Systems And Methods Of Reduced Condensation Microscopy

A system for sample imaging includes a control unit for delivering conditioned air, and a specimen chamber that receives the conditioned air from the control unit. The specimen chamber includes a chamber housing having an upper face, a lower face opposite the upper face and configured to face an imaging lens, and walls that extend vertically between the upper and lower faces. The walls define an interior volume of the specimen chamber. The specimen chamber includes an air actuator unit configured to direct conditioned air to a target location alongside the lower face for inhibiting or at least reducing condensation accumulation on the imaging lens.

TECHNICAL FIELD

The present disclosure relates to the field of microscopy, in particular the field of components for effecting localized environmental conditions.

BACKGROUND

To date, those in the field have encountered difficulty in designing an on-stage incubation chamber that is compatible with a variety of sample containers (e.g., vessel plates) while also creating an air-tight seal with the various sample containers. This tends to be problematic because gaps between the bottom edge of the vessel plate and the incubation chamber can allow humid gas to escape. The microscope objective lenses are often located directly beneath the part of the stage where the incubation chamber sits and are typically much colder in temperature than the escaping humid gas. In such instances, humid air that escapes the incubation chamber (e.g., through gaps between the vessel plate and the chamber) and contacts the objective lenses tends to condense on the lenses, which can cause imaging problems.

One option to combat condensation is to use a heating jacket which warms the objective to raise its dewpoint above the point of condensation. Such an approach, however, is primarily used with water-immersion optics to keep the sample from being cooled by the objective, and the solution thus is not useful with other kinds of microscopy. Heating jackets can also require physical access to the objective area of the microscope and will not work on microscopes which have rotating objective turrets. Other options to address fogging including stopping an experiment to dry the objective and/or to use an anti-fogging agent, but the efficacy of these methods is limited. Accordingly, there is a long-felt need in the art for improved systems for incubation chambers that can be used with minimal or even no fogging of the objective lens used to observe sample within the incubation chamber.

SUMMARY

In meeting the described needs, the present disclosure provides a system for sample imaging, the system comprising: a specimen chamber configured to contain a local environment therein; and an air actuator, the specimen chamber being configured such that air encouraged by the actuator is directed between the specimen chamber and an objective lens, the air being directed so as to reduce or eliminate condensation on the objective lens.

In certain aspects, a specimen chamber for use with a sample imager includes a chamber housing that has a first face and a second face opposite each other along a first direction, wherein the first face is configured to face an imaging lens, the second face is configured to mount with a lid having a window, and the first face is spaced from the second face in a lens-facing direction along the first direction. The chamber housing includes first and second endwalls opposite each other along a second direction substantially perpendicular to the first direction. The chamber housing also includes first and second sidewalls opposite each other along a third direction substantially perpendicular to the first and second directions, so that the first and second endwalls and first and second sidewalls substantially enclose an interior volume with respect to the first and second directions. The interior volume is configured to contain a local environment therein. The chamber housing includes an air actuator unit that is configured to direct conditioned air to a target location alongside the first face and spaced from the first face in the lens-facing direction. The conditioned air is configured to inhibit or at least reduce condensation accumulation on the imaging lens.

In certain aspects, a system for sample imaging includes a control unit for delivering conditioned air, and a specimen chamber that receives the conditioned air from the control unit. The specimen chamber includes a chamber housing having an upper face, a lower face opposite the upper face and configured to face an imaging lens, and walls that extend vertically between the upper and lower faces. The walls define an interior volume of the specimen chamber. The specimen chamber includes an air actuator unit configured to direct conditioned air to a target location alongside the lower face for inhibiting or at least reducing condensation accumulation on the imaging lens.

In certain aspects, the specimen chamber comprises a feature that supports a sample container disposed within the specimen chamber. In some embodiments, the specimen chamber comprises a lower region for engaging with a specimen container. In some embodiments, the lower region comprises a first heating element. In some embodiments, the first heating element is configured to heat the specimen chamber to a range of about 30° C. to about 40° C. In some embodiments, the heating element further heats the air encouraged by the air actuator. In some embodiments, the specimen chamber comprises a lid. In some embodiments, the lid comprises a second heating element. In some embodiments, the second heating element is configured to heat the specimen chamber to a range of about 30° C. to about 40° C.

In certain aspects, the chamber comprises a manifold and an outlet, wherein the manifold is configured to direct air encouraged by the actuator to the outlet. In some embodiments, the outlet is in register with the objective lens and in proximity to the objective lens such that air exiting the outlet impinges on the objective lens. In some embodiments, the outlet is moveable. In some embodiments, the outlet is slidable, rotatable, or both. In some embodiments, the manifold further comprises one or more sensors. In some embodiments, the one or more sensors comprise a temperature sensor.

In certain aspects the system further comprises a control unit that delivers a conditioned air to the specimen chamber. In some embodiments, the control unit comprises a gas mixing manifold. In some embodiments, a gas mixture within the gas mixing manifold comprises one or more gases selected from oxygen, carbon dioxide, nitrogen, and standard air, the gases balanced to a selected mixture of concentrations. In some embodiments, the control unit comprises a pump configured to encourage the conditioned air to a fluid inlet of the specimen chamber. In some embodiments, the pump is positioned internal to the control unit. In some embodiments, the conditioned air comprises humidified air or heated humidified air. In some embodiments, the conditioned air comprises dried air or heated dried air. In some embodiments, the humidified air comprises a humidity of about 50% to about 90% humidity. In some embodiments, the selected mixture of concentrations comprises from about 5% to about 12% carbon dioxide. In some embodiments, the selected mixture of concentrations comprises up about 21% oxygen. In some embodiments, the selected mixture of concentrations comprises from about 67% to about 95% nitrogen.

In certain aspects, the system of the present disclosure as described above is configured for operation such that the minimum temperature among all locations of a sample container located within the specimen chamber is within about 15% of the maximum temperature among all locations of the sample container.

Also provided is a method comprising: operating a system for improved imaging as disclosed herein so as (1) reduce or eliminate condensation on the objective lens, (2) maintain a first environment within the specimen chamber that differs in one or more of temperature, humidity, and gas mixture composition from an ambient environment exterior to the specimen chamber, or both (1) and (2).

In some embodiments, the method further comprises operating the system as disclosed herein so as to maintain a second environment within the specimen chamber that differs in one or more of temperature, humidity, and gas mixture composition from an ambient environment exterior to the specimen chamber, the second environment differing from the first environment. In some embodiments, wherein the operating is performed such that during the operating, the minimum temperature of all locations of a sample container located within the specimen chamber is within about 15% of the maximum temperature of all locations of the sample container.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present disclosure may be understood more readily by reference to the following detailed description of desired embodiments and the examples included therein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

Unless indicated to the contrary, the numerical values should be understood to include numerical values which are the same when reduced to the same number of significant figures and numerical values which differ from the stated value by less than the experimental error of conventional measurement technique of the type described in the present application to determine the value.

All ranges disclosed herein are inclusive of the recited endpoint and independently of the endpoints. The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values.

As used herein, approximating language can be applied to modify any quantitative representation that can vary without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term or terms, such as “about” and “substantially,” may not be limited to the precise value specified, in some cases. In at least some instances, the approximating language can correspond to the precision of an instrument for measuring the value. The modifier “about” should also be considered as disclosing the range defined by the absolute values of the two endpoints. For example, the expression “from about 2 to about 4” also discloses the range “from 2 to 4.” The term “about” can refer to plus or minus 10% of the indicated number. For example, “about 10%” can indicate a range of 9% to 11%, and “about 1” can mean from 0.9-1.1. Other meanings of “about” can be apparent from the context, such as rounding off, so, for example “about 1” can also mean from 0.5 to 1.4. Further, the term “comprising” should be understood as having its open-ended meaning of “including,” but the term also includes the closed meaning of the term “consisting.” For example, a composition that comprises components A and B can be a composition that includes A, B, and other components, but can also be a composition made of A and B only. Any documents cited herein are incorporated by reference in their entireties for any and all purposes.

Referring now toFIG.1, an incubator system100according to the present disclosure includes a control unit102and a specimen incubation chamber106(also referred to herein as a “specimen chamber”106) connected to the control unit102. The incubator system100can also be referred to herein as an “incubator”100. The control unit102is configured to control and adjust a local environment contained within the specimen chamber106. As shown, the control unit102can be connected via at least one connector104to at least one inlet107of the specimen chamber106.

Referring now toFIG.2, the control unit102can include a gas mixing manifold110, which can allow for mixing of gases received from one or more sources. In some embodiments, the one or sources can include one or more external sources, that is, external to the manifold110, including for example, one or more gas tanks, gas generators, or other external gas supplies. In some embodiments, the one or more sources can include one or more sources internal to the manifold110. The manifold110can include a plurality of gas regulator valves and can be connected to external gas supplies, which external gases can be mixed within the manifold to the required experimental conditions. The gas supplies can include but are not limited to nitrogen (N2), carbon dioxide (CO2), oxygen (O2), air, and the like, including one or more combinations thereof. Valves connected to the external gas supplies can be actuated to allow and stop the passage of the external gases into the manifold110; the manifold110can include one or more sensors configured to detect one or more gas levels. In this way, a user can connect the manifold110to the desired gases and then, by actuation of the valves, blend the gases to arrive at a mixture within the manifold110that is to the required experimental conditions. For example, the user can set the O2level to a value in the range of from about 0% to about 21% including any and all intermediate values. Similarly, the user can set the CO2level to a value in the range of from about 0% to about 20% including any and all intermediate values.

The composition of the gas mixture within the manifold110can be changed over time, that is, from a first blend at a first time to a second blend at a second time, which can in turn allow a user to expose a sample in fluid communication with the gas manifold110to different gas conditions at different times. A pump108, such as an air pump108, can be used to take in air from exterior to the control unit and/or gases from the gas mixing manifold110of the control unit. Control unit102can include a water reservoir114, which can be located within the control unit. In some instances, the lower portion of the reservoir can be aluminum or another thermally conductive material, and the upper portion can be polycarbonate or other plastic. By way of non-limiting examples, the lower portion of the reservoir can be a material that conducts heat efficiently, and the upper portion of the reservoir can be clear to allow the user to see the water level therein. A heater, including for example a heater plate116can be used to heat the water reservoir114, which can in turn give rise to a humidified air. The heater plate116can be a 60 Watt (W) heater, by way of a non-limiting example, and can include a digital temperature sensor. Conditioned air, which can include at least some of the contents of the gas mixing manifold110and/or a humidified air derived from heating of the water reservoir114, can be delivered via the connector104(seeFIG.1) from the control unit102to the specimen chamber106. For example, the humidity in the specimen chamber106is controlled by one or more valves on the gas mixing manifold110. A valve can be actuated to allow the gas mixture to flow over the water reservoir114, thereby humidifying the gas mixture. If less humidity is preferred, the valve is at least partially closed and a second valve opens, allowing an amount of the gas mixture to bypass the heated water reservoir114. In some embodiments, the connector104can also include a heating element that heats air and allows a user to deliver to the specimen chamber106air at a desired temperature. The pump108can be located within control unit102and can deliver conditioned air (or non-conditioned air) from the control unit102via the connector104to the specimen chamber106. In some embodiments, humidified air provides for an environment where sample media either does not evaporate or evaporates at a reduced rate, allowing the user to perform longer-term experiments while maintaining sample viability and more accurate sample media volume levels in the absence of evaporation.

As shown inFIG.2, the control unit102can include one or more of various sensors, such as an oxygen sensor118and a carbon dioxide (CO2) sensor112, by way of non-limiting examples. The control unit102can also include one or more gas filter/regulators120. The gas filter/regulators120can be used to, for instance, modulate the delivery of gases to the gas mixing manifold110from exterior sources of gas and to remove particulate from the circulating air via filtration. In some embodiments, the filter/regulators remove particulate greater than about 5 μm from the circulating air. The filter/regulators operate to control the pressure of gas to the manifold110. In some embodiments, the circulating gas is regulated to a pressure of about 35 psi. The control unit102can include one or more sensors that report one or more of a temperature, a humidity, and a gas content of a conditioned air delivered from the control unit102via connector104. The sensors can be mounted on or adjacent to the manifold110, or in fluid communication with the manifold110.

FIG.3provides an exterior view showing a user interface panel126, which can be located at the rear of the control unit102. As shown, the user interface panel126of the control unit102can include an air filter126athat filters air drawn in by the air pump108shown inFIG.2. The control unit102can include one or more gas inlets126b, which gas inlets126bcan be connected to external gas supplies, for example, supplies of O2, N2, CO2gas and/or air. The control unit102can also include a power supply connection126c, a data and/or power connection126dto the specimen chamber106(seeFIG.1), and a data port126e, for example, a USB port, that allows for connection between the control unit102and an external computer or other control device.

Referring now toFIG.4A, a partial exploded, top perspective view of the specimen chamber106is shown according to an exemplary embodiment. As shown, the specimen chamber106can include a chamber housing150that defines an interior volume135for holding therein one or more sample containers having sample media. The interior volume135can also be referred to herein as the “interior”135of the specimen chamber106. The specimen chamber106also preferably includes a cover or “lid”122for coupling with the chamber housing150and enclosing a portion of the interior volume135. The lid122preferably includes a window124for viewing sample media located in the interior volume135of the specimen chamber106. The lid122is discussed in more detail below. The chamber housing150includes at least one inlet107, which can receive conditioned air from the control unit102via the at least one connector104(seeFIG.1) for delivery to the interior volume135.

The chamber housing150has a first face152and a second face154opposite the first face152along a first direction Z. The first face152is configured to face an imaging lens, such as an objective lens142, along the first direction Z. The second face154is configured to mount with the lid122. The lid122can be securably mountable to the second face154. Alternatively, the lid122can be a lift-off lid that is not otherwise secured to the chamber housing150.

The first face152is spaced from the second face154of the chamber housing150in a lens-facing direction Z1along the first direction Z, while the second face154is spaced from the first face152in a lens-away direction Z2opposite the lens-facing direction Z1. It should be appreciated that the lens-facing direction Z1and the lens-away direction Z2are each mono-directional components of the first direction Z, which is bi-directional. The chamber housing150also includes first and second endwalls156,158opposite each other along a second direction X substantially perpendicular to the first direction Z. The chamber housing150further includes first and second sidewalls160,162opposite each other along a third direction Y substantially perpendicular to the first and second directions. The first and second endwalls156,158and the first and second sidewalls160,162substantially enclose the interior volume135with respect to the second and third X, Y directions. The interior volume135is configured to contain a local environment therein, preferably an incubated local environment for receiving one or more sample containers. The specimen container106is configured so that a user can remove the lid122from the chamber housing150, place one or more sample containers within the interior volume135, and replace the lid122atop the second face154, thereby enclosing the interior volume135with respect to the lens-away direction Z2.

In the illustrated embodiment, the specimen chamber106is configured to be placed atop a stage (e.g., a movable x-, y-stage) of a microscopic imaging system. Accordingly, during use, the first direction Z is the vertical direction and the second and third directions X, Y are each horizontal directions when the specimen chamber106is placed at such an orientation. In such embodiments, the lens-facing direction Z1can be characterized as the “downward” direction Z1, and the lens-away direction Z2can be characterized as the “upward” direction Z2. It should be appreciated that, as used herein with reference to the illustrated embodiments (e.g., when referring to spatial relationships between various features), directional terms can be used to indicate spatial relationships between various features of the specimen chamber106. For example, the terms “downward”, “down”, “under”, “bottom”, “beneath”, and derivatives thereof refer to the downward direction Z1; and the terms “upward”, “upper”, “above”, “top”, “atop”, and derivatives thereof refer to the upward direction Z2. By way of some specific, non-limiting examples, when referring to the illustrated embodiments herein, the first face152of the housing body150can also be referred to as a “lower” face152; and the second face154of the housing body150can also be referred to as an “upper” face154. Similar such directional terms are also used herein to describe other features of the illustrated embodiment. In other embodiments, however, that specimen chamber106can be adapted so that the first direction Z is offset from vertical (and by extension, one or both of the second and third directions can be offset from horizontal) during use. It should be appreciated that, unless stated otherwise herein, the foregoing spatial relationships of the various described features also indicate spatial relationships between various features in embodiments where the first direction Z is offset from vertical. By way of two such examples, the specimen chamber106can be adapted for use with a microscopic imaging system in which the imaging lens faces downward instead of upward, or alternatively faces horizontally instead of vertically. The reader will appreciate that, in such alternative configurations, it is the “bottom face”152of the chamber housing150that faces the imaging lens, even if the lower face152is positioned above the upper face154(in the case of a downward facing lens), or even if the lower face152and the upper face154are spaced from each other horizontally instead of vertically (in the case of a horizontally facing lens). Summarized differently, the directional terms used herein indicate spatial relationships between various features and, unless stated otherwise herein, those spatial relationships will also apply regardless of the specific orientation in which the specimen chamber106is oriented in three-dimensional space.

Referring now toFIG.4B, the chamber housing150can include a first or “lower” housing body150aand a second or “upper” housing body150bthat are connectable to each other. In the illustrated embodiment, the lower housing body150adefines the lower face152, the upper housing body150bdefines the upper face154, and the lower and upper housing bodies150a,bare connectable to each other in a sandwich-like fashion. As shown, the lower and upper housing bodies150a,bcan each include respective first and second body endwalls156a,b,158a,band first and second body sidewalls160a,b,162a,b, which combine to form portions of the first and second endwalls156,158and first and second sidewalls160,160of the chamber housing150when the lower and upper housing bodies150a,bare coupled together. The lower and upper housing bodies150a,balso preferably each define a central aperture155a,bextending therethrough along the first direction Z. The central aperture155aof the lower housing body150aprovides an open, unobstructed space between the specimen chamber106and the imaging lens for obtaining clear images of sample media placed in the specimen chamber106. The central aperture155bof the upper housing body150bprovides an opening through which a user can place one or more sample containers into the interior volume135of the specimen chamber106while the lid122is removed.

The lower housing portion150apreferably also has an interior support surface153, which can extend around an interior periphery of the endwalls156a,158aand sidewalls160a,162ain a rim-like fashion. The interior support surface157can be configured to support various features of the specimen chamber106, as described in more detail below. The lower housing portion150aalso preferably has a platform surface157, which can be located on a side of the first endwall156aopposite the central aperture155aalong the second direction X. The platform surface157can be configured to support various structural features of the chamber housing150, such as circuitry (e.g., one or more printed circuit boards (PCBs) and the like), and air delivery components, as described in more detail below. The upper housing portion150bpreferably has a canopy portion159, which can overlay at least a portion of the platform surface157and can be configured to cover some or all of the various structure features supported by the platform surface157.

The chamber housing150can also include a seat member150cthat is disposed between the lower and upper housing bodies150a,b. The seat member150chas a top end164and a bottom end166that are spaced from each other along the first direction Z. The seat member150calso includes first and second member endwalls156c,158dand first and second member sidewalls160c,162c, which form portions of the first and second endwalls156,158and first and second sidewalls160,160of the chamber housing150when the seat member150cis coupled together with the lower and upper housing bodies150a,b. Interior surfaces170of the member endwalls156c,158cand member sidewalls160c,162cdefine respective portions of the interior volume135. The first member endwall156ccan define an aperture172for passage of one or more vents128into the interior volume135, as described in more detail below. The seat member150calso includes a sample support surface168located vertically between the top and bottom ends164,166and facing upward (i.e., along the upward direction Z2). The sample support surface168is configured to hold one or more sample containers175placed in the internal volume135of the specimen chamber106(seeFIG.5F). As shown, the sample support surface168can extend around an entire inner perimeter of the seat member150cin a rim-like fashion and is spaced inwardly from the member endwalls156c,158cand member sidewalls160c,162c. The seat member150cdefines a central aperture155cextending therethrough along the first direction Z. Similar to the lower housing body150a, the central aperture155cof the seat member provides an open, unobstructed space between the specimen chamber106and the imaging lens. Moreover, similar to the upper housing body150b, the central aperture155cof the seat member150calso provides an upper opening through which the user places the one or more sample containers into the interior volume135and atop of the sample support surface168. The seat member150ccan be made of a material providing favorable thermal conduction and insulation for incubating the interior volume135, including aluminum, steel, titanium, by way of non-limiting examples. In such embodiments, the seat member150ccan also be referred to as a “heat spreader”150c. It should be appreciated that the seat member150ccan optionally be made of virtually any material that is machinable, physically stable, and thermally conductive.

The specimen chamber106includes the at least one inlet107, which can be in communication with a chamber manifold130configured to distribute the air delivered to inlet107to the one or more vents128of the specimen chamber106. In the illustrated embodiment, the chamber manifold130defines the inlet107at one end thereof, and also defines an interior vent128at an opposite end thereof. The interior vent128is configured to extend through, or at least reside within, the aperture172in the first endwall156cof the seat member150cand to communicate air received from the inlet107to the interior volume135of the specimen chamber106.

With continued reference toFIG.4B, the specimen chamber106includes various thermal regulation features, which can be used to heat the environment within interior volume135to a desired temperature, such as for incubating sample media placed therein. For example, the thermal regulation features can be configured to heat the environment within the interior volume135to a temperature in a range of about 30° C. to about 40° C., and more particularly in a range of about 35° C. to about 40° C., and more preferably about 37° C. The thermal regulation features can also be employed for maintaining and/or adjusting temperature within the interior volume135as needed. Examples of such thermal regulation features will now be described. For example, the specimen chamber106can include a first heater138, which can extend around a periphery of the interior volume135. The first heater138can have a base heater portion144that extends around the periphery of the interior volume135and defines a central aperture155d. The base heater portion144can be configured to underlay and heat the bottom end166of the seat member150cas needed to maintain the desired temperature within the interior volume135. The first heater138preferably also has one or more wall heater portions146that extend upwards along a respective sidewall or endwall of the chamber housing150. In the illustrated embodiment, the first heater138includes a pair of wall heater portions146that extend upwardly from the base portion138alongside the first and second sidewalls160a-c,162a-c. The pair of wall heater portions146can be configured for heating the sidewalls160c,162cof the seat member to further regulate the temperature of the seat member150cas needed to maintain the desired temperature within the interior volume135.

The specimen chamber106can also include an inlet heater148for heating the air delivered through the chamber manifold130. The inlet heater148can include lower and upper panels174,176that contact upper and lower portions of the chamber manifold130. In this manner, the inlet heater148can work together with (or provide redundancy to) the first heater138as needed to maintain the desired temperature within the interior volume135. The first heater138and the inlet heater148also include circuitry features, such as flex circuits, tracers, connecting pins, headers, and the like, for electronically connecting the heaters138,148to the control unit102, which can include a processor executing computer readable instructions stored in computer memory for controlling operation of the heaters138,148, among other things. Alternatively, the heaters138,148can be electronically connected to a separate electronic control unit, which can be located on-board the specimen chamber106.

It should be appreciated that the lid122(seeFIG.4A) can also include one or more thermal regulator features, such as thermally insulating film(s) and/or a conductive heater, by way of non-limiting examples. The thermally insulating film(s) and/or the conductive heater can be superimposed on part or all of the window124of lid122. Thus, the window124can also be characterized as a heater or heat insulator. The conductive heater and/or heat insulator of the lid122can be used, alone or in connection with the first heater138and/or the inlet heater148, to heat the environment within specimen chamber106to a desired temperature. The thermally insulating film(s) of the lid122can include one or more suitable thermally insulating films, such as an indium tin oxide resistive film, by way of a non-limiting example. The specimen chamber106also preferably includes one or more sensors that report one or more of a temperature, a humidity, and a gas content of the environment within the interior volume135to the control unit102, as described in more detail below.

With continued reference toFIG.4B, the specimen chamber106includes an air circulator or “actuator” unit132for directing air to a target location between the interior volume135of the specimen chamber106and the imaging lens with respect to the first direction Z. Such target location is preferably alongside and underneath the lower face152of the housing body150, which location can be referred to as “beneath” the specimen chamber106. Preferably, the air actuator unit132is configured to direct conditioned air, that is, air having a conditioned (e.g., heated) temperature, to the target location between the interior volume135and the imaging lens. By heating such air and directing it to the target location, the specimen chamber106can effectively warm or heat the imaging lens in a manner inhibiting or at least reducing the formation of condensation thereon, thereby improving image quality. Additionally or alternatively, the heated air can be directed toward or adjacent the imaging lens in order to dry or remove any existing condensation that may have accumulated on the imaging lens, thereby improving image quality. The air actuator unit132includes an air actuator180, which can be a fan180or other mechanism that acts to draw air, such as ambient air adjacent the specimen chamber106, and directs the drawn air through the air actuator unit132. The air actuator unit132preferably also includes a duct member182that directs the drawn air from the air actuator180along one or more channels184to one or more outlet vents or ports140facing the target location. The air actuator unit132can include an air actuator filter134, which filter acts to filter air drawn into the duct member182by the air actuator180. As shown, the air actuator filter134can be carried by a filter support137, which can be couplable with and decouplable from the duct member182. The air actuation unit132can reside in a compartment188defined by portions of the lower and upper housing bodies150a,b, such as along the first sidewalls160a,bthereof, as in the illustrated embodiment. It should be appreciated that the air actuation unit132can be removeable and/or replaceable from the specimen chamber106, as described in more detail below.

Referring now toFIG.5A, a partial view of the specimen chamber106is shown, with the upper housing body150bremoved for visualization purposes to show the air actuator unit132. As shown, the air actuator180can draw or otherwise induct air (e.g., external, ambient air, shown by arrows145) into the duct member182, particularly through an opening181defined in an exterior face183of the duct member182. The housing body150preferably defines vent ports190adjacent the air actuator180, allowing passage of the external air into the air actuator unit132. As shown inFIG.5B, once drawn into the duct member182via the air actuator180, the inducted air is then directed through the duct member182as indicated by arrows, toward the one or more outlet ports140, which are preferably located beneath a sample container present within the interior volume135of the specimen chamber106, as described in more detail below. As shown inFIG.5C, the air actuator filter134and the filter support137can reside within a filter receptacle192defined by the chamber housing150, such as by both the lower and upper housing bodies150a,bthereof. The filter receptacle192and the filter support137can be configured for case of filter replacement by pulling the filter support137upward from the receptacle192, as shown.

Referring now toFIGS.5D-5E, an interior face185of the duct member182is shown, which interior face185faces toward the interior volume135of the specimen chamber106. The duct member182defines one or more channels184that direct the flow of inducted air to the one or more outlet vents or ports140. In the illustrated embodiment, the duct member182defines a single channel184that extends therethrough and defines a flow path that leads toward the one or more outlet ports140. As shown, the air actuation unit132can include an outlet member194connectable adjacent the duct member182, particularly adjacent an underside196of the duct member182. The channel184can be characterized as having multiple portions, such as: a first or opening portion184a, which can be aligned with the air actuator180along the third direction Y; a second or main portion184b, which can extend along the second direction X and alongside one of the wall heater portions146of the first heater138(seeFIG.4B); and a third or re-direction portion184c, which re-directs the flow of inducted air behind and under the lower face152of the housing body150. Some exemplary air flow paths are shown inFIG.5Efor illustrative purposes. The second main portion184bof the channel184can be defined between an interior face198of an inner wall200of the duct member182and the associated wall heater portion146(seeFIG.4B). The associated wall heater portion146can be positioned in close proximity to, or in contact with, the interior face185of the duct member182. In this manner, air inducted into the channel184is directed along the second direction X alongside the associated wall heater portion146, which can heat the inducted air to a temperature sufficient to inhibit or at least reduce the formation of condensation of the imaging lens. At the re-direction portion184cof the channel184, the inducted air is directed generally in the third direction Y around a bend located downstream of an end surface202of the inner wall200. Within the re-direction portion184cdownstream of the bend, the inducted air is further re-directed downward (direction Z1) by an interior face204of an exterior wall206of the duct member182. The downwardly re-directed air passes through a bottom opening208of the duct member182(shown inFIG.5E) and an associated bottom opening210of the lower housing body150a(shown inFIG.5F) and into an outlet passageway212at least partially defined by the outlet member194.

Referring now toFIG.5F, the outlet passageway212is partially defined by an outlet base surface214of the outlet member194and can be further partially defined by the lower face152of the lower housing body150a. In the illustrated embodiment, the outlet passageway212is defined vertically between the outlet base surface214of the outlet member194and the lower face152of the lower housing body150a. As shown, the outlet base surface214has a bend portion, which re-directs the inducted air passing through the bottom openings208,210generally along the third direction Y. The outlet base surface214also has a flat portion, which directs the inducted air along the outlet passageway212in the third direction Y and toward the target location underneath the lower face152of the chamber housing150. As shown, the flat portion of the outlet base surface214is preferably substantially parallel with the lower face152of the chamber housing150. This causes the inducted air to exit from the outlet member194in respective air flow directions that are substantially parallel with the lower face152. Additionally, the outlet member194preferably includes guide members, such as baffles or fins216, that protrude within the outlet passageway212and are configured to spread the inducted air exiting the outlet port(s)140along respective directions having directional components along the second direction X, thereby expanding the target location to extend alongside a larger portion of the lower face152of the chamber housing150. This provides a more uniform area of conditioned (e.g., heated) air between the specimen chamber106and the imaging lens, thereby beneficially enhancing the inhibition (or at least reduction) of condensation formation on the imaging lens. As shown inFIG.5G, the fins216are elongated along respective directions D1-D4that diverge from each other in a downstream direction of air flow DO, thereby causing the inducted air to spread outwardly as it exits the outlet port140. As shown inFIG.5F, the fins216can extend upwardly and into contact with, or at least close proximity to, the lower face152of the chamber housing150, thereby providing a plurality of outlet ports140between the fins216along the second direction X.

Referring now toFIGS.6A and6B, exemplary embodiments of an air actuator180are shown for use with the air actuation unit132. In these embodiments, the air actuator180comprise a fan180, which has a front face230(FIG.6A) configured to be located adjacent the exterior face183of the duct member182. The fan180also has a rear face232(FIG.6B) configured to be adjacent the interior face198of the inner wall200of the duct member182. It should be appreciated that the fan180provides numerous benefits for use in the air actuation unit132. One benefit, as the inventors have discovered, is that the fan180is effective to inhibit or at least reduce condensation accumulation and/or fogging of objective lenses when used with various sizes and configurations of sample containers. Another such benefit is that the fan180also helps maintain the sample container (and the sample media therein) at a uniform temperature, thus enhancing the incubation environment of the sample media within the specimen chamber106.

Thus, according to the embodiments described above, air, such as ambient air, can be inducted into the air actuator unit132and can be heated by the heater138, and the heated air can then be directed out of one or more outlet vents or ports140and against, along, or nearby to an imaging lens (e.g., an objective lens142) that is present beneath the specimen chamber106. This heated air can in turn inhibit or at least reduce fogging and/or accumulation of condensation on the imaging lens. It should be appreciated that the air actuation unit132can be configured to direct heated air directly toward the imaging lens or to a region between the imaging lens and the specimen chamber106.

Referring now toFIG.7, the specimen chamber106preferably includes at least one sensor, such as a temperature/humidity sensor136, that monitors the environment of the target location beneath the specimen chamber106, such as the temperature and/or humidity of the target location. The output of the temperature/humidity sensor136can be used to control the activity of the air actuator unit132, heater(s)138,148, and/or other features of the specimen chambers106disclosed herein to. In this way, the disclosed incubators100can (1) give rise to a locally modulated environment, where the environment includes, for example, a specified temperature, humidity, within the interior of the specimen chamber106while (2) reducing or eliminating fogging of an imaging lens, such as an objective lens142, that is imaging a sample or samples disposed within the specimen chamber106.

Referring now toFIGS.8A and8B, photographs of an objective lens142are shown having condensation thereon as a result of a temperature difference between the objective lens142and a prior art specimen container. As shown in these photographs, the objective lens142has accumulated a significant amount of condensation thereon, which can significantly obstruct the images obtainable by the objective lens142, and can require time and effort to remove based on prior art techniques.

Referring now toFIG.9, a process flow is shown for an exemplary method according to the present disclosure. As shown, at step1, a user can set a first set of desired conditions for the interior135of the specimen chamber106for instance, temperature, humidity, gas levels to which a sample within the specimen chamber106will be exposed. The user can then, for example, in an automated fashion at step2, actuate the gas manifold110, pump108, and other features of the disclosed incubator100to achieve the desired set of conditions. The user can then, at step3, image a sample located within the interior135of the specimen chamber106following exposure to the set of conditions for the desired period of time. The user can then, at step4, set a next set of desired conditions such as environmental conditions for the interior of the specimen chamber106. For example, the environmental conditions include a specified temperature, humidity, and/or gas mixture levels or gas mixture composition to which a sample within the specimen chamber106is exposed, and then perform the foregoing steps as needed.

Referring now toFIG.10, a schematic is shown depicting an exemplary airflow provided by the air actuation unit132according to another embodiment of the present disclosure. In particular, in this embodiment, ambient air99ais inducted into and through the air actuation unit132, and is directed out of the one or more outlet ports140, as expelled air99b, in a direction that intersects an objective lens142of a microscope that is present beneath the specimen chamber106. As in the embodiments above, the inducted ambient air99aof the present embodiment can be heated by heater138(seeFIG.4B) as the air travels through the air actuation unit132, and can then be directed as expelled air99btoward, across, or adjacent the objective lens142in order to dry or remove any condensation that may have accumulated and/or or prevent the accumulation of any condensation on the objective lens142, thereby improving image quality.

An advantage to the disclosed design of the specimen chamber106includes improved uniformity in heating across the incubated samples located in the chamber. Table 1 provides test data showing an array of temperature measurements taken at various positions within a 96-well sample plate incubated within a specimen chamber106as described herein.

TABLE 1Array of Temperature Measurements from various wells of a sample plateincubated in a chamber as contemplated by the present disclosure.Sample Plate Column Labels123456789101112SampleA35.8° C.36.3° C.35.6° C.PlateB35.9° C.36.5° C.RowCLabelsD36.3° C.37.1° C.E36.7° C.35.8° C.FG35.8° C.36.4° C.H35.6° C.35.9° C.35.9° C.

The test results shown in Table 1 demonstrate that comparatively uniform heating is obtained and maintained within a sample plate incubated within a specimen chamber106of the present disclosure. The maximum temperature measured in the sample plate was 37.1° C. and the minimum temperature measured was 35.6° C., with a difference of 1.5° C. The greater uniformity of heating achieved with the specimen chamber106of the present disclosure provides improvements in experimental design and experimental results obtained from samples contained within a specimen chamber106as disclosed herein.

It should be appreciated that the specimen chamber106can be adapted to as needed to receive sample containers of various sizes and shapes.

It should also be appreciated that in additional embodiments, the incubator system100can be provided in a kit that includes a specimen chamber106and a plurality of sample containers (e.g., vessel holders or plates) having different sizes and shapes, and which can be interchangeable with each other in the specimen chamber106.

In should further be appreciated that, in additional embodiments, an air actuation unit can employ compressed air, as an alternative to a fan180, to inhibit or at least reduce accumulation of condensation and/or fogging on the imaging lens. In such embodiments, the air actuation unit can include a reservoir of compressed air and a nozzle through which the compressed air can be released and directed toward, across, or adjacent the imaging lens. In this manner, the compressed air can create a pressure differential in the target location between the specimen chamber and the imaging lens. Such a pressure differential can be employed to lower the dew point temperature in the target location, thereby inhibiting or at least reducing the accumulation of condensation on the imaging lens and/or fogging of the imaging lens.

It should be appreciated that, according to yet additional embodiments herein, the air actuation unit132can be said to include a means for adjusting air conditions at a target location across, at, or adjacent an imaging lens. In such embodiments, the means for adjusting the air conditions in the target location can include a fan180, compressed air, or other features and techniques. The air actuation unit132according to such embodiments can also include means for directing the conditioned air to the target location, which means can include a duct member, such as the duct member182described above, and can also include an outlet member, such as the outlet member194described above. The means for directing the conditioned air to the target location can also include fins or baffles, such as the fins216described above.

It should further be appreciated when a numerical preposition (e.g., “first”, “second”, “third”) is used herein with reference to an element, component, dimension, or a feature thereof (e.g., “first” sensor, “second” sensor), such numerical preposition is used to distinguish said element, component, dimension, and/or feature from another such element, component, dimension and/or feature, and is not to be limited to the specific numerical preposition used in that instance. For example, a “first” sensor can also be referred to as a “second” sensor in a different context without departing from the scope of the present disclosure, so long as said elements, components, dimensions and/or features remain properly distinguished in the context in which the numerical prepositions are used.

Aspects

The following Aspects are illustrative only and do not limit the scope of the present disclosure or the appended claims. Any part or parts of any one or more Aspects can be combined with any part or parts of any one or more other Aspects.Aspect 1. A system for improved imaging, the system comprising:a specimen chamber configured to contain a local environment therein; andan air actuator,the specimen chamber being configured such that air encouraged by the actuator is directed between the specimen chamber and an objective lens configured for imaging a sample within the specimen chamber, the air being directed so as to reduce or eliminate condensation on the objective lens.Aspect 2. The system of Aspect 1, wherein the specimen chamber comprises a feature that supports a sample container disposed within the specimen chamber.Aspect 3. The system of Aspect 1, wherein the specimen chamber comprises a lower region for engaging with a specimen container.Aspect 4. The system of Aspect 3, wherein the lower region comprises a heating element.Aspect 5. The system of Aspect 4, wherein the heating element is configured to heat the specimen chamber to about 37° C.Aspect 6. The system of Aspect 4, wherein the heating element further heats the air encouraged by the air actuator.Aspect 7. The system of Aspect 1, wherein the specimen chamber comprises a lid.Aspect 8. The system of Aspect 7, wherein the lid comprises a heating element.Aspect 9. The system of Aspect 8, wherein the heating element is configured to heat the specimen chamber to 37° C.Aspect 10. The system of Aspect 1, wherein the chamber comprises a manifold and an outlet, wherein the manifold is configured to direct air encouraged by the actuator to the outlet.Aspect 11. The system of Aspect 10, wherein the outlet is in register with the objective lens.Aspect 12. The system of Aspect 11, wherein the outlet is moveable.Aspect 13. The system of Aspect 12, wherein the outlet is slidable, rotatable, or both.Aspect 14. The system of Aspect 10, wherein the manifold further comprises one or more sensors.Aspect 15. The system of Aspect 14, wherein the one or more sensors comprise a temperature sensor.Aspect 16. The system of Aspect 1, further comprising a control unit that delivers a conditioned air to the specimen chamber.Aspect 17. The system of Aspect 16, wherein the control unit comprises a gas mixing manifold.Aspect 18. The system of Aspect 17, wherein a gas mixture within the gas mixing manifold comprises one or more gases selected from oxygen, carbon dioxide, nitrogen, and standard air, the gases balanced to a selected mixture of concentrations.Aspect 19. The system of Aspect 16, wherein the control unit comprises a pump configured to encourage the conditioned air to a fluid inlet of the specimen chamber.Aspect 20. The system of Aspect 19, wherein the pump is positioned internal to the control unit.Aspect 21. The system of Aspect 16, wherein the conditioned air comprises humidified air or heated humidified air.Aspect 22. The system of Aspect 16, wherein the conditioned air comprises dried air or heated dried air.Aspect 23. The system of Aspect 21, wherein the humidified air comprises a humidity of about 50% to about 90% humidity.Aspect 24. The system of Aspect 18, wherein the selected mixture of concentrations comprises from about 5% to about 12% carbon dioxide.Aspect 25. The system of Aspect 18, wherein the selected mixture of concentrations comprises up about 21% oxygen.Aspect 26. The system of Aspect 18, wherein the selected mixture of concentrations comprises from about 67% to about 95% nitrogen.Aspect 27. The system of any one of Aspects 1-26, wherein the system is configured for operation such that the minimum temperature among all locations of a sample container located within the sample chamber is within about 15% of the maximum temperature among all locations of the sample container.Aspect 28. A method, comprising: operating a system according to any one of Aspects 1-27 so as (1) reduce or eliminate condensation on the objective lens, (2) maintain a first environment within the sample chamber that differs in one or more of temperature, humidity, and gas mixture relative to an ambient environment exterior to the sample chamber, or both (1) and (2).Aspect 29. The method of Aspect 28, further comprising operating the system according to any one of Aspects 1-27 so as maintain a second environment within the sample chamber that differs in one or more of temperature, humidity, and gas mixture relative to an ambient environment exterior to the sample chamber, the second environment differing from the first environment.Aspect 30. The method of any one of Aspects 28-29, wherein the operating is performed such that during the operating, the minimum temperature of all locations of a sample container located within the sample chamber is within about 15% of the maximum temperature of all locations of the sample container.Aspect 31. An incubator, comprising: a specimen chamber, the specimen chamber comprising an interior and (i) a fluid inlet, (ii) a surface for receiving a specimen sample container, and (iii) one or more heating elements; and a control unit configured to engage with the specimen chamber, wherein the fluid inlet is configured to receive a conditioned air from the control unit, the specimen chamber being configured to deliver the conditioned air to the interior of the specimen chamber.

A specimen chamber can be placed on a microscope stage and/or a microscope slide. A gasket can be used to seal the specimen chamber to the microscope stage and/or slide.Aspect 32. The incubator of Aspect 31, wherein the control unit comprises a gas mixing chamber, which is also termed a gas mixing manifold, in some instances. The gas mixing chamber can include any one or more of a valve, sensor, a filter, and/or a regulator for generating a gas mixture for delivery to the fluid inlet. One or more sensors can be positioned to monitor any one or more of a gas content, a temperature, and a humidity of the contents of the gas mixing manifold.Aspect 33. The incubator of Aspect 32, wherein a gas mixture within the gas mixing chamber comprises one or more gases selected from oxygen, carbon dioxide, nitrogen, and standard air, the gases balanced to a selected mixture of concentrations. One or more such gases can be received from a gas source exterior to the gas mixing chamber, for example, a tank of gas. By modulating the introduction of different gases to the gas mixing chamber, the user can achieve within the gas mixing chamber a mixture according to a set specification of gas levels.Aspect 34. The incubator of Aspect 31, wherein the conditioned air comprises humidified air or heated humidified air. A user can, for example, set a desired humidity level, which humidity level can be achieved by humidifying air drawn into the control unit, by mixing humidified air developed within the control unit with air that is drawn into the control unit, or both. It should be understood that the contents of the gas mixing manifold can be mixed with humidified air and/or non-humidified air.Aspect 35. The incubator of Aspect 31, wherein the conditioned air comprises dried air or heated dried air. An incubator can include a dehumidifier, for example, a dehumidifier configured to dehumidify air that is delivered via the connector to the inlet of the specimen chamber.Aspect 36. The incubator of Aspect 31, further comprising a lid.Aspect 37. The incubator of Aspect 36, wherein the lid comprises (i) a thermally insulating film coating, (ii) a conductive heater, or both. A lid can be connected to a power supply, which power supply in turn acts to heat the lid. A lid can also comprise a power source, for example, a battery, which power source is used to energize a heater of the lid.Aspect 38. The incubator of Aspect 31, wherein the control unit comprises a pump configured to encourage the conditioned air into the fluid inlet.Aspect 39. The incubator of Aspect 38, wherein the pump is positioned internal to the control unit.Aspect 40. The incubator of Aspect 34, wherein the humidified air is humidified to from about 50% to about 90% humidity, preferably about 80% humidity. Humidity levels of from 50% to 90%, 55% to 85%, 60% to 80%, or even 70% are all suitable.Aspect 41. The incubator of Aspect 33, wherein the selected mixture comprises from about 5% to about 12% carbon dioxide, preferably about 5% carbon dioxide. Carbon dioxide levels of from about 5% to about 12%, from about 6% to about 11%, from about 7% to about 10%, or even from about 8% to about 9% are all considered suitable.Aspect 42. The incubator of Aspect 33, wherein the selected mixture comprises up to about 21% oxygen. The mixture can have less than 21% oxygen, e.g., from about 0.5% to about 20%, from about 1% to about 19%, from about 2% to about 18%, from about 3% to about 17%, from about 4% to about 16%, from about 5% to about 15%, from about 6% to about 14%, from about 7% to about 13%, from about 8% to about 12%, from about 9% to about 11%, or even about 10%.Aspect 43. The incubator of Aspect 33, wherein the selected mixture comprises from about 67% to about 95% nitrogen, preferably about 75% nitrogen. The mixture can have from about 67% to about 95% nitrogen, or from about 70% to about 90% nitrogen, or from about 75% to about 85% nitrogen, or even about 80% nitrogen.Aspect 44. The incubator of Aspect 34 or 35, wherein the conditioned air is heated to about 37° C. This is not a requirement, however, as conditioned air can be heated to, for example, about 20° C., about 25° C., about 30° C., or even about 35° C.Aspect 35. The incubator of Aspect 31, wherein the one or more heating elements are configured to heat the specimen sample container to about 37° C.Aspect 46. The incubator of Aspect 31, further comprising a manifold configured to receive the conditioned air.Aspect 47. The incubator of Aspect 36, wherein the manifold is configured to distribute the conditioned air within the interior of the specimen chamber. The manifold can extend around part of or even around the entirety of a perimeter (inner or outer) of the interior of the specimen chamber.Aspect 48. The incubator of any one of Aspects 46-47, further comprising at least one vent in fluid communication with the manifold, the at least one vent being configured to direct conditioned air received from the control unit to the specimen sample container. The conditioned air directed to the specimen sample container can be heated by the one or more heating elements of the specimen chamber.Aspect 49. The incubator of Aspect 31, further comprising an air circulator embodied in the specimen chamber, the air circulator configured to encourage air beneath the specimen sample chamber. Such air can be used to reduce or even eliminate fogging of an objective lens positioned beneath the specimen sample chamber.Aspect 50. The incubator of Aspect 49, wherein the air actuator comprises a fan.Aspect 51. The incubator of any one of Aspects 31-50, further comprising at least one sensor configured to detect a humidity, a gas level, or both.Aspect 52. The incubator of any one of Aspects 31-51, further comprising a water reservoir, the water reservoir being in fluid communication with and/or embodied within the control unit, the water reservoir is in fluid communication with the fluid inlet.Aspect 53. The incubator of any one of Aspects 31-53, wherein the incubator is configured to include in the conditioned air (i) the gas mixture, (ii) humidified air, or both (i) and (ii).Aspect 54. The incubator of any one of Aspects 31-53, wherein the incubator modulates an atmosphere within the interior of the specimen chamber to at least one specified level of any one or more conditions selected from: oxygen level, carbon dioxide level, nitrogen level, percent humidity, or temperature.Aspect 55. The incubator of Aspect 31, wherein the gas mixing chamber mixes gases received from external to the incubator.Aspect 56. The incubator of Aspect 31, wherein the incubator is configured for mounting on a microscope stage.Aspect 57. The incubator of Aspect 31, wherein the chamber comprises one or more ports configured for introducing or removing one or more materials to or from the interior of the specimen chamber.Aspect 58. A method for imaging a specimen in a controlled environment, comprising: positioning the specimen within the specimen chamber of an incubator according to any one or Aspects 31-57; and acquiring one or more images of the specimen in the specimen chamber. Image acquisition can be, e.g., via an inverted microscope.Aspect 59. The method of Aspect 58, further comprising exposing the specimen positioned within the specimen chamber to a first set of environmental conditions in an atmosphere of the interior of the specimen chamber for a first interval of time.Aspect 60. The method of Aspect 59, wherein the first set of environmental conditions comprises a first specified level of one or more of: oxygen, carbon dioxide, nitrogen, humidity, temperature, or one or more combinations thereof.Aspect 61. The method of Aspect 59, wherein the first set of environmental conditions includes a condition that differs from a corresponding condition of an atmosphere exterior to the specimen chamber.Aspect 62. The method of any one of Aspects 58-61, wherein the first interval of time is in the range of from about 1 minute to 72 hours or more. Intervals of, e.g., from about 1 minute to 72 hours or more, from about 1 minute to 48 hours, from about 5 minutes to 24 hours, from about 10 minutes to 20 hours, from about 30 minutes to 10 hours, and from 1 hour to 5 hours are all considered suitable.Aspect 63. The method of any one of Aspects 58-62, further comprising exposing the specimen to a second set of environmental conditions in the interior of the specimen chamber for a second interval of time.Aspect 64. The method of Aspect 63, wherein the second set of environmental conditions includes a condition, including for example, temperature, humidity, and/or gas mixture, that differs from a corresponding condition of the first set of environmental conditions. The second interval of time can differ from the first interval of time.Aspect 65. The method of any one of Aspects 58-64, further comprising introducing one or more reagents into the specimen chamber.Aspect 66. The method of any one of Aspects 58-65, further comprising extracting one or more samples from the specimen chamber.