Serial crystallography experiment device based on conveyor belt technology

Abstract

The invention discloses a serial crystallography experimental device based on a conveyor belt technology. The device comprises an angle measuring head end, a bottom film end and a crystallization plate storage disc, an angle measuring head groove is formed in the bottom end of the angle measuring head end and is used for being matched with an angle measuring head base on a synchrotron radiation beam line station; the top end of the angle measuring head end is provided with two angle measuring head end supporting rods used for being matched with the bearing short rods, and an angle measuring head end waste liquid tank used for collecting waste liquid irradiated by X rays. The bottom film end is assembled with the crystallization plate storage disc and is used for supporting and turning over the crystallization plate storage disc; in an experiment, a crystallization plate storage disc provided with a sitting drop in-situ crystallization plate is assembled on the bottom film end, and a low background scattering film on the back surface of the sitting drop in-situ crystallization plate is connected with a bearing short rod through an adhesive film to form a conveyor belt film; when the bearing short rod is driven by the motor to convey a sample to the angle measuring head end, the conveying belt film is torn down, diffraction data collection is carried out, and waste sample liquid drops are extruded out, flow down along with the angle measuring head end supporting rod and are converged into the waste liquid tank.

Description

Technical Field

[0001] The invention relates to the field of protein crystal loading, and more specifically to an experimental device and method based on conveyor belt loading technology. Background Art

[0002] As the main technology for protein structure analysis, the research focus of crystallography has gradually shifted to serial crystallography in recent years. Serial crystallography has become a new research focus due to its potential for high-throughput loading and time resolution. As an important technology in serial crystallography, serial loading technology has developed rapidly, including fixed target technologies such as chips, in-situ plates, nylon rings, and mobile phase technologies such as high-viscosity nozzles, electrospinning, and capillaries.

[0003] In serial crystallography, membrane-based in-situ plate technology has been widely studied due to its high-throughput potential. The membrane-based in-situ plate technology is not only compatible with existing high-throughput automated platforms, but also avoids the tedious work of crystal retrieval by using an in-situ method. Existing membrane-based in-situ plate technology mainly loads fixed targets by cutting the in-situ small plate from the in-situ plate, but this method limits the loading efficiency of the sample. At the same time, this loading method also limits the use scenarios of time-resolved technology, and conventional mobile phase time-resolved technology cannot be applied. The existing serial crystallography based on conveyor belt technology also has some limitations. The first is how to efficiently culture protein crystals, and the second is how the conveyor belt-based technology can efficiently transfer crystals to a conveyor belt with low background scattering. Summary of the invention

[0004] In order to solve the problems of efficiency of existing membrane-based in-situ plate technology and conveyor belt technology, the present invention provides a conveyor belt-based serial crystallography experimental device and method, which integrates the advantages of the two technologies, improves the in-situ sampling efficiency and time resolution potential, and at the same time improves the sample preparation efficiency of the conveyor belt method itself and the efficiency of sample transmission to the conveyor belt.

[0005] The conveyor belt loading device according to the present invention mainly includes an angle measuring head end 10, a bottom film end 20, a crystallization plate storage plate 30, a sitting drop in-situ crystallization plate 40, a bearing short rod 50, a bearing long rod 60, and a bearing rod cap 70.

[0006] The crystallization plate storage disk 30 can be assembled on the bottom film end 20 through a bearing long rod 60. One end of the bearing long rod 60 is provided with a long rod thread 62 which can pass through the two storage disk bearing holes 31 and the two bottom film end bearing holes 21. The bearing rod cap 70 completes the fixation of the crystallization plate storage disk 30 and the bottom film end 20 by assembling the rod cap thread 71 on its inner wall with the long rod thread 62.

[0007] The sitting drop in-situ crystallization plate 40 is assembled with the crystallization plate storage plate 30 through the storage plate groove 32 on the crystallization plate storage plate 30 . The crystallization plate storage plate 30 can be assembled with one or more sitting drop in-situ crystallization plates.

[0008] One end of the bearing long rod 60 is provided with a long rod thread 62 which can pass through the two bottom film end through-film bearing holes 23, and the bearing rod cap 70 is assembled with the long rod thread 62 through the rod cap thread 71 on its inner wall. Similarly, one end of the bearing short rod 50 is provided with a short rod thread 52 which can pass through the two angle measuring head end through-film bearing holes 13 on the angle measuring head end 10, and the bearing rod cap 70 is assembled with the short rod thread 52 again through the rod cap thread 71. The angle measuring head end has two functions: one is to be assembled with the angle measuring head, so as to facilitate the use of the angle measuring head base to adjust the spatial position of the conveyor belt and the sample droplet; the other is to serve as a fixed end of the conveyor belt, and cooperate with the bottom film end to form a whole conveyor belt device.

[0009] The goniometer head end 10 is assembled with the goniometer head base on the synchrotron radiation beamline station through the goniometer head groove 14 .

[0010] The goniometer head end 10 can be connected to the low background scattering film of the sitting drop in situ crystallization plate 40 on the bottom film end 20 through an adhesive film to form a conveyor belt film. The crystallization plate storage disk 30 is rotated 180° clockwise, and the motor drives the short rod head 51 of the goniometer head end 10 to continuously transport the sample on the sitting drop in situ crystallization plate 40 to the goniometer head end 10 and collect diffraction data in the process.

[0011] The conveyor belt film is continuously rolled onto the bearing short rod 50 , and the discarded sample droplets are squeezed out and flow down along the goniometer head end support rod 12 and finally flow into the goniometer head end waste liquid tank 11 .

[0012] The crystallization plate storage tray 30 is rotated 180° counterclockwise to complete the reset, and the conveyor belt film is reconnected with the low background scattering film of the sitting drop in-situ crystallization plate 40 to form a new conveyor belt film.

[0013] Preferably, the goniometer head end 10 is provided with two goniometer head end support rods 12, on which there are two goniometer head end through-membrane bearing holes 13 for assembling the bearing short rod 50. The device is also provided with a goniometer head end waste liquid tank 11 and a goniometer head groove 14, which are respectively used to collect waste liquid after being irradiated by X-rays and fix the goniometer head base.

[0014] Preferably, the bottom film end 20 is provided with two bottom film end bearing holes 21 and two bottom film end through-film bearing holes 23 for assembly with the bearing long rod 60. The device is also provided with a bottom film end protrusion 22 for correcting and supporting the crystallization plate storage tray 30.

[0015] Preferably, the crystallization plate storage plate 30 is provided with two storage plate bearing holes 31 for assembly with the bearing long rod 60, and the positions of the two storage plate bearing holes 31 are located at the non-center upper position of the side of the crystallization plate storage plate 30 so that when the crystallization plate storage plate 30 is rotated 180° clockwise, the crystallization plate storage plate 30 is turned over and supported by the bottom film end protrusion 22. The device is also provided with a storage plate groove 32 for assembly with the sitting drop in-situ crystallization plate 40.

[0016] Preferably, the bearing short rod 50 is provided with a short rod head 51 and a short rod thread 52 , and an external motor can be assembled with the short rod head 51 of the bearing short rod 50 to drive the bearing short rod 50 to rotate, and the short rod thread 52 is assembled with the bearing rod cap 70 through the rod cap thread 71 .

[0017] Preferably, the bearing long rod 60 is provided with a long rod head 61 and a long rod thread 62. The long rod thread 62 is assembled with the bearing rod cap 70 through a rod cap thread 71.

[0018] The present invention also provides a protein crystal loading method, the steps of which include:

[0019] 1) The front side of each sitting drop in situ crystallization plate 40 is sealed with a transparent tape, and the back side of each sitting drop in situ crystallization plate 40 is sealed with a plurality of low background scattering films, with a certain length left at both ends of each low background scattering film as a film head;

[0020] 2) Assembling a plurality of the sitting drop in-situ crystallization plates 40 with their reverse sides facing upward onto the crystallization plate storage tray 30;

[0021] 3) connecting the film heads of the low background scattering films at the same position on each of the sitting drop in-situ crystallization plates 40 to each other and then connecting them to an adhesive film to form a conveyor belt film;

[0022] 4) Connect the i-th conveyor belt film to the bearing short rod 50, rotate the crystallization plate storage disk 30 180° clockwise, and use the motor to drive the bearing short rod 50 to rotate clockwise. During the process of continuously conveying the sample sealed by the i-th conveyor belt film on each sitting drop in-situ crystallization plate 40 to the goniometer head end 10, the i-th conveyor belt film is gradually torn off, and X-rays are used to pass through the low background scattering film and irradiate the sample to collect diffraction data. After the sample collection is completed, the conveyor belt film is continuously rolled on the bearing short rod 50, and the discarded sample droplets are squeezed out and flow down along the goniometer head end support rod 12 and merge into the goniometer head end waste liquid tank 11;

[0023] 5) Rotate the crystallization plate storage disk 30 180° counterclockwise, connect the (i+1)th conveyor belt film to the (i)th conveyor belt film through the film belt head, and continue to collect samples.

[0024] Compared with the prior art, the present invention has the following positive effects:

[0025] The device and method are compatible with the advantages of membrane-based in-situ plate technology and conveyor belt technology: the membrane-based in-situ plate technology is compatible with existing high-throughput automation platforms, can efficiently cultivate protein crystals, and its in-situ method avoids the tedious work of crystal retrieval. The conveyor belt technology can efficiently transfer crystals to the X-ray path, and the technology has the potential of mobile phase time resolution technology. The method creatively solves the problem of how to quickly splice low-background scattering membranes to form a conveyor belt based on the membrane-based in-situ plate. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of an experimental device for conveyor belt loading technology according to a preferred embodiment of the present invention.

[0027] Figure 2 1 is a schematic structural diagram of a goniometer head according to a preferred embodiment of the present invention.

[0028] Figure 3 It is a schematic diagram of the bottom surface structure of the angle measuring head according to a preferred embodiment of the present invention.

[0029] Figure 4 It is a schematic structural diagram of the bottom film end according to a preferred embodiment of the present invention.

[0030] Figure 5 It is a schematic structural diagram of a crystallization plate storage disk according to a preferred embodiment of the present invention.

[0031] Figure 6 It is a schematic structural diagram of a sitting drop in-situ crystallization plate according to a preferred embodiment of the present invention.

[0032] Figure 7 It is a schematic diagram of the bottom structure of a sitting drop in-situ crystallization plate according to a preferred embodiment of the present invention.

[0033] Figure 8 It is a schematic structural diagram of a bearing short rod according to a preferred embodiment of the present invention.

[0034] Fig. 9 It is a schematic structural diagram of a bearing long rod according to a preferred embodiment of the present invention.

[0035] Fig.10 It is a schematic structural diagram of a bearing rod cap according to a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0036] The preferred embodiments of the present invention are given below in conjunction with the accompanying drawings and described in detail.

[0037] Example

[0038] like Figure 1As shown, the serial crystallography experimental device based on the conveyor belt technology mainly includes an angle measuring head end 10, a bottom film end 20, a crystallization plate storage plate 30, a sitting drop in situ crystallization plate 40, a bearing short rod 50, a bearing long rod 60, and a bearing rod cap 70.

[0039] like Figure 2 , Figure 3 As shown, a goniometer head groove 14 is provided at the bottom end of the goniometer head end 10 for assembly with a goniometer head base on a synchrotron radiation beamline station; two goniometer head end support rods 12 are provided at the top end of the goniometer head end 10 for assembly with a bearing short rod 50; a goniometer head end waste liquid tank 11 is provided at the top end of the goniometer head end 10 for collecting waste liquid after being irradiated by X-rays; the top end of the goniometer head end 10 is a groove, a cylinder is provided at the center of the groove, and two goniometer head end support rods 12 are provided on the cylinder; the goniometer head end waste liquid tank 11 is formed between the cylinder and the side wall of the groove.

[0040] like Figure 4 As shown, the bottom film end 20 includes two supporting plates and a rear end plate connecting the two supporting plates; the crystallization plate storage disk 30 is assembled on the bottom film end 20 through a first bearing long rod and a bearing rod cap; wherein, storage disk bearing holes 31 are respectively provided on the two side plates of the crystallization plate storage disk 30, and bottom film end bearing holes 21 are respectively provided on the two supporting plates of the bottom film end 20, and a long rod thread is provided at one end of the first bearing long rod, and the long rod thread passes through the two storage disk bearing holes 31, the two bottom film end bearing holes 21 and the rod cap thread on the inner wall of the bearing rod cap to complete the assembly of the bottom film end 20 and the crystallization plate storage disk 30.

[0041] like Figure 5 As shown, the crystallization plate storage plate 30 includes two side plates and a front end plate connecting the two side plates, the bottom end of the side plate is provided with an inwardly protruding edge, the bottom end of the front end plate is provided with an insertion port, the insertion port and the protruding edge constitute a storage plate groove 32, the sitting drop in situ crystallization plate 40 is inserted into the crystallization plate storage plate 30 through the storage plate groove 32, so as to realize the assembly of the sitting drop in situ crystallization plate 40 and the crystallization plate storage plate 30.

[0042] When conducting crystallographic experiments, a conventional sitting drop in situ crystallization plate 40 is used. The sitting drop in situ crystallization plate 40 has 96 crystallization chambers 41 in 12 rows and 8 columns, each of which has a mother liquid tank 43 and a crystallization hole 42. Figure 6 As shown, the front side of the sitting drop in-situ crystallization plate 40 is sealed with a transparent tape, as shown in FIG. Figure 7As shown, the reverse side of the sitting drop in situ crystallization plate 40 is sealed by a low-background scattering film, which is different from the front side of the sitting drop in situ crystallization plate which is sealed by a whole piece of transparent tape. In this experiment, four strip-shaped low-background scattering films are used to seal the reverse side of the sitting drop in situ crystallization plate 40. Each strip-shaped low-background scattering film can seal two columns of crystallization holes 42 on the reverse side of the sitting drop in situ crystallization plate. Four strip-shaped low-background scattering films can seal eight columns of crystallization holes on the reverse side of the sitting drop in situ crystallization plate. A certain amount of excess length of film is left at both ends of the four strip-shaped low-background scattering films, which is called a membrane head.

[0043] After the sitting drop in situ crystallization plate 40 has finished culturing the sample, its reverse side faces upward and can be assembled with the crystallization plate storage plate 30 through the storage plate groove 32, such as Figure 1 As shown, the reverse side of the sitting drop in situ crystallization plate 40 is assembled on the crystallization plate storage tray 30 with the reverse side facing upward, and the viscous low background scattering film is also facing upward. The crystallization plate storage tray 30 can be assembled with one or more sitting drop in situ crystallization plates 40. In this experiment, we take the crystallization plate storage tray device that can store two sitting drop in situ crystallization plates 40 as an example to describe the entire experimental method. After the two sitting drop in situ crystallization plates 40 are assembled on the crystallization plate storage tray 30, the crystallization plate storage tray 30 is placed as shown in the following figure. Figure 1 The method shown is assembled with the bottom film end 20, at which time the right end of the crystallization plate storage plate 30 is against the bottom film end protrusion 22 and is located on the left side thereof. The two storage plate bearing holes 31 and the two bottom film end bearing holes 21 are aligned, and the long rod thread 62 of the bearing long rod 60 is passed through the two storage plate bearing holes 31 and the two bottom film end bearing holes 21, and the bearing rod cap 70 is assembled with the long rod thread 62 through the rod cap thread 71 to complete the fixation of the crystallization plate storage plate 30 and the bottom film end 20.

[0044] Then, the long rod thread 62 of a bearing long rod 60 is passed through the two bottom film end through-film bearing holes 23, and the bearing rod cap 70 is again assembled with the long rod thread 62 through the rod cap thread 71 on its inner wall. Similarly, the short rod thread 52 of the bearing short rod 50 is passed through the two angle measuring head end through-film bearing holes 13, and the bearing rod cap 70 is again assembled with the short rod thread 52 through the rod cap thread 71, and finally the angle measuring head end 10 is assembled with the angle measuring head base through the angle measuring head groove 14.

[0045] After the device is assembled, select a sticky film with the same width as the strip of low background scattering film, such as Figure 1As shown, the sitting drop in situ crystallization plate at the left end of the crystallization plate storage tray 30 is used as plate No. 1, and the sitting drop in situ crystallization plate at the right end of the crystallization plate storage tray 30 is used as plate No. 2. The sticky end of the sticky film is downward and connected to any one of the four film heads at the right end of plate No. 2, and the film head at the left end of plate No. 2 can also be connected to the corresponding film head at the right end of plate No. 1. At this time, the sticky film is connected to the low background scattering film on plates No. 1 and No. 2 to form a conveyor belt film. The conveyor belt film passes through the bottom film end 20, that is, it is located under the two bearing long rods 60 on the bottom film end 20. The sticky end of the conveyor belt film is downward, and its right end passes over the top of the bearing short rod 50 on the goniometer head end 10 and adheres to the bearing short rod 50, and rotates 180 degrees clockwise. °Rotate the crystallization plate storage disk 30, and the crystallization plate storage disk 30 is flipped and supported by the protrusion 22 at the bottom film end. At this time, the positions of plate No. 1 and plate No. 2 are interchanged, and the external motor can be assembled with the short rod head 51 of the bearing short rod 50. Drive the external motor to make the short rod head 51 rotate clockwise, and the conveyor belt film on plate No. 2 and plate No. 1 is torn off, and the device can continuously transport the sample on the sitting drop in-situ crystallization plate to the goniometer head end 10. In this process, X-rays can pass through the low background scattering film and irradiate the sample to collect diffraction data. After the sample collection is completed, the conveyor belt film is continuously rolled on the bearing short rod 50, and the discarded sample droplets are squeezed out and flow down with the goniometer head end support rod 12 and finally merge into the goniometer head end waste liquid tank 11.

[0046] When the transmission of samples on a conveyor belt membrane is completed, that is, the transmission of two rows of samples on plates No. 1 and No. 2 is completed, the crystallization plate storage disk 30 is rotated 180° counterclockwise to reset it, and the conveyor belt membrane is reconnected to the other membrane heads remaining on the right end of plate No. 2. Repeat the operation to form a new conveyor belt membrane and finally transmit all samples on the sitting drop in situ crystallization plate 40 to the X-ray path.

[0047] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiment of the present invention can also be modified in various ways. That is, all simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A serial crystallography experimental device based on conveyor belt technology, characterized in that: It comprises an angle measuring head end (10), a bottom film end (20), and a crystallization plate storage tray (30); The bottom end of the goniometer head end (10) is provided with a goniometer head groove (14) for being assembled with a goniometer head base on a synchrotron radiation beamline station; the top end of the goniometer head end (10) is provided with two goniometer head end support rods (12) for being assembled with a bearing short rod (50); the top end of the goniometer head end (10) is provided with a goniometer head end waste liquid tank (11) for collecting waste liquid after being irradiated by X-rays; The crystallization plate storage plate (30) is used to store the sitting drop in-situ crystallization plate (40); The bottom film end (20) is assembled with the crystallization plate storage tray (30) and is used to support and flip the crystallization plate storage tray (30); During the experiment, the crystallization plate storage plate (30) equipped with the sitting drop in situ crystallization plate (40) is assembled on the bottom film end (20), and the low background scattering film on the back side of the sitting drop in situ crystallization plate (40) is connected to the bearing short rod (50) through an adhesive film to form a conveyor belt film. The bearing short rod (50) is driven by a motor to transport the sample on the sitting drop in situ crystallization plate (40) to the goniometer head end (10), tear off the conveyor belt film and collect diffraction data, and the discarded sample droplets are squeezed out and flow down along the goniometer head end support rod (12) and merge into the goniometer head end waste liquid tank (11).

2. The serial crystallography experimental device according to claim 1, characterized in that: The crystallization plate storage tray (30) comprises two side plates and a front end plate connecting the two side plates, the bottom ends of the side plates are provided with inwardly protruding edges, the bottom end of the front end plate is provided with an insertion port, the insertion port and the protruding edge form a storage tray groove (32), the sitting drop in-situ crystallization plate (40) is inserted into the crystallization plate storage tray (30) through the storage tray groove (32), so that the sitting drop in-situ crystallization plate (40) is assembled with the crystallization plate storage tray (30).

3. The serial crystallography experimental device according to claim 2, characterized in that: The bottom film end (20) comprises two supporting plates and a rear end plate connecting the two supporting plates; the crystallization plate storage disk (30) is assembled on the bottom film end (20) through a first bearing long rod and a bearing rod cap; wherein, storage disk bearing holes (31) are respectively provided on the two side plates of the crystallization plate storage disk (30), and bottom film end bearing holes (21) are respectively provided on the two supporting plates of the bottom film end (20); a long rod thread is provided at one end of the first bearing long rod, and the long rod thread passes through the two storage disk bearing holes (31), the two bottom film end bearing holes (21) and the rod cap thread on the inner wall of the bearing rod cap to complete the assembly of the bottom film end (20) and the crystallization plate storage disk (30).

4. The serial crystallography experimental device according to claim 3, characterized in that: A bottom film end protrusion (22) is provided at the bottom end of the rear end plate of the bottom film end (20) for correcting and supporting the crystallization plate storage tray (30).

5. The serial crystallography experimental device according to claim 4, characterized in that: The two storage plate bearing holes (31) are located at a non-center upper position on the side of the crystallization plate storage plate (30) so that when the crystallization plate storage plate (30) is rotated 180 degrees clockwise, the crystallization plate storage plate (30) is turned over and supported by the bottom film end protrusion (22).

6. The serial crystallography experimental device according to claim 3, characterized in that: Two support rods are provided on the rear end plate of the bottom film end (20), and each of the support rods is provided with a bottom film end through-film bearing hole (23) for assembly with the second bearing long rod; the sticky film is connected to the bearing short rod (50) via the second bearing long rod to form a conveyor belt film.

7. The serial crystallography experimental device according to any one of claims 1 to 6, characterized in that: Each goniometer head end support rod (12) is provided with a goniometer head end through-membrane bearing hole (13), and one end of the bearing short rod (50) is provided with a short rod thread (52); the short rod thread (52) passes through the two goniometer head end through-membrane bearing holes (13) and is connected to the rod cap thread (71) on the inner wall of the bearing rod cap (70), thereby completing the assembly of the goniometer head end support rod (12) and the bearing short rod (50).

8. The serial crystallography experimental device according to claim 7, characterized in that: The top end of the goniometer head end (10) is a groove, a cylinder is provided at the center of the groove, and two goniometer head end support rods (12) are provided on the cylinder; The angle measuring head end waste liquid tank (11) is formed between the cylinder and the side wall of the groove.

9. The serial crystallography experimental device according to claim 1, characterized in that: The crystallization plate storage disk (30) stores a plurality of side-by-side sitting drop in-situ crystallization plates (40); a plurality of rows of crystallization holes (42) on the reverse side of each sitting drop in-situ crystallization plate are sealed by a low-background scattering film, and the film tape heads of the low-background scattering films at corresponding positions on adjacent sitting drop in-situ crystallization plates (40) are connected together and then connected to the bearing short rod (50) via an adhesive film to form a conveyor belt film.

10. A protein crystal loading method based on the serial crystallography experimental device of claim 1, comprising the steps of: 1) using a transparent tape to seal the front side of each of the sitting drop in-situ crystallization plates (40), and using a plurality of low-background scattering films to seal the back side of each of the sitting drop in-situ crystallization plates (40), with a certain length left at both ends of each of the low-background scattering films as film tape heads; 2) assembling a plurality of the sitting drop in-situ crystallization plates (40) with their reverse sides facing upward onto the crystallization plate storage tray (30); 3) connecting the film heads of the low background scattering films at the same position on each of the sitting drop in-situ crystallization plates (40) to each other and then connecting them to an adhesive film to form a conveyor belt film; 4) Connecting the i-th conveyor belt film to the bearing short rod (50), rotating the crystallization plate storage plate (30) clockwise by 180 degrees, and using a motor to drive the bearing short rod (50) to rotate clockwise, the i-th conveyor belt film on each of the sitting drop in-situ crystallization plates (40) is continuously transported to the goniometer head end (10), and the i-th conveyor belt film is gradually torn off during the process, and X-rays are used to pass through the low background scattering film and irradiate the sample to collect diffraction data. After the sample collection is completed, the conveyor belt film is continuously rolled on the bearing short rod (50), and the discarded sample droplets are squeezed out and flow down along the goniometer head end support rod (12) and merge into the goniometer head end waste liquid tank (11); 5) Rotate the crystallization plate storage disk (30) 180° counterclockwise, connect the (i+1)th conveyor belt film to the (i)th conveyor belt film via the film belt head, and continue to collect samples.

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