ea- mi cro sy stem s com/products/Iight-rni scop
e slp/ieicawater
and
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immersion html, such systems have various drawbacks, for example their design
adds weight to
the objective, decreases scanning area, and reduces the throughput of the
microscopy system.
[0006] There is thus a need in the art for a system and a method for automatic
high-content
microscopy using an immersion liquid.
DETAILED DESCRIPTION
[0007] Embodiments of the invention will be better understood from the
following detailed
description, as well as with reference to the figures, in which:
[0008] Fig. 1 is a schematic diagram of an embodiment of a microscopy system
which can be
adapted for use in accordance with to an embodiment of the present invention;
[0009] Fig. 2 is an isometric view of an apparatus constructed and operative
to be adapted for
use in accordance with an embodiment of the present invention;
[0010] Fig. 3 is a cross-sectional view of an objective lens mounted on an
objective lens holder
and attached to a kinematic base, useable in accordance with an embodiment of
the present
invention;
[0011] Fig. 4 is an exploded isometric view from above of the components in
Fig. 3;
[0012] Fig. 5 is an exploded isometric view from below of the components in
Fig. 3;
[0013] Fig. 6 is an isometric view of the kinematic of Figs. 4 and 5;
[0014] Fig. 7 shows how the lower portion of the objective lens unit of Figs.
3-5 fits into the V-
shaped grooves of the kinematic base of Fig. 6;
[0015] Figs. 8 and 9 are isometric views from above and below, respectively,
showing an
objective lens mounted in an objective lens holder and a kinematic base having
magnets attached
thereto;
[0016] Figs. 10 and 11 show how objective lenses may be stored and changed, in
accordance
with embodiments of the present invention;
[0017] Fig. 12 is a partially exploded view of an oil loading subassembly,
having an immersion
oil cartridge, disposed above a lens changing subassembly, in accordance with
embodiments of
the present invention;
[0018] Fig. 13 is an exploded view illustration of the oil loading
subassembly, in accordance
with embodiments of the present invention;
[0019] Figs. 14A, 14B, 14C, and 14D, are, respectively, a perspective view, a
planar front view,
a planar side view, and a sectional view of the oil loading subassembly of
Fig. 13, when
constructed;
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[0020] Figs. 15A, 15B, and 15C are sectional illustrations of steps of
insertion of an immersion
oil cartridge into the oil loading subassembly of Figs. 13, 14A, 14B, 14C and
14D;
[0021] Figs. 16A and 16B are sectional illustrations illustrating the oil flow
path within the oil
loading subassembly of Figs. 13, 13, 14A, 14B, 14C, 14D, 15A, 15B and 15C;
[0022] Fig. 17 is a perspective view illustration of the lens changing
subassembly of Figs.1-11
and the oil loading subassembly of Figs. 12, 13, 14A, 14B, 14C and 14D,
including an objective
lens in an immersion oil loading position of the lens changing subassembly;
[0023] Figs. 18A and 18B are, respectively, a front view illustration and a
side view illustration
of loading of immersion oil onto the objective lens in the structure of Fig.
17;
[0024] Figs. 19A, 19B, and 19C illustrate the spreading of the immersion oil
as the objective
lens moves relative to a viewing surface;
[0025] Fig. 20 is a flow chart outlining a method for scanning a sample using
an oil immersion
lens, and reloading immersion oil onto the oil immersion lens, in accordance
with embodiments
of the present invention;
[0026] Figs. 21A, 21B, and 21C are flow charts outlining a method for
automatically focusing
an oil-loaded oil-immersion lens on a biological sample, in accordance with
embodiments of the
invention;
[0027] Figs. 22A, 22B and 23 are flowcharts outlining a method for scanning a
sample, in
accordance with embodiments of the invention;
[0028] Fig. 24 is a perspective view illustration of the oil loading
subassembly, according to
another embodiment of the present invention;
[0029] Fig. 25 is a sectional illustration of an immersion oil cartridge,
according to another
embodiment of the present invention; and
[0030] Figs. 26A and 26B are sectional illustrations of steps of insertion of
the immersion oil
cartridge of Fig. 25 into the oil loading subassembly of Fig. 24.
[0031] It will be appreciated that despite the prevalence of color drawings
and photographs in
the scientific literature and the ease of presentation of such in electronic
format, PCT rules
remain mired in the 19th century and still do not permit the filing of color
drawings or
photographs, and the USPTO only allows color drawings or photographs pursuant
to a petition.
Therefore Figs. 13, 14A, 14B, 14C, 14D, 15A, 15B, 15C, 16A, 16B, 17, 18A, 18B,
19B, 19C,
24, 25, 26A and 26B are being filed with this PCT application as black-and-
white drawings.
However, the original drawings are in color and, for the purpose of making
them publicly
available, have been uploaded to a publicly-available picture sharing service,
Shutterfly, and can
be accessed over the internet by anyone using the
link
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http s ://d ri pp eip a ten tcol ofdrawings. sliu itertly com/pi c itife s4n 5
; the link is being shared for the
first time with the filing of this patent application, and the color drawings
are incorporated
herein by reference.
[0032] Reference is now made to Fig. 1, which is a block diagram showing
schematically an
apparatus 10 constructed and operative so as to be adapted for use in
accordance with
embodiments of the invention. Apparatus 10 contains a holder 12, which may be
configured to
hold a sample plate 13, for example a 6-, 24-, 96-, 384 or 1536-well plate
commonly used to
hold biological samples and having lower and upper faces 13A and 13B,
respectively,
containing a sample or multiple samples to be observed, as is known in the
art. Holder 12 may
also be configured to hold a microscope slide, a Petri dish, or another
substrate having a bottom
that is transparent to electromagnetic radiation of a wavelength or
wavelengths of interest. For
reference, sample plate 13, which is not itself part of the apparatus, lies in
the XY-plane, so that
through its lower face 13A, samples contained therein will be opposite the
components of
apparatus 10 that will be described below.
[0033] A turretless objective lens, viz, a single objective lens 14, which is
part of a linear XYZ
scanner 16, is arranged so that the lens lies facing the sample holder (and,
when a sample plate
13 is present, facing the lower face 13A of sample plate 13), and the optical
axis of objective
lens 14 lies along the Z-axis with respect to the sample holder. By "linear
XYZ scanner" is
meant a mechanism constructed and operative to move the objective lens 14 in
three mutually
perpendicular directions, wherein the "Z" direction is used to denote movement
along the optical
axis. Such scanners per se are known in the art, for example from Israel
Patent No. 143836, filed
June 19, 2001 and entitled "Compact Linear Scanner System" or U.S. Patent No.
6850362, the
contents of both of which are incorporated herein by reference. It will be
appreciated that for the
sake of simplicity, only some of the components of XYZ scanner 16 are shown in
Fig. 1; a more
detailed description of this component follows below. Among the components of
the XYZ
scanner that are shown in Fig. 1 are mirrors 18 and 20, which work together to
redirect light
along the optical axis of objective lens 14, such as when the XYZ scanner is
arranged to operate
in an inverse microscope configuration, to reflect light from illumination
unit 22 and from
autofocus unit 24 through objective lens 14. Mirror 18 is constructed and
operative to move
together with objective lens 14 in the X- and Y-directions, and mirror 20 is
constructed and
operative to move together with objective lens 14 and mirror 18 in the X-
direction, in order to
ensure that light is able to travel along the optical axis of objective lens
14. Mirrors 18 and 20
also reflect light received from the sample, including reflected incident
light from illumination
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unit 22 or autofocus unit 24, or light which results from fluorescence of the
sample, along the
optical axis of objective lens 14 and away from the sample.
[0034] As shown in Fig. 1, apparatus 10 also comprises an autofocus unit 24.
Autofocus units
per se for focusing non-fluid immersion objectives, viz, objectives that do
not use oil or water,
are known in the art. For use as shown in Fig. 1, the autofocus is preferably
an autofocus unit
that is usable in high-resolution, high-throughput microscopy applications,
for example the
autofocus unit and method described in PCT patent publication WO 03/077008
entitled "Auto-
focusing method and device" and filed March 13, 2003, or U.S. Patent no.
7109459 of the same
title, the contents of both of which are incorporated herein by reference.
[0035] In Fig. 1, autofocus unit 24 emits a beam of laser light at a
wavelength at which the
vehicle carrying the sample(s) is transparent, e.g. 635 nm, which is then
reflected by a beam-
splitting device (dichroic filter 26) onto the optical axis of objective lens
14, reflects off mirrors
18 and 20 through objective lens 14 and onto the vehicle in the sample holder.
It is then reflected
back along the same pathway, and reflected by dichroic filter 26 back into the
autofocus unit,
where it is sensed by a sensor (not shown), and a controller (not shown),
programmed to adjust
the focus of the objective lens along the Z-axis, if necessary. When the
autofocus unit 24 is used
with samples containing fluorescent labels, the wavelength of the autofocus
light may be chosen
so as not to elicit a fluorescent response in the sample, although this is
generally not critical, as
typically the autofocus process will be completed before the image capture
process begins. A
similar process can be employed when using e.g. an oil-immersion lens, with
certain changes to
account for the use of the oil, as will be described in more detail below.
[0036] Also shown in Fig. 1 is an illumination unit 22. Illumination unit 22
includes an
illumination source (not shown), such as a mercury lamp, LED lamp, a laser, or
other suitable
radiation source. If necessary, the illumination unit 22 includes collimating
optics. In the case
where the sample contains one or more fluorescent probes or the like, a
suitable beam splitting
device is arranged so as to reflect the excitation light onto the optical axis
of the objective lens
14. This beam splitting device could be a quad filter 28 that reflects light
of the excitation
wavelength generated by the illumination unit but allows light of other
wavelengths, in
particular light generated by fluorescence of fluorescent probes in the
sample, to pass through. It
will be appreciated that illumination unit 22 may be configured to generate
electromagnetic
radiation of more than one wavelength, or that more than one illumination unit
may be
employed in order to generate electromagnetic radiation of more than one
wavelength, if for
example multiple fluorescent probes are employed in the samples being
observed, provided that
appropriate beam splitting devices are also employed to ensure reflection of
the excitation light
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onto the optical axis of the objective lens 14 and passage of light of
wavelengths of interest, e.g.
fluorescent light generated by the fluorescent probes in the samples. Also, it
will be appreciated
that although Fig. 1 shows the autofocus unit 24 being located between the
illumination unit 24
and the objective 14, in principle the positions of illumination unit 22 and
autofocus unit 24
could be reversed, provided that appropriate optics are provided to ensure
that only light of
wavelengths of interest passes through to the image capturing devices 30.
[0037] As shown in Fig. 1, light which is either reflected off of or generated
by the sample (by
fluorescence), or, if the sample is illuminated from the side of upper face
13b, light which is
transmitted through the sample, travels along the optical axis of objective
lens 14 and passes
through dichroic filter 26 and quad filter 28 before being detected by one or
more image
capturing devices 30. Fig. 1 depicts an arrangement in which three such image
capturing
devices, viz. three CCD cameras 30, 30' and 30" are present, and in which
after passing through
beam-splitting devices 26 and 28 but before impinging on the CCD cameras, the
light passes
through a tube lens 32, reflects off a fold mirror 34 and is split by an RGB
prism 36 before
passing through emission filters 38, 38' and 38" that filter out all light but
that of the emission
bands of the fluorescent probes in the sample; in the Fig. 1, emission filter
38 allows passage of
red light, emission filter 38' allows passage of green light and emission
filter 38" allows passage
of blue light. It will be appreciated that prism 36 may be other than an RGB
prism and filters 38,
38' and 38" may consequently filter in different ranges of wavelengths.
[0038] The operation of the system shown in Fig. 1 is controlled by one or
more controllers (not
shown) which are collectively programmed to control the operation of the
autofocus unit, the
illumination unit, and the movement of the XYZ scanner. An analysis unit (not
shown) for
analyzing the images obtained by the image capturing device(s), which may also
be part of the
one or more controllers or may be a separate unit, may also be provided, and
may be configured
to provide feedback to the one or more controllers. In addition, as will be
appreciated by those
skilled in the art, input and/or output devices, such as a keyboard, optical
or magnetic storage
reader and/or writer, printer, and display device such as a plasma or LCD
display, as well as
storage devices, may also be provided.
[0039] It will be appreciated by those skilled in the art that variations on
the arrangement shown
in Fig. 1 may be employed in accordance with embodiments of the invention; at
least one such
variation, for implementing an auto-focus method when using an oil-immersion
lens, is
described below.
[0040] An XYZ scanner such as is shown in Fig. 1 can be incorporated into an
apparatus in
accordance with embodiments of the invention. See, e.g., Fig. 2, which shows
in isometric view
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portions of an apparatus 810 constructed and operative such that it can be
adapted for use in
accordance with embodiments of the invention. Apparatus 810 contains a sample
holder 812
which holds a 96-well sample plate 813. Sample plate 813 lies in a plane
perpendicular to the
optical axis of objective lens 814, which is part of scanner 816 and moveable
in three mutually
orthogonal directions, viz, the X, Y and Z directions. Scanner 816 includes
mirror 818 mounted
in mirror mount 819 which is moveable in the X and Y directions, and mirror
820 mounted in
mirror mount 821 which is moveable in the X direction.
[0041] Fig. 2 also shows autofocus unit 824 and a mirror 827 which directs
light from the
autofocus unit to dichroic filter 826 and vice versa; dichroic filter 826
directs light from
autofocus unit 824 to the optical axis of objective lens 814. Also shown is
part of an illumination
unit 822, including a bundle of fiber optic cables 822a and collimation optics
822b, and quad
filter 828, arranged to reflect light from the illumination unit to the
optical axis of objective lens
814. Light that reflects off of samples or is generated by the fluorescence in
the sample plate
(e.g. the fluorescence of fluorescent probes) and that is not filtered out by
quad filter 828 is then
focused by tube lens 832 and reflected by fold mirror 834 to camera 830;
filter wheel 837
contains filters 838, 838' and 838" which can be selected to filter light
entering camera 830.
[0042] In accordance with some embodiments of the present invention, the
apparatus is fitted
with a coupling mechanism to facilitate the changing of objective lenses,
although it will be
appreciated that this mechanism can be employed in other optical instruments.
Reference is now
made to Figs. 3-9, which show one embodiment of such a mechanism. As shown in
Figs. 3, 8
and 9, an objective lens 1010 is permanently mounted on objective lens base
1020. Objective
lens 1010 and objective lens base 1020 together form objective unit 1060.
Objective unit 1060 is
attached to a kinematic base 1040. In accordance with some embodiments of the
invention,
kinematic base 1040 may be mounted permanently on top of the Z-axis component
of the XYZ
scanner, so that objective unit 1060 containing an objective lens such as lens
338 may be
brought to rest thereupon, as will presently be described; alternatively; the
kinematic base may
be formed as part of the top of the Z-axis component of the XYZ scanner.
[0043] As will presently be described, the attachment between the objective
lens base 1020 and
the kinematic base 1040, as depicted in the figures, uses a specific kinematic
mount
configuration, which provides positioning precision in the range of 50
nanometers or better.
Objective lens base 1020 contains a plurality of coupling balls 1030 (three
such balls are
depicted in Figs. 3-9) and is machined such that after being set in position,
there is high accuracy
in the spatial position of coupling balls 1030 relative to the optical axis of
objective lens 1010.
Coupling balls 1030 are of high stiffness (e.g. hardness of 53-58 RC SS or
more) and of suitable
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diameter, e.g. 3-3.5 mm. Each of coupling balls 1030 is held in place in one
of holes 1020a
formed in the lower surface of objective lens base 1020 such that 30-40% of
the ball diameter
protrudes downwardly out of the lower surface of objective base 1020. Holes
1020a may
penetrate only partially into objective lens base 1020, whereby to form a
cylinder having one
end closed, or they may run completely through the bottom of objective lens
base 1020. Each of
holes 1020a has an interference diameter tolerance with the coupling ball 1030
placed therein,
whereby to hold the ball firmly in place. Objective lens base 1020 is made of
ferromagnetic steel
such as 17-4PH.
[0044] As depicted in the figures, kinematic base 1040 is of generally annular
ring shape, and
has several indentations in the form of V-shaped grooves 1040a formed in the
upper side
thereof. The spacing of coupling balls 1030 and grooves 1040a is such that the
coupling balls fit
into three of the grooves, as shown in simplified form in Fig. 7. Kinematic
base 1040 may also
be built of high performance ferromagnetic steel such as 17-4PH, which is
thermally treated to
reach a surface hardness of 39RC or higher, so that kinematic base 1040 will
maintain
attachment precision over cycling load and unload operations of the lens unit
1060. To ensure
that kinematic base 1040 has the required surface hardness, the following
manufacturing process
is observed: (a) manufacture parts to final dimensions leaving 50 microns for
the final groove
1040a grinding process; (b) perform thermal hardening process; (c) grind the V
groove 1040a to
the final dimensions.
[0045] As shown in the figures, kinematic base 1040 is formed with three holes
1040b
therethrough, spaced approximately evenly around the base. A magnet 1050 is
inserted into each
hole 1040b and glued in place. Together magnets 1050 cause a magnetic
attachment force with
the objective unit 1060 when the parts are in close proximity. This attachment
force both
positions the objective in place, by balancing the attachment forces applied
on the kinematic
coupling, as well as maintains the objective unit 1060 in place while the
optical system moves at
high acceleration. It will be appreciated that the magnets need not
necessarily come into contact
with the object base 1060 nor, as can be see e.g. in Fig. 9, even protrude
through holes 1040b.
Thus, in this application, when it is stated that such magnets are "installed
within" a surface that
is opposite a ferromagnetic surface, the magnets may protrude out from, or be
embedded within
or even below the surface that they are "installed within", since magnetic
attractive force
operates without the need for direct physical contact.
[0046] Although a particular embodiment of the coupling mechanism has been
shown in Figs.
3-9, it will be appreciated that variations on what is shown therein are
possible. This is because
the collective effect of the balls 1030 and grooves 1040a is to both precisely
locate the objective
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unit 1060 in the XY plane and to restrict the motion of the objective unit
1060 in the XY-plane
and the negative Z-direction, and the addition of the magnetic force restricts
motion of the
objective unit in the positive Z direction, and these effects can in principle
be achieved with
other arrangements. Thus, for example, more or fewer holes 1040b and,
accordingly, more or
fewer magnets could be employed, and the magnets could installed within the
bottom of the lens
objective unit as well as the kinematic base, or the magnets could be
installed exclusively within
the bottom of the lens objective unit. Similarly, the positions of the
indentations and balls could
be reversed, so that the bottom of the objective unit 1060 contains
indentations, such as grooves,
and the upper surface of the kinematic base has balls protruding therefrom, or
both the objective
unit and the kinematic base could have indentations and balls, or the
kinematic base could have
two grooves and a protrusion and the bottom of objective unit 1060 could have
two
corresponding protrusions and a corresponding groove.
[0047] Moreover, the indentations may be in a shape other than V-shaped
grooves, for example
one or more of the indentations could be in the shape of a well that provides
three points of
contact for a ball 1030 resting therein rather than two points of contact as
in a V-shaped groove.
One such well, in combination with a single V-shaped groove and the surface of
the kinematic
base and appropriately positioned magnets of sufficient strength, could have
the same effect as
three V-shaped grooves. Furthermore, protrusions other than a ball shape that
fit into those
indentations may be employed. Thus, for example, persons skilled in the art
will appreciate that
although in the figures a plurality of balls 1030 are shown held in holes,
other construction
arrangements are possible, for example round-headed nails may be used.
[0048] The relative positions of the indentations may also differ from what is
shown in Figs. 3-
9: the indentations may be arranged so that there is only one way for the
protrusions from the
opposite piece to fit therein, thus providing only a single way for the
objective unit to set in
place on the kinematic base. Alternatively, three protrusions may be used (to
ensure that the
piece with the protrusions sits on a plane), for example as described above
with respect to the
objective lens unit, but instead of three or six V-shaped grooves being
presented in the opposite
piece (e.g. as shown above with respect to the kinematic base), a larger
number of evenly-
spaced, radially-oriented V-shaped grooves may be employed, such as 9 or 12,
to facilitate
easier placement of the lens unit, for example when used in an objective lens
changer like the
lens changer described below.
[0049] In addition, although Figs. 3-9 show an objective unit 1060 which is
formed from an
objective lens 1010 and objective lens base 1020, it will be appreciated that
objective lens 1010
may be formed in a way that obviates the need for objective lens base 1020,
for example if the
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bottom of objective lens 1010 is made from a ferromagnetic material and
machined so as to have
coupling balls 1030 protruding therefrom. It will be appreciated that where it
is stated in the
description or claims that the objective lens has a surface "associated
therewith", such surface
may be a surface of the objective lens assembly itself, or it may be a surface
of a lens holder or
base on which the objective lens is mounted, such as is shown in Figs. 3-9.
[0050] The coupling mechanism described above enables an objective lens to
repeatedly be
inserted and removed into the optical device, with sufficient accuracy to
enable high precision
observations to be obtained using the objective lens. Consequently, the
mechanism described
herein facilitates the use of a multiplicity of objective lenses in the
optical device, since lenses
can be swapped in and out; in the case of the device shown in the figures,
this can be
accomplished without burdening the XYZ scanner with the weight of the full
complement of
objective lenses, thus facilitating higher accelerations than could be
employed, and quicker
settling times than could be achieved, if the XYZ scanner were to bear the
weight of the entire
set of objective lenses. Instead, the objective lenses may be stored elsewhere
in the apparatus
and exchanged as necessary when it is desired to change magnification.
Additionally, while
turrets bearing multiple objectives are known, the objectives in such turrets
are arranged so that
the objective in use is aligned with the focal path along the Z-axis, but the
objectives not in use
are at an angle relative to the Z-axis, and thus switching out one oil
immersion lens for another
would result in loss of oil as the turret is rotated and the lens is moved to
an angle away from the
vertical. Thus, as shown in Figs. 10 and 11, a lens changing subassembly 1000
includes a
magazine 1080 which holds a plurality of objective units 1060 in a plurality
of stations 1070.
Each objective unit 1060 in magazine 1080 is roughly aligned with the Z-axis
of XYZ scanner
1090 and roughly perpendicular to the plane of kinematic base 1040, which is
attached to the
upper portion of XYZ scanner 1090, and furthermore the ball couplings 1030
protruding from
the bottom of each objective unit 1060 are positioned so that they will engage
the V-shaped
grooves 1040a in the kinematic base when the XYZ scanner is raised to contact
the objective
unit, as described below. Each station 1070 includes a pair of arms 1072, so
that each pair of
adjacent arms can hold an objective unit 1060. Although as shown in Figs. 10
and 11, magazine
1080 holds three objective units 1060, in principle an optical device may be
designed to hold
more such objective units.
[0051] To illustrate how the coupling mechanism may be employed to change
objective lenses,
assume that scanner 1090 initially has no objective unit attached thereto, as
depicted in Fig. 10,
and that at least one objective unit 1060 is loaded on one station 1070 of
magazine 1080.
Scanner 1090 is first moved to a Z-position which is sufficiently low to
enable it to move under
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a selected objective 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 resting in station 1070
is roughly aligned
with the optical axis that will be required of the objective lens once
positioned for use on XYZ
scanner 1090. The Z stage is then moved upwardly so as to come into contact
with the lower
portion of selected objective unit 1060. This results in the coupling balls
1030 resting in grooves
1040a. The proximity of the magnets 1050 to the bottom of objective unit 1060,
which is made
of ferromagnetic material and thus attracted to the magnets, results in
coupling balls 1030
settling in the grooves and being held there. As depicted in Figs. 3-11, when
the coupling balls
and the V-shaped grooves are machined with sufficient precision (e.g. within
50 nm tolerance),
the use of three coupling balls at approximately 120 separation and V-shaped
grooves oriented
radially from the center of the kinematic base and into which the coupling
balls fit results in the
optical axis of the objective lens being set with sufficient accuracy to
enable use of the objective
lens without further calibration. The Z stage is moved further upward,
sufficiently to lift the
objective unit 1060 off arms 1072. The XY stage may then be moved out of the
station 1070.
The optical system is now ready to operate using the selected objective.
[0052] A similar process is repeated when changing from the first objective to
a second one.
The scanner is moved to an open station 1070 and, operating in the reverse
sequence from that
described above, places the first objective unit 1060 in the station 1070. The
XYZ stage then
moves to a different station of magazine 1080 and loads the second objective
in a manner
analogous to that described above for the first objective.
[0053] It will be understood that the movements of the XYZ scanner may be
automated,
effected by appropriate motors and controlled by a microprocessor that has
been programmed
for such purpose.
[0054] It will also be appreciated that although in Figs. 10 and 11, an XYZ
optical scanner 1090
is shown as the device for facilitating transfer of an objective on and off
the optical system, the
method described is not restricted to use with an XYZ optical scanner and can
be used in any
system having moving optical elements controlled by a combination of motors,
encoders,
sensors, servo controller or other automation elements. Furthermore, in cases
in which the
moving optical system cannot reach all objective units, a secondary motion
system (not shown)
may be employed to move the magazine 1080 so that the particular lens in
question is in a
position that is accessible to the optical system.
[0055] The lens changing subassembly 1000, described herein with respect to
Figs. 3-11, is
particularly advantageous for conducting automated high-content or high-
throughput screening
of a biological sample using a liquid immersion objective lens. However, it
will be appreciated
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that in some embodiments of the invention, the apparatus or device does not
have a lens
changing subassembly, using only a single objective, the device or apparatus
being large enough
in the X- and/or Y-direction that (a) the objective lens can be moved away
from the sample
holder along the Z axis while maintaining its three dimensional orientation
(e.g. in terms of
pitch, roll, and yaw), and moved in the XY plane to an oil-replenishment
position, then after
replenishment of the oil returned to the same place in the XY plane, and,
after re-focusing in the
Z-direction, scanning continued from where it was interrupted, (b) while
maintaining its
orientation in the XYZ coordinate space, the sample holder can be moved from
its stationary
position for scanning, in order to facilitate replenishment of the oil, and
then moved back to the
same place from which scanning was interrupted, so that, after re-focusing of
the objective in the
Z-direction, scanning can be continued from where it was interrupted, or (c) a
combination of
both (a) and (b).
[0056] One of the challenges with liquid immersion scanning of biological
samples is the need
to periodically replenish the liquid on the objective lens. This is
particularly important when
scanning in a setting where the objective moves relative to the sample, while
close to, or in
contact with, a surface of the sample holder (e.g. multi-well plate, petri
dish, or sample bearing
slide), because the relative motion between the objective and the sample
holder results in
spreading of the immersion liquid, to the point where the layer of immersion
liquid is
insufficient for properly observing the sample. As such, scanning is limited
to the distance that
the objective can move before the immersion liquid must be replenished.
[0057] As noted in the background discussion above, prior art systems for the
replenishment of
oil suffer from various drawbacks, e.g. they require the placement of
additional items on the
objective itself, thus adding to the weight of the objective lens assembly, as
well as increasing its
effective size, which can reduce the area of the sample that can be scanned,
and limit the degree
to which the lens itself can be brought close to the sample holder.
Additionally, when this is
done manually, a human user must stop the operation of the system and manually
replenish the
immersion liquid on the objective lens, either manually, or with the
assistance of a specially-
designed apparatus. This greatly delays the operation of the system, and
requires periodic
attention of the human operator. As such, it is also more error prone.
Additionally, the need for
manual replenishing of the immersion liquid limits, or prevents, conducting of
large screening
sessions with many locations, disposed at long distances from one another,
being scanned.
[0058] Another challenge in manual application of immersion oil to the
objective, is that the
human operator may apply too little oil, resulting in frequent stopping of the
microscopy system.
Alternatively, the human operator may apply too much oil, causing the oil to
spill off the
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objective unit during motion thereof, for example from a location at which the
human operator
applies the immersion liquid to the scanning location. An additional issue is
that, in order to
avoid moving the lens from its in-focus position, hitherto it has been
necessary to effect oil
replenishment while leaving the objective in its location adjacent to the
surface of the sample
holder (e.g. close to the bottom of a multi-well plate), leaving little room
in which to maneuver
in order to add oil to the lens.
[0059] In accordance with embodiments of the present invention, as described
hereinbelow with
respect to Figs. 12-19B, a lens changing subassembly forming part of the
microscopy system
ensures that there is a fixed position, to which the objective lens can be
automatically moved,
and in which the objective lens will not be disposed beneath the sample. The
existence of such a
fixed position facilitates the construction of the inventive immersion liquid
loading
subassembly, discussed hereinbelow, directly above a predetermined position of
the magazine of
the lens changing assembly. As described hereinbelow and according to
embodiments of the
present invention, in use, the immersion liquid objective is periodically
moved to the
predetermined position of the magazine of the lens changing assembly, and oil
is automatically
applied onto the objective from the liquid loading subassembly, following
which the objective
lens is automatically returned, by the system, to the scanning position in
order to continue the
scanning process.
[0060] It is appreciated that the immersion liquid subassembly and processes
of use thereof,
described hereinbelow, are suitable for use with any suitable immersion
liquid, such as synthetic
hydrocarbon-based oil, water, glycerol, silicon oil, and the like, and the
scope of the disclosure
herein should be construed to include all types of immersion liquids suitable
for microscopy, and
specifically for microscopy of biological samples. However, for brevity and
for clarity, the
following discussion relates to a synthetic hydrocarbon-based immersion oil,
i.e. to an oil
objective, loading of immersion oil, and the like.
[0061] Reference is now made to Fig. 12, which is a partially exploded view of
an oil loading
subassembly 200, having an immersion oil cartridge 300, disposed above a lens
changing
subassembly 400, in accordance with embodiments of the invention. Lens
changing
subassembly 400 is substantially similar, in construction and operation, to
lens changing
subassembly 1000 described hereinabove. As seen, lens changing subassembly 400
includes a
lens holding magazine 410 having a plurality of positions for holding
objective lenses, here
shown as three positions. Leftmost position 412 of magazine 410 is illustrated
as temporarily
housing an oil immersion objective unit 500, similar to the objective units
described hereinabove
with respect to Figs. 3-9. Leftmost position 412 is predetermined to be the
oil-loading position,
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such that an oil outlet of oil loading subassembly 200 is disposed above oil-
loading position 412,
in a position to drip oil onto an objective lens of objective unit 500.
Immersion oil cartridge 300,
which is described in further detail hereinbelow, is positioned above a
corresponding well in oil
loading subassembly 200, and is adapted to be inserted thereinto, as explained
herein. It will be
appreciated that because position 412 is reserved for oil loading, lens
changing subassembly 400
as shown may effect changing between two lenses, which may be stored in the
other two
positions to the right of position 412. It will also be appreciated that lens
changing subassembly
400 may be formed with additional positions to facilitate changing between a
larger number of
lenses. Alternatively, as mentioned previously, in principle there need not be
a lens changing
subassembly, e.g. if there is enough room in the apparatus to provide an oil-
loading position
sufficiently far from the sample so as to not limit scanning to only a portion
of the sample.
[0062] Oil loading position 412 is selected such that the position is always
outside of the
scanning path of the lens scanning a sample.
[0063] In some embodiments, oil-loading position 412 of lens changing
subassembly 400 may
include a weighing mechanism, for determining the weight of objective unit 500
when it is
disposed in that position. For example, the weighing mechanism can obtain a
baseline weight, or
be tared, when the objective unit 500 is placed in oil-loading position 412.
As such, the
weighing mechanism can identify a change in weight of the objective unit, for
example
following application of the immersion oil thereonto. Based on the properties
of the immersion
oil, a controller associated with the weighing mechanism can determine how
much immersion
oil was applied to the objective unit 500, and whether or not additional
application of oil is
required.
[0064] The weighing mechanism may be any suitable mechanism for weighing the
objective
unit 500. In some embodiments, the weighing mechanism may include a flexible
leaf 420,
attached to a frame structure 402 of lens changing subassembly 400. A pair of
flexible arms 422
extend from leaf 420, the arms being disposed about objective unit 500, when
the objective unit
is in the position 412, such that objective unit 500 rests against arms 422.
Attached to one or
more surfaces of each arm 422, for example in a free region of the arm but, in
some
embodiments, near leaf 420 and/or near frame structure 402, there is a thin,
flat strain gauge (not
explicitly shown). Each of the strain gauges is electrically connected (e.g.
by thin wires, not
shown) to a thin, flat electronic card (not explicitly shown) located below
arms 422. The
electronic card(s) may be positioned so as to minimize the length of the
connections between the
strain gauges and the card(s). The electronic card(s) are electrically coupled
to a processor (not
shown). It will be appreciated that use of strain gauges and electronic cards
allows for
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correlation of the deflection of the arms 422 to a change in electrical
resistance in a circuit, for
example measured using a Wheatstone Bridge, which is also located on the
electronic card,
allowing calculation of the change in the weight of the objective unit 500,
and thus of the mass
of immersion oil applied to the objective lens. As such, arms 422 together
with the strain
gauge(s) form a signal provider, for providing a signal indicative of an
amount of immersion oil
applied to the objective unit. If the density of the immersion oil is known,
this facilitates
computation of the volume of immersion oil added. For example, it is possible
to iteratively
calculate the amount of immersion oil applied to the objective lens after each
application
thereof, and therefore to identify in real time, whether or not a sufficient
volume of immersion
oil has been applied. As such, the controller associated with the weighing
mechanism may be
functionally associated with an oil pump of oil loading subassembly 200,
described hereinbelow,
such that operation of the pump to apply immersion oil is started and stopped
based on
information relating to the weight of the objective unit.
[0065] In some embodiments, an overflow tray (not explicitly shown) may be
disposed beneath
magazine 410, or at least beneath oil loading position 412, to collect any
overflow of immersion
oil dripped from oil loading subassembly 200 onto objective unit 500, or which
may spill while
no objective is disposed in the oil loading position. It will also be
appreciated that the presence
of a holder for the lens, such as objective lens base 1020 in Fig. 5, may
provide a trap for oil if it
runs off the objective lens.
[0066] Alternatively, the amount of oil added can be calculated by calibrating
the average
amount of oil in 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
means of a beam of light connected to a sensor that provides a signal each
time a drop interrupts
the beam; or by means of a camera coupled to an automatic image analysis
program that detects
and counts falling oil drops. Alternatively, the amount of oil removed from
the oil reservoir can
be calculated each time the oil on the objective is replenished, e.g. by
measuring the change in
weight in the reservoir, to calculate the amount of oil applied to the lens.
[0067] Whether the amount of oil added is determined by direct measurement of
weight, or by
counting 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 a lens, can be calculated
and optionally
displayed on a screen or other output device.
[0068] Reference is now made to Fig. 13, which is an exploded view
illustration of oil loading
subassembly 200, in accordance with embodiments of the invention, and to Figs.
14A, 14B,
14C, and 14D, which are, respectively, a perspective view, a planar front
view, a planar side
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view, and a sectional view of oil loading subassembly 200 when constructed and
having
immersion oil cartridge 300 disposed therein. Such a cartridge may be made of
any suitable
material that does not significantly interact with the oil or other liquid
contained therein or lead
to degradation thereof. In the embodiments shown in Figs. 13-18B, the
cartridge is preferably
made of metal, such as aluminum. In the embodiments shown in Figs. 24-26B, the
cartridge is
preferable made of plastic.
[0069] As seen, oil loading subassembly 200 includes a main body portion 210
having a central
oil basin 212 in fluid communication with an oil viewing opening 214, and a
pump-seat bore
216, terminating in a pump inlet conduit 217 (see Fig. 14D). As seen in Fig.
14D, an oil flow
conduit 218 extends from the base of oil basin 212 and adjacent pump inlet
conduit 217, and
terminates at an edge of main body portion 210, where the oil flow conduit is
sealed by a stopper
220. An observation window 222 is disposed within, and seals, oil viewing
opening 214. As
seen clearly in Fig. 14B, observation window 222 includes a generally circular
transparent
portion 224, allowing an operator to observe the oil level in oil loading
subassembly 200, and to
determine when immersion oil cartridge 300 must be replaced, as explained in
further detail
hereinbelow. In a variation on this, the window may be provided with a cover
to keep light out
of the window when the user is not directly observing the oil level; or the
oil loading
subassembly 200 can be provided with neither viewing opening 214 nor window
222.
[0070] A cartridge puncturing element 230 is seated within central oil basin
212, and is attached
thereto by fasteners 232, such as screws or bolts. However, fasteners 232 may
be replaced by
any other suitable attachment mechanism, such as soldering, adhesion, and the
like. Cartridge
puncturing element 230 includes a base 234 and a puncturing pin 236, having a
hollow channel
238 extending longitudinally therethrough, the channel including a bore 239
(see Figs. 15B-
15C) and terminating in a sharp tip. When immersion oil cartridge 300 is
installed in central oil
basin 212, as shown in Figs. 14A-14D, hollow channel 238 is in fluid
communication with
central oil basin 212 as well as with oil flow conduit 218 (see Figs. 14D and
16A)
[0071] A diaphragm pump 250 is disposed within pump-seat bore 216, such that
an inlet 252
thereof is seated within pump inlet conduit 217, and an outlet conduit 254
thereof extends
downwardly out of main body portion 210. Diaphragm pump 250 is adapted to
aspirate oil from
oil flow conduit 218, through outlet conduit 254, onto an item disposed
beneath the outlet
conduit, which, in use, is an immersion oil objective lens.
[0072] Oil loading subassembly 200 further include a base mount 260, mounted
onto a bottom
side of main body portion 210, and adapted for attachment of oil loading
subassembly 200 to
other components of the microscopy system, above the lens changing subassembly
400. A back
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mount 265 is mounted to main body portion 210, rearwardly of central oil basin
212. Both base
mount 260 and back mount 265 may be connected to main body portion 210 by
fasteners 268,
such as screws, bolts, and the like. However, any other suitable way of
connection, such as
adhering, soldering, and the like, is considered within the scope of the
invention.
[0073] Reference is now made to Figs. 15A, 15B, and 15C, which are sectional
illustrations of
steps of insertion of immersion oil cartridge 300 into oil loading subassembly
200. As seen in
Fig. 15A, immersion oil cartridge 300 includes a housing 301 formed of a
generally cylindrical
wall portion 302 defining a hollow 304 having a first internal diameter, and
terminating at a lip
306. At one end thereof, distal to lip 306, wall portion 302 extends to a
generally transverse
shoulder 310, which narrows the internal diameter of the wall portion to form
a generally
cylindrical hollow neck portion 312, having a second internal diameter. The
second internal
diameter of neck portion 312 may be smaller than the first internal diameter
of wall portion 302.
At an end of neck portion 312, distal to shoulder 310, the thickness of the
wall of neck portion
312 is reduced, defining a first chamber having a third internal diameter,
which is greater than
the second internal diameter. The first chamber includes an annular shoulder
316, and terminates
at a lip 318. On an exterior surface thereof, neck portion includes snap fit
engagement
protrusions and/or grooves 319, adapted for snap fit engagement with
corresponding grooves
and/or protrusions 219 in the internal circumference of central oil basin 212.
[0074] A generally annular transverse wall 320 extends radially inward from
wall portion 302 at
a location disposed between lip 306 and neck portion 312, significantly closer
to lip 306 than to
neck portion 312. Annular transverse wall 320 terminates radially inwardly in
a cowl portion
322, which is substantially concentric with wall portion 302. A volume between
annular
transverse wall 320 and lip 306 defines a second chamber. A fluid flow path
exists, between the
first chamber and the second chamber, via the hollow defined by neck portion
312, the hollow
defined by wall portion 302, and the hollow of cowl portion 322.
[0075] A puncturing piston 330 is disposed within housing 301. Puncturing
piston 330 includes
a generally circular base 332 from which extends a central shaft 334, which
terminates at a
pointed edge 336, which may include one or more points. At an upper portion of
shaft 334, close
to edge 336, shaft 334 defines a hollow 338, and includes one or more bores
340 connecting the
hollow 338 with an environment surrounding shaft 334. The remainder of shaft
334 is non-
hollow.
[0076] In the initial, closed, operative orientation of immersion oil
cartridge 300, illustrated in
Fig. 15A, a first seal 350, which may be a material that is inert to the oil,
such as nylon or
aluminum foil, is disposed within the first chamber, and engaging annular
shoulder 316. Base
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332 of puncturing piston 330 is disposed within the hollow of neck portion 312
and engages first
seal 350, such that shaft 334 extends through the hollows defined by neck
portion 312, wall
portion 302, and cowl portion 322. Pointed edge 336 does not extend beyond an
end of annular
transverse wall 320, and may be flush therewith. A second seal 352, which may
be a material
that is inert to the oil, such as nylon or aluminum foil, is disposed in the
second chamber, and
engages transverse wall 320. As such, in the closed operative orientation of
immersion oil
cartridge 300, the cartridge is sealed, with oil disposed therein (for
clarity, the oil is not shown in
Fig. 15A).
[0077] In order to use the immersion oil in cartridge 300, the user moves the
cartridge in the
direction of arrow 360, such that first seal 350 faces toward central oil
basin 212 and puncturing
element 230 disposed therein.
[0078]
Turning to Fig. 15B, it is seen that as the user pushes the oil immersion
cartridge 300 into
central oil basin 212, puncturing element 230 thereof, and particularly
puncturing pin 236,
punctures through first seal 350, and pushes base 332 of puncturing piston 330
toward second
seal 352. Consequently, the entirety of puncturing piston 330 moves toward
wall portion 320, in
the direction of arrow 362, and pointed edge 336 of shaft 334 punctures
through second seal
352. Snap fit engagement protrusions and/or grooves 319 of oil immersion
cartridge 300 engage
corresponding grooves and/or protrusions 219 in the internal circumference of
central oil basin
212, ensuring that the cartridge is snap fit within the oil well. In this
orientation, the seals 350
and 352 are punctured, air can flow into oil immersion cartridge 300, and oil
can flow out of the
cartridge, as shown in Fig. 15C. As such, oil flows out of the cartridge,
around the base 332 of
puncturing piston 330, and through channel 238 and bore 239 of puncturing
element 230, into
central oil basin 212.
[0079] Reference is now additionally made to Figs. 16A and 16B, which are
sectional
illustrations illustrating the oil flow path within oil loading subassembly
200. As seen, following
unsealing of immersion oil cartridge 300 as shown in Figs. 15A to 15C, oil
flows from central
oil basin 212, via oil flow conduit 218 to pump inlet conduit 217. It will be
appreciated that in
order to reduce the likelihood of dust or other material contaminating the
oil, oil cartridge 300
may be left in place after it has been emplaced and punctured to release the
oil into basin 212, or
the apparatus may be provided with a cover (not shown) that covers basin 212
when oil cartridge
300 is removed.
[0080] Operation of diaphragm pump 250, for example in response to an input or
a signal
received from a controller, causes the pump to draw oil thereinto via inlet
252 seated in pump
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inlet conduit 217, as shown in Fig. 16A. The oil drawn into diaphragm pump 250
is then dripped
out of the pump via outlet conduit 254 of the pump, as shown in Fig. 16B. In
operation, an oil
immersion objective unit is disposed beneath outlet conduit 254, such that the
oil drips onto the
objective lens, as described in further detail hereinbelow.
[0081] Reference is now made to Fig. 17, which is a perspective view
illustration of lens
changing subassembly 400 and oil loading subassembly 200, with an objective
unit 500
disposed in immersion oil loading position 412. Reference is additionally made
to Figs. 18A and
18B, which are, respectively, a front view illustration and a side view
illustration of loading of
immersion oil onto the objective lens in the structure of Fig. 17.
[0082] As seen, immersion oil cartridge 300 is installed in oil loading
subassembly 200, which
is mounted above lens changing subassembly 400. Objective unit 500, including
an oil
immersion lens 502, is located in oil loading position 412 of lens changing
subassembly, such
that outlet conduit 254 of diaphragm pump is directly above oil immersion lens
502. As seen in
Fig. 18A, upon receipt of a suitable control signal triggering operation of
diaphragm pump 250,
a drop 504 of immersion oil is emitted from outlet conduit 254 onto immersion
lens 502. In
some embodiments, the oil drop contains 20-30 11.1 of oil. The drop remains as
a mound 506 on
the lens, due to the surface tension of the immersion oil. In some
embodiments, lens unit 500
may be raised, for example by motion of lens changing subassembly 200, so that
lens 502 is
close to the outlet opening of outlet conduit 254. This may ensure that the
emitted oil is dripped
directly onto the lens, and is not wasted.
[0083] In some embodiments, multiple drops of oil may be applied to lens unit
500 at each oil
loading occurrence. Once a sufficient amount of oil has been applied to lens
unit 500, the lens
unit may be returned to its scanning location, for example as described
hereinabove with respect
to Figs. 10 and 11, As explained in further detail hereinbelow, the amount of
oil dripped onto the
lens may be estimated, for example based on the expected spreading of oil in
view of motion
already carried out by the lens, or may be calculated, for example by a
weighing mechanism as
described hereinabove.
[0084] Reference is now made to Figs. 19A, 19B, and 19C, which illustrate the
spreading of the
immersion oil as the objective unit moves relative to a viewing surface,
during use of the
microscopy system.
[0085] As seen in Figs. 19A and 19B, a multi-well plate 600 is disposed within
a plate holder
602. Multi-well plate has a lower surface 604, and a plurality of wells 606,
each including a
biological sample. An oil immersion lens unit 500 is disposed beneath lower
surface 604, and is
initially positioned to scan a first sample in well 606a. As seen in the
enlarged portion of the
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figure, at this stage, which is the initial stage after loading immersion oil
onto lens unit 500, the
drop of oil that was disposed on the lens of lens unit 500 is spread by the
close proximity of the
lens unit to lower surface 604, forming an oil layer 610. Any excess oil may
flow to sloped
edges 510 of the objective unit, and may be caught in a 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 in engagement with the objective unit on the one side
and with the plate
on the opposing side. As will be discussed more below, the oil is usually
formulated so that it
has the same index of refraction as the bottom of the multi-well plate or
other sample holding
vessel; often that bottom is made of glass.
[0086] Fig. 19B illustrates lens unit 500 after it has moved along plate 600,
scanning wells 606b
and 606c, and has now reached well 606d. At this stage, the thickness of oil
layer 610 is reduced
relative to its thickness illustrated in Fig. 19A. This is because the
immersion oil has been spread
along lower surface 604 of the plate, by the motion of the objective unit. As
explained in further
detail hereinbelow, a controller associated with the objective unit, such as a
controller of the
microscopy system or of the XYZ movement discussed hereinabove, may be
configured to
determine that the motion distance of the objective unit has reached a
threshold value, at which
point it is expected that the immersion oil will be too thinly spread, and to
control operation of
the objective unit to return the objective unit to the oil loading position
for application of
additional oil thereto.
[0087] Fig. 19C illustrates five different states of the oil drop, disposed at
the top of lens unit
500. As seen, at an initial stage (i), which typically occurs immediately
after application of oil to
the lens unit, the oil drop forms a dome, based on the surface tension of the
oil. In fact, at this
time, the oil drop is actually in the shape of a drop.
[0088] At (ii), the lens unit approaches the surface 604 of the plate, but is
not yet spread along
the surface. At this time, the drop 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 detachment of
the oil drop from the
plate.
[0089] At (iii), the lens unit has come closer to surface 604, such that the
oil drop is spread
along a portion of the plate surrounding the area of the lens unit. However,
the oil is still
contained only in the region of the lens unit. In some embodiments, the
configuration of the oil
drop as shown in (iii) may be at a height equivalent to the initial scanning
height, as described
hereinbelow with respect to Figs. 21A and 21B.
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[0090] Image (iv) shows the configuration of the oil following focusing of the
objective, as
described in detail hereinbelow, and during scanning of an initial region or
well of the plate. As
seen, the oil is more spread out relative to (iii), but is still located only
above the objective lens,
and is substantially concentric thereto. After the lens unit moves, for
example to scan another
field of the well, or another well, some oil remains on the portion of the
plate previously scanned
by the objective, and some oil travels with the objective to the new region,
resulting in spreading
of the oil also to regions not disposed directly above the objective, as shown
in (v), where the
objective has moved to the right relative to the plate, and the oil now
extends above the
objective and to the left of the objective.
[0091] Fig. 20 is a flow chart outlining a method for scanning a sample using
an oil immersion
lens, and reloading immersion oil onto the oil immersion lens, in accordance
with embodiments
of the invention. The following description is provided with respect to plate
600 shown in Figs.
19A and 19B, which has a lower surface 604, and each well has an upper surface
upon which the
biological sample is disposed. However, the disclosure is equally relevant for
any other sample
carrying structure, having a lower surface and an upper surface.
[0092] As seen in Fig. 20, at a first setup step 700, the Z-axis location of
lower surface 604 of
plate 600 is approximated. The approximated value is stored, and will be used
as the
approximated value of the plate height for scanning of the entire plate. Such
an approximation
step may be carried out for each individual plate checked, or may be carried
out once for a
plurality of plates or sample carriers.
[0093] In some embodiments, the approximation of the Z-axis location of the
lower surface of
the plate may be carried out using a quick laser scan, identifying the bottom
surface of the plate
at several locations of planned scanning. In some other embodiments, multiple
focus points, e.g.
3 or 4, may be determined, using laser scanning, for the plate or for a region
of the plate, and an
approximate plane of the lower surface of the plate is determined based on the
multiple focus
points.
[0094] As explained hereinbelow, the approximated height of the lower surface
of the plate is
used as a basis for subsequent focusing operations for the plate, which are
conducted using a
search of a maximal signal of laser beam reflection, as explained herein.
There are several ways
this initial approximation may be carried out. One option is to be determine
the approximate
height of the lower surface of the plate using an air objective, and then
switching to the oil
immersion objective for actual focusing and scanning. Another option is to
determine the height
on the basis of the structure of the plate and apparatus, knowing that the
average distance of the
bottom of a particular model plate from a particular manufacturer will be a
given distance above
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the bottom edge of the plate, and knowing the dimensions of the apparatus. In
some
embodiments, the initial approximation of the height of the lower surface of
the plate may be
carried out by an oil immersion objective lens having a lower magnification
than the oil
immersion objective lens used for scanning the sample, and then switching to
the higher
magnification oil immersion objective lens for actual scanning.
[0095] In a second setup step 702, one or more scanning regions of the plate
or slide are defined,
for example using a user interface, and are provided to a controller
controlling the XYZ motion
of the objective lens, as described herein. In a third setup step 704, a
maximal threshold amount
of oil that can be lost by the oil immersion objective lens, prior to
replenishing the oil thereon, is
determined; this amount can be tracked in accordance with the total movement
of the objective,
by weight loss, or by oil thickness, as described below. In some embodiments,
the system can be
programmed to adjust this threshold dynamically after each instance of oil
replenishment, in
accordance with the amount of oil applied to the lens.
[0096] Although setup steps 700, 702, and 704 are described in a specific
order in Fig. 20, they
may also be carried out in a different order, provided that they occur before
scanning of the
biological sample is initiated.
[0097] At step 706, which also occurs prior to initiation of scanning, the
objective unit is placed
in the oil loading position of the lens changing assembly, and an initial
amount of oil is dripped
onto the objective lens from the oil loading subassembly, as described herein.
For example, the
controller associated with the diaphragm pump provides a signal for the pump
to drip immersion
oil onto the objective lens, when the objective lens is in the right position.
[0098] In some embodiments, the initial amount of oil is greater than
subsequent amounts of oil,
to be loaded at later stages as described herein.
[0099] In some embodiments, the exact amount of oil loaded onto the objective
lens is
determined using a weighing system as described hereinabove. In some other
embodiments, the
amount of oil loaded onto the objective is approximated, based on the number
of oil drops
applied to the objective lens, the volume of each drop of immersion oil
aspirated by the pump,
which is a known value, and the desired volume of oil to be aspirated.
[00100] At step 708, the controller controls XYZ motion of the oil-loaded
objective lens to get
the lens to the appropriate XY position for scanning, as determined in step
702. When initiating
scanning, the XY position is the position at which scanning should begin.
Following later
iterations of replenishing oil on the objective, as described hereinbelow, the
XY position is the
XY position at which scanning had stopped in order to replenish the immersion
oil, or the next
XY position. At step 710, the controller causes raising of the objective lens
to a predetermined
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initial scanning height, typically several microns from the approximate Z-axis
location of the
lower surface of the plate, as established at step 700. The predetermined
height is selected to be
close enough to the lower surface of the plate so that the oil drop on the
objective lens is spread
onto the bottom surface of the plate, as shown in Fig. 19A.
[00101] The distance from the plate at which the objective lens is in focus,
with respect to the
sample, is automatically calculated at step 712, for example using the method
described
hereinbelow with respect to Fig. 21, and the objective is moved to the focus
distance. Once the
objective lens is at the established focus distance, scanning of the sample
begins, at step 714.
[00102] During the scanning, the objective lens is moved relative to the
sample, or to plate
600, between different positions, as described hereinabove with respect to
Figs. 19A and 19B.
The distance traversed by the objective lens is tracked, for example by a
controller associated
therewith, including both movement when the oiled objective is in contact with
the surface of
the plate and movement when the objective is not in contact with the plate.
The controller can
be configured to calculate the amount of oil lost from the objective as a
result of such
movement, as well as the amount of oil that is lost when the objective is
lowered along the Z-
axis so that it is brought out of contact with the plate. In some embodiments
the controller also
takes into account the effect of differences in the plate material on the
amount of oil lost as the
objective is moved along the plate or removed from contact with the plate.
Alternatively or
additionally, the amount of oil lost may be tracked by monitoring the change
in the weight of the
oiled objective from the time it is oiled, and/or by inspection of the
thickness of the oil on the
objective, for example with a camera or by measuring with a laser.
[00103] At step 716, which may occur continuously or periodically, the
controller checks
whether the amount of oil lost as a result of objective lens movement is
greater than maximal
threshold amount established at step 704. If the traversed distance is smaller
than the maximal
threshold oil loss, scanning continues at step 714.
[00104] Otherwise, if the calculated amount of oil lost is equal to, or
greater than, the maximal
threshold amount, at step 718 the objective is lowered relative to plate 600,
and is moved, in a
XY plane, to the oil loading position. A replenishing amount of oil is then
loaded onto the
objective at step 720, substantially as described hereinabove with respect to
step 706, with the
exception that the replenishing amount of oil may be smaller than the initial
amount of oil. As
discussed hereinabove, the amount of oil required for replenishing the oil
drop on the objective
may be computed, for example based on the weighing mechanism, or may be
approximated
based on the distance traversed by the objective lens, or based on any other
relevant parameter.
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The flow then returns to step 708, for moving the objective lens back to the
scanning position
for continued scanning of the sample.
[00105] Reference is now made to Figs. 21A, 21B, and 21C, which are flow
charts outlining
embodiments of a method for automatically focusing an oil-loaded oil-immersion
lens on a
biological sample, in accordance with embodiments of the invention. The method
of Figs. 21A
to 21C assumes that an initial scanning height, typically of several microns
from the
approximate Z-axis location of the lower surface of the plate, is known. The
initial scanning
height is typically based on the height of the lower surface of the plate, as
established, for
example, at step 700 of Fig. 20. This automatic focusing is conducted each
time the lens is
repositioned at a different XY position, including after oil replenishment.
[00106] The initial scanning height is one in which it is known that the oil
drop, disposed on
the oil objective, engages the oil objective and the lower surface of the
plate, and is spread
therebetween. As noted above, typically the oil is formulated to have the same
index of
refraction as the material of which the bottom of the plate is constructed,
which is often glass.
The immersion oil, and the initial scanning height, are thus selected to
ensure that there are no
unwanted reflections, or refractions, when light passes between the oil and
the material of the
multi-well plate, viz, to ensure that the oil is in contact with both the lens
and the bottom of the
plate. As such, when a light beam is passed from the objective into the
sample, via the oil and
the material of the plate, the light will only be refracted once, at the
transition between the
material of the plate and the material of the sample, which occurs at the
upper surface of the
wells.
[00107] Turning to Fig. 21A, it is seen that in an initial step 750 of the
focusing process, the
objective is brought to the predetermined initial height, which is already
known, so that the oil is
brought into contact with the lower surface of the plate. This is equivalent
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. At step
752, the reflection of a laser signal, emitted via the objective, is measured.
[00108] At step 754, the objective is moved to another height along the Z-
axis, closer to the
lower surface of the plate, and the reflection of the laser signal at the new
position is measured at
step 756. The reflection value, at the new Z-axis height, is recorded. This
process is then
repeated, essentially continuously. The measurement is expected to increase,
until the focus
distance is reached, and then to begin decreasing.
[00109] At step 758, the controller evaluates the collected reflection data
and evaluates
whether there has been a decrease in the measured reflection of the signal
over the last several
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WO 2022/229906 PCT/IB2022/053972
iterations. If not, the flow returns to step 754 for measurement of the signal
at a new Z-axis
position. If however the measured reflection decreases, at step 760 the
objective is moved to the
Z-axis position at which the signal was maximal. This position along the Z-
axis is considered
the focus distance of the objective, with respect to the sample.
[00110] In some embodiments, the decay is considered sufficiently significant
if it is a
continuous decay of 0.1V in the laser signal.
[00111] The method of Fig. 21 is used to find the focus distance of the sample
each time a
new sample, or plate, is scanned, and following each replenishing of immersion
oil.
[00112] In some embodiments, a similar process is used to ensure that the
objective is focused,
for example when moving to a new field of the plate or when the objective has
been standing in
a single position, imaging for a long time in the same position (e.g. to avoid
the problem of drift
from the predetermined position). However, when moving to a new field, or
recalculating when
the objective has been standing a single position for a long time, the
previous focus distance is
known. As such, in this situation, at step 752 the objective is moved between
heights in the
range of known focus distance predetermined search range. Here too, the
focus distance is
determined to be at the point at which the maximal signal is obtained.
[00113] Turning to Fig. 21B, it is seen that in an initial step 770 of the
focusing process, the
objective is brought to the predetermined initial height, which is already
known, so that the oil is
brought into contact with the lower surface of the plate. This is equivalent
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. At step
772, the reflection of a laser signal, emitted via the objective, is
repeatedly measured while the
objective is moved a pre-determined distance along the Z-axis. The pre-
determined distance
along the Z-axis is selected such that it is expected that the measured
reflection of the signal will
reach a peak, and then decrease, during the Z-axis motion of the objective.
[00114] At step 774, the best fitted Gaussian (or polynomial) curve of the
signal peak
collected over all measurements taken at step 772 is calculated to determine
the peak maximum,
and the objective is moved to the Z-axis position at which the Gaussian (or
polynomial) was
maximal. This position along the Z-axis is considered the focus distance of
the objective, with
respect to the sample.
[00115] At step 776, the objective is moved to the focus distance determined
at step 774, and
scanning is initiated. As part of the scanning, images of the sample are taken
at the focus
position, until the scanning of the region is complete, or until a maximal
scanning duration has
passed.
CA 03218518 2023-10-30
WO 2022/229906 PCT/IB2022/053972
[00116] At step 778, the controller evaluates whether the scanning of the
region is complete. If
the scanning of the region is complete, the objective is lowered and is moved
to a new XY
position at step 780. The flow then returns to step 770, for refocusing of the
objective at the new
location.
[00117] Otherwise, if the scanning of the region is not complete, at step 782,
the controller
evaluates whether the maximal scanning duration has passed. If the maximal
scanning duration
has not passed, flow returns to step 776 and scanning is continued. If the
maximal scanning
duration has passed, the flow returns to step 772 for refocusing of the
objective, and ensuring
that there has been no drift of the objective or sample during the scanning
process. However,
when moving to a new field or remaining in the same position for a long time,
the previous
focus distance is known. As such, in this situation, at step 772 the objective
is moved between
heights in the range of known focus distance predetermined search range.
Here too, the
focus distance is determined to be at the point at which the maximal signal is
obtained.
[00118] In some embodiments, the decay is considered sufficiently significant
if it is a
continuous decay of 0.1V in the laser signal.
[00119] The method of Fig. 21B is used to find the focus distance of the
sample each time a
new sample, or plate, is scanned, and following each replenishing of immersion
oil.
[00120] Reference is now made to figs. 22A, 22B and 23, which show flowcharts
outlining
methods of scanning, as well as image processing and analysis, in accordance
with embodiments
of the invention. The flowchart in Fig. 22A outlines the scanning process
according to some
embodiments of the present invention. Initially, a first scanning operation of
a target is
performed according to user-defined parameters, such as size (e.g. ignore
objects larger than
and/or smaller than a particular size), shape (ignore non-circular objects or
non-semi-circular
objects), intensity, etc. The scanned image so obtained is then processed to
identify objects of
interest and their characteristics; this processing may be effected using
image processing
algorithms that are presently known in the art or that may be developed in the
future. The image
processing results are then analyzed according to predefined rules for
determining the optimal
parameters for performing further scanning operations of the same area. Based
on the results of
this analysis, new parameters are defined for scanning to acquire a new image
of the same target.
At least one second scanning operation is then performed using the new
scanning parameters.
Thus, for example, the system may scan a biological sample plate having 96
wells of 6 mm
diameter each. During the scan, the image processing algorithm recognizes each
living cell
present in the plate; if two or more cells are attached together, or if there
are individual cells
larger than a certain size, the system may note this as an unusual event which
should be
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observed further using higher magnification optics; if an objective lens
changer such as the one
described above is present, the lens can be changed to facilitate such
observation. The system
then determines the parameters that define the proper image quality for the
high magnification
scan. In some embodiments, the image processing is performed while the first
scanning
operation is still running, in which case the results of the analysis may
affect the operations of
the scanning in real time, in accordance with the newly-defined parameters. In
some
embodiments the at least one second scanning operation is performed at higher
magnification
than the first scanning operation; in some embodiments, in which the present
method is utilized
in conjunction with an apparatus equipped with an objective lens changer as
described above,
the second scanning operation at higher magnification is performed by changing
to a higher
magnification objective lens using the objective lens changer and then
scanning the areas of
interest at higher magnification.
[00121] Fig. 22B is a flowchart of the scanning process in accordance with
some embodiments
of the present invention. At the first stage of the scanning, a low
magnification image at a
defined location is acquired; this process is repeated until images at a
plurality of defined
locations are acquired. Upon acquisition, each scanned image is transferred to
an image
processing and analysis module, which begins image processing and analysis
upon receipt of the
first image; this module may be incorporated into the software controlling 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 which includes information about areas of
interest in accordance
with objects identified by the initial scan and their characteristics. The
location matrix may thus
be completed shortly after completion of the low magnification scan.
Alternatively, the location
matrix may be generated after all processing and analysis is complete. On the
basis this location
matrix, the second stage of scanning is performed, during which high
magnification images at
specified locations of interest are acquired.
[00122] Fig. 23 is a flowchart of the image processing and analysis process
according to some
embodiments of the present invention. At the first stage of the image
processing the following
steps are performed: acquire a small magnification image, apply a low pass
filter to the data
obtained, apply a high pass filter to the data obtained, and perform a
Watershed transformation.
On the basis of these image processing steps, objects and object
attributes/characteristics are
detected and extracted. Next, relevant objects are selected using user
parameters/
attributes/characteristics, and object centers (L[Cx,Cy]) are extracted. High-
magnification
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images of the selected objects are then obtained, from which a 3D
transformation matrix
between objects (M) is then created, and then utilized.
[00123] Fig. 24 is a perspective view illustration of an oil loading
subassembly 1200, in
accordance with another embodiment of the present invention. Oil loading
subassembly 1200 is
substantially similar to oil loading subassembly 200 of Figs. 13 to 14D, with
like numbers
representing like elements. For brevity, the following description focuses on
the distinctions
between oil loading subassembly 1200 and oil loading subassembly 200.
[00124] As seen, oil loading subassembly 1200 includes a main body portion
1210, which
includes a central oil basin 1212, which may be in fluid communication with an
oil viewing
opening, and a pump-seat bore, terminating in a pump inlet conduit, as
described hereinabove
with respect to Fig. 14D). Similarly to that shown in Fig. 14D, an oil flow
conduit extends from
the base of oil basin 1212 adjacent the pump inlet conduit, and terminates at
an edge of main
body portion 1210, where the oil flow conduit is sealed by a stopper. An
observation window, as
described hereinabove with respect to Fig. 14B, may be disposed within, and
may seal, the oil
viewing opening, although as explained above such a window is not required.
[00125] A cartridge puncturing element 1230 is seated within central oil basin
1212, and may
be attached thereto by any suitable attachment mechanism, such as fasteners,
soldering,
adhesion, and the like. Cartridge puncturing element 1230 includes a base 1234
and a central
puncturing pin 1236, having a hollow channel 1238 extending longitudinally
therethrough, the
channel terminating in a sharp tip and being in fluid communication with a
bore 1239 formed in
the side of cartridge puncturing element 1230. A pair of peripheral puncturing
pins 1240 extend
from edges of base 1234, and may be substantially parallel to each other. Each
of puncturing
pins 1240 is substantially planar, and has a first thickness for most of the
longitudinal length
thereof. An end of each puncturing pin 1240, distal to base 1234, has a second
thickness, smaller
than the first thickness, such that a shoulder 1242 is formed near the end of
the puncturing pin.
Each puncturing pin 1240 terminates in a sharp tip 1244.
[00126] When an immersion oil cartridge 1300 is installed in central oil basin
1212, as
described hereinbelow with respect to Figs. 26A and 26B, hollow channel 1238
of central
puncturing pin 1236 is in fluid communication with central oil basin 1212 as
well as with the oil
flow conduit, in a similar manner to that shown in Figs. 14D and 16A.
[00127] A diaphragm pump 1250 is disposed within the pump-seat bore, such that
an inlet of
pump 1250 is seated within the pump inlet conduit, and an outlet conduit 1254
of the pump (see
Figs. 26A and 26B) extends downwardly out of main body portion 1210,
substantially analogous
to what is shown in Figs. 13 to 14D. As described hereinabove with reference
to diaphragm
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pump 250, diaphragm pump 1250 is adapted to aspirate oil from the oil flow
conduit, through
the pump outlet conduit, onto an item disposed beneath the pump outlet
conduit, which, in use,
is an immersion oil objective lens.
[00128] Oil loading subassembly 1200 further include a base mount 1260,
mounted onto a
bottom side of main body portion 1210, and adapted for attachment of oil
loading subassembly
1200 to other components of the microscopy system, above the lens changing
subassembly 400.
A back mount 1265 is mounted to main body portion 1210, rearwardly of central
oil basin 1212.
Both base mount 1260 and back mount 1265 may be connected to main body portion
1210 by
any suitable way of connection, such as fasteners, adhering, soldering, and
the like.
[00129] Reference is now additionally made to Fig. 25, which is a sectional
illustration of an
immersion oil cartridge 1300, according to another embodiment of the present
invention.
[00130] As seen in Fig. 25, immersion oil cartridge 1300 includes a generally
cylindrical
housing 1301. Housing 1301 includes a base 1302, from which extends a first
cylindrical wall
portion 1304, having a first width, and defining a first chamber 1305 having a
first internal
diameter dl. A second cylindrical wall portion 1306 extends from first
cylindrical wall portion
1304. Second wall portion 1306 has a second width, smaller than the first
width. An external
surface of second wall portion 1306 is flush with an external surface of first
wall portion 1304,
and an internal shoulder 1308 is formed between the internal surfaces of wall
portions 1304 and
1306. A third cylindrical wall portion 1310 extends from second cylindrical
wall portion 1306.
Third wall portion 1310 has a third width, smaller than the second width, and
forms a neck
portion of cartridge 1300. An internal surface of third wall portion 1310 is
flush with an internal
surface of second wall portion 1306, and an external shoulder 1312 is formed
between the
external surfaces of wall portions 1310 and 1306. Third cylindrical wall
portion 1310 terminates
at a lip 1314, distal to base 1302. Second wall portion 1306 and third wall
portion 1310 together
define a second chamber 1315, having a second internal diameter d2. Second
chamber 1315 is in
fluid communication with first chamber 1305.
[00131] In some embodiments, an external surface of third wall portion 1310,
distal to
external shoulder 1312, may include snap fit engagement protrusions and/or
grooves, adapted
for snap fit engagement with corresponding grooves and/or protrusions in the
internal
circumference of central oil basin 1212.
[00132] A substantially cylindrical oil container 1320 is disposed within
housing 1301, and
defines a hollow 1321. A first end 1322 of oil container 1320 is disposed
within first chamber
1305 and engages an internal surface of base 1302. A second end 1324 of oil
container 1320 is
substantially flush with lip 1314, such that the majority of oil container
1320 is disposed within
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WO 2022/229906 PCT/IB2022/053972
second chamber 1315. Oil container 1320 includes a plurality of bores 1326,
which are closer to
first end 1322 than to second end 1324, while being in fluid communication
with second
chamber 1315. A cylindrical gap 1330 is formed between housing 1301 and oil
container 1320,
within second chamber 1315.
[00133] A substantially cylindrical pushing element 1340 is disposed about oil
container 1320
within gap 1330, and is movable longitudinally relative to the oil container.
A first end 1342 of
pushing element 1340 is adapted to be disposed closer to first end 1322 of oil
container 1320,
and a second end 1344 of the pushing element is adapted to be disposed closer
to second end
1324 of the oil container. Cylindrical pushing element 1340 includes a
plurality of bores 1346,
which are adapted to be radially aligned with bores 1326 of oil container
1320. The longitudinal
alignment of bores 1346 and bores 1326 depends on the position of pushing
element 1340
relative to oil container 1320, as explained in further detail hereinbelow.
[00134] First end 1342 of pushing element 1340 is adapted to push against an
annular 0-ring
1350, which is disposed within gap 1330. As explained in further detail with
respect to Figs.
26A and 26B, when 0-ring 1350 is disposed below bores 1326, it seals gap 1330
such that no
fluid path exists between hollow 1321 and gap 1330. By contrast, when 0-ring
1350 is pushed
to lie against shoulder 1308, and bores 1326 and 1346 are aligned with one
another, as shown in
Fig. 25, a fluid path is formed between gap 1330 and hollow 1321, via bores
1326 and 1346.
[00135] In a storage operative orientation of cartridge 1300, a sealing foil
1360 (see Fig. 26A),
or any other seal, is disposed against lip 1314 of housing 1301 and second end
1324 of oil
container 1320 to seal oil within the cartridge. In the storage orientation, 0-
ring 1350 is disposed
below bores 1326, and seals gap 1330 such that no fluid path exists between
hollow 1321 and
gap 1330.
[00136] Reference is now additionally made Figs. 26A and 26B, which are
sectional
illustrations of steps of insertion of the immersion oil cartridge 1300 of
Fig. 25 into the oil
loading subassembly 1200 of Fig. 24.
[00137] As seen in Fig. 26A, cartridge 1300 is in the storage orientation,
with lip 1314 of
housing 1301, second end 1324 of oil container 1320, and second end 1344 of
pushing element
1340 all being flush with one another, and disposed against sealing foil 1360.
In the storage
orientation, 0-ring 1350 is disposed below bores 1326, against first end 1342
of pushing
element 1340. The 0-ring seals gap 1330 such that no fluid path exists between
hollow 1321 and
gap 1330. Oil 1365 is disposed within hollow 1321, such that a height of the
oil does not extend
above bores 1326.
CA 03218518 2023-10-30
WO 2022/229906 PCT/IB2022/053972
[00138] To load cartridge 1300 into oil loading subassembly 1200, cartridge
1300 is moved
toward central oil basin 1212, in the direction of arrow 1370, until central
pin 1236 punctures
sealing foil 1360 and enters hollow 1321. At this initial installation step, 0-
ring 1350 remains
below bores 1326, and the fluid path between hollow 1321 and gap 1330 remains
blocked.
[00139] As cartridge 1300 continues to be moved in the direction of arrow
1370, tips 1244 of
peripheral puncturing pins 1240 puncture the periphery of foil 1360, in the
vicinity of gap 1330.
Shoulders 1242 of the peripheral puncturing pins 1240 engage second ends 1344
of pushing
element 1340, and as the cartridge continues to be lowered, pressure applied
to the second ends
1344 by shoulders 1242 moves the pushing element 1340 toward base 1302 of
housing 1301,
which in turn pushes 0-ring 1350 toward shoulder 1308.
[00140] As seen in Fig. 26B, in the installed operative orientation of
cartridge 1300, shoulders
1242 of peripheral puncturing pins 1240 and pushing element 1340 have pushed 0-
ring 1350 to
rest against shoulder 1308. In this arrangement, bores 1346 of pushing element
1340 are aligned
with bores 1326 of oil container 1320, such that a fluid path exists between
central oil basin
1212, gap 1330, hollow 1321 of oil container 1320, and channel 1238 of central
puncturing
element 1236. In this arrangement, oil can flow out of container 1320 into
central oil basin 1212,
and operation of the system continues substantially as described hereinabove.
[00141] It will be appreciated that an oil- or other liquid dispensing
apparatus such as that
described herein may also be utilized in conjunction with a confocal
microscope. Additionally,
the illumination source need not necessarily be a traditional laser but may be
a light-emitting
diode (LED) or a combination or array of LEDs. Consequently, a microscope or
scanner
equipped with an oil- or other liquid dispensing apparatus such as described
herein may be used
in high-content imaging (HCI, also sometimes called high-content screening) to
obtain images
which can then be processed in accordance with known techniques, such as photo-
activated
localization microscopy (PALM) (see e.g. Betzig, E. et al., "Imaging
intracellular fluorescent
proteins at nanometer resolution", Science 313, 1642-1645 (2006)) or
Stochastic Optical
Reconstruction Microscopy (STORM) (see e.g. Rust, M. J., Bates, M. & Zhuang,
X., "Sub-
diffraction-limit imaging by stochastic optical reconstruction microscopy
(STORM)", Nature
Methods 3, 793-795 (2006)). In one embodiment, images obtained using an
acquisition device
equipped with an oil-dispensing apparatus such as described herein can be
processed in
accordance with super-resolution radial fluctuations (SRRF), as described in
Gustafsson et al.,
"Fast live-cell conventional fluorophore nanoscopy with ImageJ through super-
resolution radial
fluctuations", Nature Communications 7:12471 (published August 12, 2016). Such
processing
may be carried out using the ImageJ software plugin, freely available at
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CA 03218518 2023-10-30
WO 2022/229906 PCT/IB2022/053972
https://henriqueslab.github.io/resources/Nanal-SRRF/. The fact that the
apparatus and methods
described herein facilitate automated dispensing of oil (or other liquid) to
the objective lens
means that this apparatus can be combined in completely automated image
acquisition:
automated scanning, automated autofocusing, automated objective exchange,
automated
dispensing of lens immersion medium, and automated detection of the target of
interest.
[00142] Unless otherwise defined, all technical and scientific terms used
herein have the same
meanings as are 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.
[00143] 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 prevail. In addition, the materials, methods, and
examples are
illustrative only and not intended to be limiting.
[00144] 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 general combination of parts that perform
the same functions
as exemplified in the embodiments, and includes both 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.
32
