NL2026627B1 - A MEMS device for transmission microscopy, and a method - Google Patents

Abstract

A MEMS device for transmission microscopy, said device comprising — a planar body, said body having an observation window, — a sample chamber located at said observation window, — a conduit extending in said body, said conduit having — an inlet at the outside of the device, and — an outlet opening up in the lumen of said sample chamber; for passing the sample in fluid form to the lumen. To conveniently fill the MEMS device, the device comprises a hollow cantilever extending from the body, said hollow cantilever comprising the inlet of the conduit.

Description

A MEMS device for transmission microscopy, and a method The present invention relates to a MEMS device for transmission microscopy, said device comprising ~ a planar body, said body having ~ a first main surface, and a second main surface, and ~ an observation window; — a sample chamber located at said observation window in said body, said chamber ~ having a lumen for containing a sample, and ~ comprising wall sections delimiting the lumen, wherein said wall sections are transparent for said transmission microscopy; ~ a conduit extending in said body, said conduit having ~ an inlet at the outside of the device, and — an outlet opening up in said lumen; for passing the sample in fluid form to the lumen.

A MEMS device according to the preamble is known from "Nanofluidic and monolithic environmental cells for cryogenic microscopy” by S. Gorelick et al (Nanotechnology 30, page 1-8 (2019) 085301), in particular for transmission electron microscopy (TEM). The device allows for holding a sample under vacuum in a transmission electron microscope. The device is in particular useful for cryo-electron microscopy, wherein a liguid sample is frozen into a vitreous (i.e. non-crystalline) state.

The device can be filled by putting a liquid sample onto the first main surface, and the liquid sample passes through the inlet at the first main surface to the chamber by capillary action. The chamber is located in a central section of the MEMS device, which central section is broken out after freezing the MEMS device, and the central section is mounted in the transmission electron microscope.

A disadvantage of the MEMS device is that it is cumbersome to ready for microscopy.

The object of the present invention is to provide a MEMS device according to the preamble to reduce the above problem.

To this end, a mems device according to the preamble is characterized in that the device comprises a hollow cantilever extending from the body, said hollow cantilever comprising the inlet of the conduit.

By contacting a liquid sample to be introduced into the lumen against the distal end of the cantilever, the liguid can be introduced conveniently and the device is not subject to breaking stress after filling, which stress could affect the integrity of the chamber.

Reduced stress allows the walls of the chamber to be thinner, which may have a beneficial effect on the resolution.

Concomitantly, a reduction of the time until microscopy can be performed may be achieved, which for certain applications may be desirable.

The amount of liquid sample required is also significantly reduced and may be for example as little as 50 picoliter or less).

The device comprises a lateral side and conveniently, the inlet is at the lateral side of the device. Typically the holiow cantilever will extend in a direction parallel to the first main surface.

Although it is possible to fill a MEMS device under reduced pressure (near vacuum), typically the device will comprise a vent passage connecting the lumen with the outside of the device.

A MEMS device comprising a cantilever may be connected to an accessory (MEMS) device in a convenient fashion. A small distal end of cantilever may be used to penetrate a biological cell deposited in blind hole of said accessory device, the blind hole having dimensions capable of receiving the distal end of the cantilever. The cell contents will be passed to the lumen.

According to a favourable embodiment, the device comprises at least two conduits and cantilevers, each cantilever comprising the inlet of a conduit.

This allows for introducing different liquids, which may comprise agents that interact upon contact of the liguids. An agent may be a biochemical agent such as an antibody, an enzyme for performing a reactant, or a chemical agent, including a pH modifying substance such as an acid.

When more than one conduit is present, it may serve as the vent passage for air. An accessory device may conveniently be used to apply negative pressure. Passing liquid through the chamber may help to get more of the compound of interest into the lumen in case the compound of interest is adsorbed to walls of the device, in particular those of the (supply) conduit.

In case of at least two conduits, it is also possible to perform electrophoresis, which may help to guide a desired molecule such as a protein into the lumen. To this end, an electrical potential can be applied between two or more conduits. Typically the electrodes will not be part of the device, so as to keep it simple and versatile. An electrode may be part of the device providing the liguid.

According to a favourable embodiment, the inner height of a conduit is larger than the inner height of the chamber.

A sample may contain a component to be studied using transmission microscopy or an agent that is absorbed to the wall of the conduit. By having a conduit having a relatively large height compared to the chamber, more component Lo be studied will reach the lumen as relatively less is lost due to absorption.

A relatively low height of the chamber allows the photons or particles (such as electrons) used for transmission microscopy Lo pass through the sample more easily, helping to achieve a better image quality.

According to a favourable embodiment, the conduit is a first conduit and the chamber comprises a second conduit having a cross-sectional surface area that is at least 10x smaller than the cross-sectional surface area of the first conduit.

A sample may contain a component to be studied using transmission microscopy or an agent that is absorbed to the wall of the conduit. By having a conduit having a relatively large height, more component to be studied will reach the lumen.

According to a favourable embodiment, the device is a device for transmission electron microscopy.

This 1s an important field of application. The windows may be of, for example, silicon nitride, silicon carbide or silicon oxide.

The lumen may comprise a plurality of support beams extending from the top window to the bottom window so as to help to keep the windows parallel to the main surfaces.

According to a favourable embodiment, the device is a device for transmission electron cryo-microscopy.

This is an important field of application.

Finally, the present invention relates to a method of providing a MEMS device comprising a sample chamber with a liquid, wherein the MEMS device is a device according to any of the claims 1 to 6, and an accessory MEMS device is used to provide the sample chamber with a compound, said accessory device comprising an accessory device body comprising at least one well for holding a liquid, wherein the number of wells, the dimensions of the wells and spacing between the wells is chosen so as to allow the at least one well to receive the distal end of the cantilever or cantilevers of the MEMS device.

This allows for a variety of possibilities, amongst which filling the sample chamber with sample in a very convenient manner.

According to a favourable embodiment, the accessory device comprises at least two wells, the at least two wells being provided with an electrode, and with the distal end of a cantilever received in at least two of the wells, electrophoresis is performed.

This allows the accessory device to be used for electrophoresis while the cantilevers are inserted in the wells. If desired, the accessory device will be removed after electrophoresis is performed and before a picture is taken.

The present invention will now be illustrated with reference to the drawing where Fig. 1A through 1D show pictures taken with an optical microscope of MEMS devices and details thereof; Fig. ZA and Fig. 2B show scanning electron micrographs of a device according to Fig. 1; Fig. 3 shows a transmission electron microscopy photo of a window of the device of Fig. 2B together with four pictures taken with said device; Fig. 4A through Fig. 40 show process steps in cross-sectional views for manufacturing a MEMS device according to the invention; Fig. 5 shows a top view of an alternative MEMS device and an accessory device; and Fig. 6 shows a top view of yet another alternative MEMS device and an accessory device for electrophoresis.

Fig. 1A through 1D show pictures taken with an optical microscope of MEMS devices and details thereof.

More specifically Fig. 1A shows a wafer 190 containing a plurality of MEMS devices 100. Each device 100 comprises a body 105 and the device 100 is attached with said body 105 by four bridges 191 to the wafer 190 from which the MEMS devices 100 are formed. At the surrounding black areas there is no material and a MEMS device to be used for holding a sample can be broken out of the wafer 190 by breaking said bridges 191.

At the center of the device 100 an observation window 110 is provided. In the embodiment shown here, the MEMS device 100 comprises two conduits 120 extending from the lateral edge 101 at the left to 5 the observation window 110, Fig. 1B shows a detail of a MEMS device 100 similar to the one of Fig. 1A, except that it has four conduits 120.

The circular structures 192 are relics of the manufacturing process and are self-closing holes ("Stiction-Induced Sealing of SurfaceMicromachined Channels" by B. Morana et al (JOURNAL OF MICROELECTROMECHANICAL SYSTEMS, VOL. 23(2), APRIL 2014459)) that allowed for etching a sacrificial material so as to create the conduits 120, as will be explained later. The use of self-closing holes is not part of this invention and is known in the art.

Similarly, through-holes 193 are relics of the manufacturing process, again used for etching.

Fig. 1C shows the window 110 of Fig. 1B in more detail. The window 110 is provided with a grid 130 supporting a membrane 140.

Supply conduits 120a join to a single conduit 120a' from which second conduits 150 formed by the membrane 140 extend to a single discharge conduit 120b'. The second conduits 150 provide chambers 150 having a lumen for holding the sample for the experiment.

The membrane 140 is very thin (about 20 nm silicon nitride in all) and is made of silicon nitride so as to allow electrons to pass, allowing transmission electron microscopy to be performed. If it is desired to halt Brownian motion, the liquid sample will be frozen such that a vitreous frozen liquid is formed, as is known in the art.

The above 1s substantially known in the art. In accordance with the present invention, the MEMS device 100 comprises a cantilever 160 {here four cantilevers 160), which extend parallel to the main plain of the device 100 transverse to body 105.

In the embodiment shown here, the cantilevers 160 comprise diminutive distal ends 161 suitable for penetrating a biological cell.

Fig. ZA and Fig. 2B show scanning electron micrographs of a device according to Fig. 1 (rotated over 180°).

Fig. 2A shows a top view on the device of Fig. 1B and Fig. 1D, with four cantilevers 160 protruding at the right and the window 110 at the left of the photo.

Fig. 2B which is rotated over 90° compared to Fig. 1B shows the window 110 with grid 130 and second conduits 150 (micro-conduits) extending from left to right from the single supply conduit 120a' to the single discharge conduit 120b'. Part of both through-holes 193 (black) are shown at the top and bottom of the photo.

Fig 3 shows a transmission electron microscopy photo of a window 110 of the device 100 of Fig. 2B, with five parallel second conduits 150 supported by the grid 130.

In the four corners, TEM pictures are shown of apoferritin (balls 20 nm; 470 kDalton). Light areas are membrane areas outside the second conduits.

Fig. 4A through Fig. 4N schematically show process steps in cross-sectional views for manufacturing a MEMS device 100 (CRYO-TEM chip) according to the invention.

The CRYO-TEM chip 100 is fabricated using a wafer-scale surface micromachining process. The fluidic components, such as hollow cantilevers 160, thick-walled supply- and discharge conduits 120, and thin-walled second conduits 150 (nanochannels), are all formed by a sacrificial polysilicon etching technique.

The fabrication process starts on a 4", <100>-oriented silicon wafer 190 having the thickness of 380 mm (Fig. 4A).

The wafer 190 is locally thinned from the backside to form silicon membranes 140 (Fig. 4B) with a thickness of 180 um, in which later the CRYO-TEM chips will be formed. The backside processing is performed by anisotropic wet etching of silicon in a 25% potassium hydroxide (KOH) solution at 75°C. A 50 nm thick stoichiometric silicon nitride (SiyN;) layer is used as the masking material for the KOH etch. After the KOH etching the masking layer is removed in a concentrated hydrofluoric acid (HF 49%).

After the local thinning of the wafer, a 300 nm thick silicon oxide layer 405 is deposited (Fig. 4C) in a LPCVD oven by pyrolysis of tetraethylorthosilicate (TEOS).

The silicon oxide layer 405 1s patterned by buffered hydrofluoric (BHF). Silicon oxide is removed (Fig. 4D) from an area 406 where ultra-thin walled second conduits 150 (nanochannels) will be formed, which second conduits 150 are chambers for holding the sample during microscopy.

On the patterned substrate, a 11 nm thin silicon-rich nitride

(SiRN) layer 410 is deposited (Fig. 4E) by LPCVD. This low-stress SiRN layer 410 serves as a bottom wall (lower half of the membrane 140) of the TEM observation openings 411 (visible in Fig. 3; openings defined by the grid 130), which will be created later.

Next, a sacrificial layer 415 of polysilicon with a thickness of 300 nm is deposited (Fig. 4F) by LPCVD. This relatively thick polysilicon layer 415 will be used to create the single supply- and discharge conduits 120a', 120b' and the large conduits 120 (fluidic channel) inside the hollow cantilevers 160.

The deposited polysilicon layer 415 is patterned by TMAH (TetraMethylAmmoniumHydroxide). The layer 415 is removed (Fig. 4G) from a region 416 where later the nanochannels 150 will be formed.

After the TMAH patterning, another sacrificial polysilicon layer 420 with a thickness of 100 nm is deposited by LPCVD (Fig. 4H). The thickness of the second sacrificial layer 420 determines the height of the nanochannels 150 and the height of the membranes 140 through which TEM observation takes place. On the other hand, the aggregate thickness of the first and the second sacrificial layers 410, 420, which is 400 nm in our design, determines the height of the larger conduits 120.

After the deposition (Fig. 41), sacrificial polysilicon 420 is patterned by TMAH etching to define an outline of the fluidic channels. A rather thin thermally grown silicon oxide layer (40 nm) is used as an etching mask. It should be noted that both large (400 nm high) and small (100 nm high) fluidic channels are etched at the same time. The underetching of the mask, due to an isotropic nature of the TMAH etch process, is used to reduce the width of the fluidic channels in which the TEM observation windows will be formed.

After patterning of the fluidic channels, the second silicon-rich nitride layer 425 is deposited (Fig. 4J) by LPCVD. The 11 nm thick low-stress SiRN layer will define a top wall of the TEM observation windows.

Thereafter, a 300 nm thick silicon oxide layer 430 is deposited (Fig. 4K) by a TEOS deposition. A multilayer of SiRN and silicon oxide will form the walls of the hollow cantilevers 160 and some fluidic channels. The relatively large thickness of these walls provides mechanical stability of these components.

After patterning of the second TEOS layer in BHF the TEM observation openings 411 are completely defined (Fig. 4L).

Subsequently, the TEOS/SiRN/SiRN/TEOS multilayer is etched by RIE. In this processing step the contour of the chip and the hollow cantilevers 160 (still containing sacrificial material at this stage) are patterned (Fig. 4M).

After the RIE etching, an access to the bulk silicon is created (Fig. 4N). By using Deep Reactive Ion Etching (DRIE) the bulk silicon is etched from the frontside to a depth of 150 um.

Finally, the wafer is placed in a 25% TMAH solution heated at 90°C. During the TMAH etching sacrificial polysilicon is removed through the access holes and the fluidic channels are created (Fig. 420). At the same time the bulk silicon is exposed to TMAH. A part of the bulk silicon underneath the TEM observation window is removed creating a suspended membrane with integrated thin-walled fluidic nanochannels 150. The bulk underneath the cantilevers 160 is also removed and the cantilevers are released. During the TMAH etching the backside of the wafer 190 remains unprotected. Due to the removal of silicon the CRYO-TEM chip 100 is further thinned down to its final thickness of 100 um.

In an alternative embodiment (Fig. 5; schematic top view), a MEMS device 100 may comprise a plurality of supply conduits 120 starting at cantilevers 160 to supply individual chambers 150, allowing more experiments to be performed, for example with different samples or samples at different stages of reaction. This saves time over performing the experiments using different devices and mounting them in the microscope consecutively for performing the experiments. Per the embodiment shown here, the chambers 150 may be filled using an accessory MEMS device 500 with wells 510 or conduits containing the liquid sample or a agent such as a reagent.

While filling the chamber 150, air is vented via vent passage

550.

Fig. 6 shows a top view of yet another alternative MEMS device 100 comprising a single chamber 150, two conduits 120 and two cantilevers 160 and an accessory device 500 having two wells 510 3% containing electrodes 610 for electrophoresis.

The method according to the present invention can be varied within the scope of the appended claims. Instead of merely relying on capillary action to transfer liquid into the chamber, use may be made of positive pressure (pumping) or negative pressure (suction). The cantilever may be a cantilever suitable for scanning across a surface,

for example to detect the presence of a cell.

To this end, the cantilever may be coated with a metal layer to detect deflection.

The method may comprises penetrating a biological cell using the distal end of the cantilever so as to transfer the contents thereof to the chamber.

Claims (8)

-10 - CONCLUSIONS A MEMS device (100) for transmission microscopy, said device (100) comprising - a flat body (105). wherein said body (105) has - a first major surface, and a second major surface, and - a viewing window (110): - a sample chamber (150) located at said viewing window (110) in said Body (105), wherein said chamber (150) - has a lumen for containing a sample, and - comprises wall sections defining the lumen, said wall sections being transparent to said transmissic microscopy; - a conduit extending into said body (105), said conduit having - an inlet on the outside of the device (100), and - an outlet opening into said lumen; for passing the sample in liquid form to the lumen: characterized in that the device (100) comprises a hollow cantilever (160) extending from the body (105), said hollow cantilever (160) said hollow cantilever (160) inlet of the conduit. The MEMS device (100) of claim 1, wherein the device (100) comprises at least two conduits (120) and cantilevers (160), each cantilever (160) comprising the inlet of a conduit. The MEMS device (100) of claim 2, wherein the internal height of a conduit (120) is greater than the internal height of the chamber (150). The MEMS device (100) of any preceding claim, wherein the conduit (120) is a first conduit (120) and the chamber (150) comprises a second conduit having a cross-sectional area (406) at least 10x smaller. is then the cross-sectional area (406) of the first conduit. The MEMS device (100) of any preceding claim, wherein the device (100) is a transmission electron microscopy device (100). 55 -11 - A MEMS device (100) according to any preceding claim. wherein the device (100) is a device (100) for transmission electron cryomicroscopy. A method of providing a MEMS device (100) comprising a sample chamber (150) with a liquid, characterized in that the MEMS device (100) is a device (100) according to any one of claims 1 6, and an additional MEMS device (100) is used to provide the sample chamber (150) with a configuration, said additional device (500) comprising an additional device (500) body (105) containing at least one well for holding a fluid, wherein the number of wells (510), the dimensions of the wells (510) and the spacing between the wells (510) are selected to allow the at least one well to reach the distal end of the cantilever (160) or cantilevers (160) of the MEMS device (100). The method of claim 7, wherein the additional device (500) comprises at least two wells (510), the at least two wells (510) having an electrode (610), and the distal end of a cantilever ( 160) contained in at least two of the wells (510), electrophoresis is performed.