ES2533144B1 - Optically transparent support for obtaining bubble-free fluid type specimens - Google Patents

Abstract

Optically transparent support for obtaining bubble-free fluid type specimens. # Optically transparent support for fluid type specimens, comprising two transparent substrates facing each other, one called slides (2) and another called coverslips (8), in the that at least one of the substrates is provided with a hydrophobic surface facing the other substrate whose contact angle with water of at least 90 °. The support makes it possible to obtain fluid-free specimens of bubbles.

Description

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DESCRIPTION

Optically transparent support for obtaining bubble-free fluid type specimens

Field of the Invention

The present invention is related to specimen supports. In particular, it is a support for avoiding air bubbles in fluid specimens that guarantees the absence of air bubbles or a reduced amount of air bubbles in any sample containing at least one fluid deposited on the substrate.

Prior art

The problem of air bubbles in the formation of optical images (microscopy)

The preparation and transfer of specimens on transparent slides is often critical for a successful inspection of them using an imaging system. Slide preparation has received a lot of attention since the invention of the microscope, being a highly developed area, which is often based on many specialized and quite sophisticated techniques.

In a dry preparation (microscopic preparation), which is the simplest form of preparation, the sample is placed, first, on the transparent slide. A coverslip can be placed on top to protect the specimen and the objective of the microscope and to keep the specimen at rest and flat. This type of preparation can be used successfully to inspect specimens such as pollen, feathers, hairs, etc. It is also used to examine particles trapped in transparent membrane filters (for example, in the analysis of suspended dust).

In a wet or temporary preparation (microscopic preparation), the specimen is placed in a drop of water or other liquid held between the slide and the coverslip by surface tension. This procedure is commonly used, for example, to observe microscopic organisms that grow in pond water or other liquid media, especially when studying their movement and behavior. Special attention is required to exclude air bubbles, which could interfere with vision as well as hinder

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the movements of organisms. It is also used for rapid investigations in which permanent registration is not required, as well as to examine physiological fluids such as blood, urine, saliva, semen (in spermiogram), and vaginal discharge (microscopic vaginal wet preparation).

In a microscopic preparation prepared or permanent for pathological or biological analysis, the specimen normally undergoes a complex histological preparation that may involve cutting it into very thin sections with a microtome, fixing it to prevent its decomposition, removing any water content, having specific parts , rinse to make it transparent, and impregnate it or infiltrate it with some transparent solid substance. As part of this procedure, the specimen usually binds firmly to the slide.

Thus, without water content, in permanent microscopic preparations there is no problem of air bubbles inside, while they are frequent when wet (temporary) microscopic preparations are used.

In a wet microscopic preparation, the specimen is in a drop of liquid (usually water) located between the slide and the glass coverslip. In water refractive index improves the image quality and also supports the specimen. In contrast to permanently mounted slides, wet microscopic preparations cannot be stored for extended periods of time, as water evaporates. For this reason, sometimes a wet microscopic preparation is called "temporary microscopic preparation" as opposed to "permanent microscopic preparations," which can be stored for longer periods. The permanently mounted slides use a solidifying mounting medium that holds the glass coverslip in place.

Preparation of the sample with little care can generate air bubbles in the microscopic preparation and often, this implies repetition of the preparation. The air bubbles have different refraction levels than the surrounding environment, for example, water. In a photo taken using an image formation system, the effect of the refractive index difference produces a thick black border around the bubbles. The spherical shape of the bubbles centers the light so that the center of the bubble appears bright.

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The material and geometry of the specimen may affect the formation of air bubbles. Some specimens produce more air bubbles than others and this depends on a wide variety of factors. For example, porous specimens, for example, plant stem cells or vascular tissue, can be filled with air, which is difficult to remove. The removal of air can be done by placing the specimen in a vacuum chamber while immersing it in the fixing solution. This procedure requires additional equipment and sample preparation, thus extending the analysis time and increasing costs.

It is true that some air bubbles can be tolerated for analysis and, unless high resolution image formation is required, the effort to prepare a specimen completely free of these bubbles may not be necessary. In some cases, there are simple practical ways to avoid the problem, such as moving the slide with respect to the image point and observing a different area of the specimen free of bubbles. However, in general, air bubbles should be avoided, especially for non-highly qualified operators, which may confuse them with the actual characteristics of a specimen. There are several reasons why air bubbles can be problematic, including:

• Bubbles prevent the free movement of organisms, such as ciliates.

• Bubbles cause optical artifacts at the interface between air and water. The air bubbles appear to be surrounded by a dark ring that hides some parts of the specimen.

• Microscopy optics are designed for an optimal resolution for a specimen surrounded by water or other liquids. If the bubble is so large that the specimen is completely surrounded by air, then the resolution decreases dramatically.

• The great variety in shape and size of air bubbles makes reading an interpretation of the specimen even more difficult.

Practical measures to minimize bubbles in microscopic slide preparations

• Placement of coverslips: The coverslip is placed over the drop of water with an angle. Using this procedure, the air is allowed to escape through one side.

• Water placement: If the specimen is not fully submerged in the water drop, another drop should be added to the top of the specimen before placing the specimen.

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coverslip.

• Immersion oil: A different medium of water can be used, for example immersion oil, which is hydrophobic.

• Breaking the surface tension: The addition of a small amount of detergent, such as soap, breaks the surface tension of the water. Because of this, water adheres better to some specimens, thus avoiding bubbles. However, soap can damage some aquatic organisms.

• Vacfo: It can be used to facilitate and accelerate the movement of the fixing solution or water in the specimen.

• Specimen dehydration: Some specimens placed in alcohol will shrink and lose water and air. When the specimen is placed back in water, the specimen will absorb it.

• Removal of oil and fats: The specimen is washed in alcohol.

• Adding water: If the air bubble is large and reaches the edge of the coverslip, more water can be added from the edge of the coverslip.

There is a clear need to avoid bubbles in the preparation of biological samples or more in general, samples of imaging systems. Although some precautions can be taken to minimize the formation of air bubbles on the slides, there is still no solution to avoid them completely or to reduce them to an acceptable level in a repeatable and economical way.

“Bubble free” concept

The term "bubble free" has a meaning that varies depending on the application. However, in general, it is used to mean that the bubbles are not visible to the naked eye and that the residue of bubbles, empty spaces or craters, is also not visible. In addition, the definitions found in patent applications EP 0690333 A1 "Optical liquid crystal element" and WO 2004/089594 A1 "Polishing apparatus and procedure for solvent casting system" add additional details to the general definition. In the first document some of the unwanted effects of the air in contact with liquid crystal material are mentioned: diffraction or refraction of light, loss of transparency, and other false optical effects. Instead, the last document focuses on plastic film products and defines bubble free as a product that has a bubble concentration less than a given threshold value based on a full width optical inspection (for example, visual ) of the film sample of approximately 10 by 14 cm.

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The lowest threshold value requirement within the same text to qualify a film as bubble free is that the number of bubbles within the range of 25 to 40 micrometers does not exceed 10.

In microscopy applications, to qualify a specimen as bubble free, the number of bubbles within the range of 0.2 to 5 micrometers in diameter must not exceed 10, and no bubble must be larger than 5 micrometers in the specimen.

EP 1466637 A2 "Bubble Detector and Bubble Detection Procedure" introduces the definition of microbubbles (bubbles with a diameter range of approximately 50 to 1000 micrometers) and macrobubbles (bubbles with a diameter greater than 1000 micrometers). A bubble-free term is used for covers of transparent adhesive film, for example for tablets.

Publications and patents related to the formation of bubbles in microfluids mainly focus on the removal of bubbles from the microfluidic channels but do not prevent their formation from the outset. The most common term used to describe such devices is "de-bubbler."

To avoid the above problems, the invention provides two optically transparent substrates, slides and coverslips, respectively in which at least one of them is hydrophobic or superhydrophobic with an angle of contact with water (3) above 90 °, preferably by above 100 °, most preferably above 120 °

Brief description of the drawings

Other features of the invention will be more apparent after consideration of the following description and the accompanying drawings, in which:

Fig. 1 shows the cross-sectional view of a fluid type specimen in an optically transparent substrate, in which its upper surface is hydrophobic, having an angle of contact with water above 90 °;

Fig. 2 shows the cross-sectional view of the optically transparent substrate of fig. 1, in which its upper surface is both nanostructured and coated;

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Fig. 3A shows the cross-sectional view of a microscopic preparation consisting of two optically transparent substrates, in which at least one of said substrates is described in fig. 2, and its hydrophobic surface is in contact with the fluid specimen between them;

Fig. 3B shows the cross-sectional view of a microscopic preparation consisting of two optically transparent substrates, in which both of said substrates are described in fig. 2, and their hydrophobic surfaces are in contact with the fluid specimen between them;

Fig. 3C shows the cross-sectional view of a microscopic preparation described in fig. 3B, from which its internal surfaces are separated by a spacer;

Fig. 4 shows a schematic view of an optical microscope that is combined with the microscopic preparation of fig. 3A or fig. 3B or fig. 3C;

Fig. 5 shows a schematic view of a lens-free microscope that is combined with the microscopic preparation described in fig. 3C, said microscopic preparation is placed in the vicinity of the set of microscope image sensors;

Fig. 6 shows a cavity 100 microns thick between two conventional slides that has air bubble formation (50 to 200 micrometers in size) inside.

Fig. 7 shows a 100 micrometer depth camera between a regular coverslip at the top and a slide at the bottom with an upper surface that is half with a coverage treatment (bottom of the photo) and half without treatment ( top of the photo). The figure shows that the formation of air bubbles in the lower part of the photo is avoided thanks to the hydrophobic properties of the STS (transparent super-wetting substrates).

Fig. 8 shows a glass substrate with a top surface that is a nanostructured and coated surface, with superhydrophobic properties, and bovine serum specimen on top.

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Fig. 9 shows a microscopic preparation with a regular coverslip at the top and a microscope glass coverslip at the bottom with a surface in contact with the sample that is superhydrophobic. The bovine serum specimen between them is bubble free.

Fig. 10 shows a diagram of the use of the preferred embodiment. Two sequential frames show how the microscopic preparation depicted in fig. 3C provides a bubble free fluid type specimen. The coverslip has a spacer to confine the fluid in a predetermined volume with a controlled optical path.

Fig. 11 shows a diagram of the preferred embodiment of the invention consisting of the microscopic preparation of fig. 3A containing a specimen of cells marked with bubble-free fluorescence, combined with a lens-free microscope to achieve a bubble-free image formation of the cells.

Detailed description of the invention

The invention comprises two optically transparent substrates, slides and coverslips respectively, in which at least one of them has a surface is hydrophobic or superhydrophobic (fig. 1) with an angle of contact with water (3) above 90 °, preferably above 100 °, most preferably above 120 °. Typically, the slide at the bottom works like a microscope slide and the coverslip forms with it a camera (slide stack) (10) as shown in fig. 3A, 3B and 3C. First, the specimen (1) is prepared on the lower slide (fig. 8), and then immobilized with the coverslip (fig. 9). When one of the two or both lower slides and coverslips are hydrophobic, the fluid propagation dynamics is more homogeneous than between regular microscope slides, since in the first case the specimen fluid propagates more slowly and homogeneously between the substrates, avoiding the formation of air bubbles, as shown in fig. 10.

The coverslip can be a regular slide; in this case its surface in contact with the fluid has natural properties, usually hydrophilic. However, it is preferred

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that the lower surface of the coverslip has hydrophobic properties.

Fig. 3A shows the embodiment in which the upper surface on the lower slide is hydrophobic, while fig. 3B includes hydrophobic or superhydrophobic surfaces for both the lower surface of the coverslip and the upper surface of the lower slide.

The coverslip can simply be placed on top of the lower slide with fluid specimens. However, it is preferred to use a spacer (9) to form a camera with a specific distance between the lower slide and the coverslip, as shown in Fig. 3C. Normally, the spacer also limits the lateral dimensions of the chamber for the specimen confined therein in a predetermined volume with a controlled optical path.

The previous embodiment will be called transparent super-wetting substrates (STS). In particular, the surfaces of the STS may be nanostructured (5), or coated with a self-contained hydrophobic monolayer (SAM), such as a fluorosilane, or both, to adapt the wetting properties of the surfaces. Surfaces must be chemically and biologically compatible with fluid specimens, that is, they should not affect the investigated properties of the specimen while the measurement is being carried out.

Given the uniform and bubble free, or at least reduced, bubble content, the STS minimizes the number of false positive errors when automatic algorithms are used to analyze or count cells / particles in the microscope specimen image. This is very important to increase the accuracy of subsequent image processing based on automatic analysis algorithms. For example, they are used in statistical feasibility studies, in which thousands of measures are taken to achieve valid statistics.

STS can be used in combination with an optical microscope (11), as shown in fig. 4. Fig. 5 shows the schematic view of a lens-free microscope combined with the STS (10) to form specimens, fluid-type, bubble-free images (7). The STS (10) is placed in the proximity of the microscope image sensor assembly (15). The proposed lens-free microscope consists of a non-coherent light source (12), which can be, for example, a light emitting diode (LED), which produces

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a non-coherent beam of light that passes through a hole (13) that converts it into a quasi-coherent beam of LED light (14), which passes through the STS slide stack (10) and strikes on the image sensor (15).

The tests show that there is a high probability of air bubble formation in a microscopic preparation composed of two regular microscope slides. As shown in fig. 6, more than 20 air bubbles of between 50 and 200 micrometers were formed when the space between the slides was filled with water.

However, using the same procedure to fill the support of the invention in the same environmental conditions, that is, the same pressure and temperature, but using a stack of slides partially treated with STS, a bubble free zone was generated (18 ) in microscopic preparation. This effect is illustrated by fig. 7, since bubbles (16) only appear on the untreated part of the slide (17).

As mentioned above, the use of spacers (9) is preferred, as shown in fig. 3C, between the coverslip (8) and the slide (2) to confine the specimen fluid (7) in a predetermined volume with a controlled optical path. If the specimen fluid volume is known, then the concentration can be calculated from the ratio of the cell or particle count in the image divided by the known volume. Consequently, it is easier to automatically calculate the concentration with images taken from a microscope if the spacers are included in the STS of the invention. For example, an STS camera (19) is observed that includes spacers ~ 100 micrometers thick, and contains fluorescently labeled blood cells, by the use of a fluorescence detection configuration consisting of a source filter, a fluorescence filter and a lens free microscope. This configuration provides a lens-free image formation (20) of fluorescently labeled blood cells, as shown in fig. eleven.

Features and fabrication of the microscope slide surface

A standard microscope slide for biological applications measures approximately 75 mm by 25 mm (3 "by 1") and is approximately 1 mm thick. Typically, microscope slides are made of glass, such as borosilicate or sodocalcic glass, but specialized plastics are also used. Often cast quartz slides are used when ultraviolet transparency is important, for

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example in fluorescence microscopy. They are biocompatible slides since the material does not affect the biological properties of the specimen. Biocompatibility is the quality of having no toxic or harmful effects on biological systems, according to the definition provided by the Dorland Medical Dictionary.

The regular microscope slides have a flat surface with the intrinsic low roughness of their material. A nanostructured surface (4) may be created in accordance with the invention in any of the support slide materials with the appropriate manufacturing process.

1. The nanostructure is transferred to an appropriate intermediate polymeric substrate by means of hot stamping lithography. In this procedure, both the original and the polymer are heated to the glass transition temperature of the polymer and then compressed together for a certain amount of time while maintaining the constant temperature. Then the glass-polymer sandwich cools below the glass transition temperature of the polymer while maintaining the pressure. Then, the pressure is released and the polymer is separated from the original.

2. A hot stamping lithography procedure is performed again, but this time using the nanostructured polymer as the original on the desired plastic substrate.

The procedure can be simplified in two stages if the original has a negative profile of the preferred nanostructure. If this is the case, the intermediate polymeric substrate is not necessary and the profile of the original is transferred directly to the target plastic surface. To improve the preparation of a specimen, coverslips with or without spacers can be placed on the microscope slide.

With respect to the prior art, the present invention allows:

• Minimize errors in image counting or image analysis (for example, counting statistics and size). It guarantees high precision measurements due to the distribution and homogeneous concentration of the specimen in the microscope analysis area.

• Savings in the cost of expensive specimen materials, as well as reagents.

• Provides a homogeneous and controlled propagation of specimen material over the microscope analysis area.

Claims (5)

ES 2 533 144 A1 1. Optically transparent support for fluid type specimens, comprising two transparent substrates facing each other, one called a slide (2) and the other 5 called coverslip (8), in which at least one of the substrates is provided with a hydrophobic surface facing the other substrate. 2. Optically transparent support according to claim 1, wherein said hydrophobic surface is a nanostructured surface (4). 10 3. Optically transparent support according to any of the preceding claims, wherein the substrates are biocompatible. 4. Optically transparent support according to any one of the preceding claims, comprising a spacer (9) between the two transparent substrates to form a camera with a specific distance between the slide (2) and the coverslip (8). 5. Optically transparent support according to any of the preceding claims, wherein at least the upper surface of the slide (2) is hydrophobic.