1 user has reported that he/she has successfully carried out the experiment using this protocol.
A Microfluidic Device for Massively Parallel, Whole-lifespan Imaging of Single Fission Yeast Cells
一种用于单裂变酵母细胞大量平行的整个生命期成像的微流体装置   

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eLIFE
Jan 2017

 

Abstract

Whole-lifespan single-cell analysis has greatly increased our understanding of fundamental cellular processes such as cellular aging. To observe individual cells across their entire lifespan, all progeny must be removed from the growth medium, typically via manual microdissection. However, manual microdissection is laborious, low-throughput, and incompatible with fluorescence microscopy. Here, we describe assembly and operation of the multiplexed-Fission Yeast Lifespan Microdissector (multFYLM), a high-throughput microfluidic device for rapidly acquiring single-cell whole-lifespan imaging. multFYLM captures approximately one thousand rod-shaped fission yeast cells from up to six different genetic backgrounds or treatment regimens. The immobilized cells are fluorescently imaged for over a week, while the progeny cells are removed from the device. The resulting datasets yield high-resolution multi-channel images that record each cell’s replicative lifespan. We anticipate that the multFYLM will be broadly applicable for single-cell whole-lifespan studies in the fission yeast (Schizosaccharomyces pombe) and other symmetrically-dividing unicellular organisms.

Keywords: Cellular aging (细胞衰老), Lifespan (生命期), Microdissection (显微解剖), Microfluidics (微流体), Lithography (光刻), Fabrication (制造)

Background

Cellular aging results in the cumulative decline of cellular function that eventually leads to mortality. Most studies of cellular aging focus on the replicative lifespan of model unicellular organisms, such as budding yeast Saccharomyces cerevisiae (Nyström and Liu, 2014; Wasko and Kaeberlein, 2014; Wierman and Smith, 2014; Ruetenik and Barrientos, 2015). The replicative lifespan (RLS) of a cell is defined as the number of daughters produced by a mother cell over the course of its life (Henderson and Gottschling, 2008; Sutphin et al., 2014). RLS studies have greatly expanded our understanding of cellular aging in mitotically active cells. For example, in budding yeast, old mothers preferentially retain misfolded proteins and other cellular senescence factors from the budding daughter cells (Aguilaniu et al., 2003; Hughes and Gottschling, 2012; Liu et al., 2010; Saka et al., 2013; Zhou et al., 2014; Paoletti et al., 2016). This feat is achieved by restricting the flow of these ‘senescence factors’ across the bud septum, preventing their accumulation in the rejuvenated daughters (Shcheprova et al., 2008; Higuchi-Sanabria et al., 2014). Whether senescence factors are also segregated in symmetrically dividing cells is unclear (Wang et al., 2010; Coelho et al., 2013; Nakaoka and Wakamoto, 2017). Indeed, relatively little is known about the mechanisms and causes of aging in symmetrically dividing cells.

Whole-lifespan cellular aging studies require the separation of aging cells from their progeny. Pioneering, early studies in budding yeast removed daughter cells from their mothers via manual microdissection (Mortimer and Johnston, 1959). Since the first such study in 1959, manual microdissection still remains a popular, albeit laborious method for studying replicative aging in most unicellular organisms (Mortimer and Johnston, 1959; Kennedy et al., 1994; Barker and Walmsley, 1999; Fu et al., 2008). However, the low-throughput and laborious nature of this assay limits our current understanding of replicative aging. Most recently, removal of progeny cells has been automated in microfluidic devices that capture and retain individual aging cells (Wang et al., 2010; Lee et al., 2012; Xie et al., 2012; Zhang et al., 2012; Tian et al., 2013; Crane et al., 2014; Nobs and Maerkl, 2014; Jo et al., 2015; Liu et al., 2015; Nakaoka and Wakamoto, 2017; Spivey et al., 2017). Using such devices, relatively large cohorts of individual cells (100 s to 1,000 s of cells) can then be tracked independently from one another. However, most of these approaches focused on prokaryotic cells or the asymmetrically dividing budding yeast (Spivey and Finkelstein, 2014; Chen et al., 2017).

Here, we describe the fabrication and assembly of a microfluidic device for capturing and imaging thousands of fission yeast cells over their entire replicative lifespans. The multiplexed fission yeast lifespan microdissector (multFYLM) enables the experimentalist to track the lifespan of over a thousand fission yeast cells (Spivey et al., 2017). The cells may be continuously imaged for up to six independent populations for over a week, yielding high-resolution imaging over each cell’s replicative lifespan. The multFYLM is constructed of silicone elastomer using templates manufactured via UV photo-lithography. The protocol contained herein details construction of the multFYLM, loading with fission yeast cells, and image collection using a fluorescent microscope. We anticipate that this protocol will be broadly useful for long-term imaging of rod-shaped eukaryotic cells and will shed light on diverse biological processes, including cell cycle regulation, chromatin dynamics, proteome homeostasis, and cellular aging.

Materials and Reagents

  1. Microfabrication
    1. SU-8 2005 photoresist (Microchem)
    2. SU-8 2010 photoresist (Microchem)
    3. P-doped silicon wafers (University Wafers, catalog number: 452 ; 100 mm-diameter; test-grade)
    4. Custom quartz photomasks (Compugraphics)
      Photomask design files available at:
      https://github.com/finkelsteinlab/FYLM_mask_files/raw/master/l1-151030.gds
      https://github.com/finkelsteinlab/FYLM_mask_files/raw/master/l2-151030.gds
    5. SU-8 developer (Microchem)
    6. Acetone (Pharmco-Aaper, Midland Scientific, catalog number: 329000000CSGF )
    7. Isopropanol (Fisher Chemical, catalog number: BP26184 )
    8. Cyclopentanone (Sigma-Aldrich, catalog number: W391018-1KG-K )

  2. multFYLM assembly
    1. 50 ml conical tubes (Genesee Scientific, Olympus Plastics, catalog number: 21-108 )
    2. Large Petri dish (150 mm; Fisher Scientific, catalog number: FB0875714 )
    3. 200 µl pipette tips (Genesee Scientific, catalog number: 23-150RL )
    4. Biopsy punch (P125; 1 mm Acu-punch; Acuderm)
    5. Glass coverslips (48 x 65 mm #1; Gold Seal, Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3335 )
    6. Aluminum foil (Fisher Scientific, catalog number: 01-213-102 )
    7. Razor blades (duridium style, single edge; Gem/Star)
    8. Lab tape (Fisher Scientific, catalog number: 15-901-10R )
    9. Nanoports (IDEX Health & Science, catalog number: N-333 ; 12 x; 10-32 thread; headless knurled head)
    10. Polydimethylsiloxane (PDMS; Dow Corning, Sylgard 184, Fisher Scientific, catalog number: 50-366-794)
      Manufacturer: Electron Microscopy Sciences, catalog number: 2423610 .
    11. Hellmanex III (Hellma Analytics)
    12. Ethanol (Pharmco-Aaper, catalog number: 111000200CSPP )

  3. Microscope and microfluidics setup
    1. PFA Tubing (IDEX Health & Science, catalog number: 1512L ; 1/16” OD)
    2. Coned nut and ferrule (IDEX Health & Science, catalog number: F-333N ; 12 x; 10-32 thread; headless knurled head)
    3. Inline filter (IDEX Health & Science, catalog number: P-272 ; 6 x)
    4. Luer adapter (IDEX Health & Science, catalog number: P-658 ; 6 x; ¼-28 thread; Luer-Lok thread)
    5. Flangeless nut (IDEX Health & Science, catalog number: P-215 ; 6 x; ¼-28 thread)
    6. Union (IDEX Health & Science, catalog number: P-704-01 ; 6 x; 10-32 thread)

  4. Cell loading and image acquisition
    1. Test tubes (14 ml; Corning, catalog number: 352051 )
    2. Large gauge syringe needles (16 G 1.5”; BD, catalog number: 305198 )
    3. Large syringes (100 ml; Veterinary Concepts, catalog number: 60271 )
    4. 10 ml syringes (Luer-Lok tip; BD, catalog number: 309604 )
    5. Steriflip vacuum filtration tubes (50 ml; 20 μM nylon net; Millipore Sigma, catalog number: SCNY00020 )
    6. Petri dishes (100 mm; Fisher Scientific, catalog number: FB0875713 )
    7. Stericup-GP filter sterilizing module (500 ml; 0.22 µm PES; Millipore Sigma, catalog number: SCGPU05RE )
    8. Yeast strains
    9. Bovine serum albumin (Sigma-Aldrich, catalog number: A2153 )
    10. Agar powder (Sigma-Aldrich, catalog number: A1296 )
    11. YES 225 powder (250 g; Sunrise Science, catalog number: 2011-250 )
    12. YES 225 agar media (Recipe 1)
    13. YES 225 liquid media (Recipe 2)

Equipment

  1. Microfabrication
    1. 1 L flask (No. 1000; Corning, Pyrex®, catalog number: 1000-1L )
    2. Suss MA-6 Mask Aligner (Suss MicroTec Lithography GmbH)
    3. Spin Coater (Laurell Technologies)
    4. Hot plate (Cimarec+; Thermo Fisher Scientific, Thermo Scientific, catalog number: HP88857100 )
    5. Anisotropic RIE Plasma Etcher (Nordson March, catalog number: CS170IF )
    6. Hot-Hand Protector Mitt (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: F38000-0001 )

  2. multFYLM assembly
    1. Mini labroller (Labnet)
    2. Plasma Cleaner (Harrick Plasma, catalog number: PDC-32G )
    3. Laboratory oven (Ecotherm, Precision)
    4. Dissection microscope (AmScope, catalog number: SM-1T-PL )
    5. Fine tweezers (Fisher Scientific, catalog number: 16-100-103 )
    6. Sonicator (Bransonic, catalog number: 2510R-DTH )
    7. Rocker/agitator (Belly Dancer; Stovall Life Science)
    8. Bunsen burner (accuFlame; Fisher Scientific, catalog number: 03-902Q )
    9. Centrifuge (Beckman Coulter, model: Avanti® J-26XP )
    10. Centrifuge rotor (Beckman Coulter, model: JLA-16.250 )

  3. Microscope and microfluidics setup
    1. Epifluorescence imaging microscope (Eclipse Ti; Nikon)
    2. Focus maintenance system (Nikon Perfect Focus, Nikon)
    3. CMOS camera (Andor, model: Zyla 5.5 sCMOS )
    4. 10x, 0.3 NA objective (Plan Fluor; Nikon)
    5. 60x, 0.95 NA objective (Plan Apo Lambda; Nikon)
    6. Computer-controlled microscope stage (Proscan III motorized stage; Prior)
    7. Objective heater (Bioptechs, catalog number: 150819-19 )
    8. Appropriate filters for fluorescent imaging
      1. eGFP (Chroma, catalog number: 49002 )
      2. mKO (Chroma, catalog number: 49010 )
      3. E2Crimson (Chroma, catalog number: 49015 )
    9. Light source shutter (SmartShutter; Sutter Instrument)
    10. Shutter controller (Lambda SC; Sutter Instrument)
    11. Computer-controlled syringe pump (KD Scientific, model: LEGATO® 210 )
      Note: This pump is configured for two syringes. If more than two syringes are required, either multiple pumps can be used, or adapters can be fabricated (Figure 4) to allow additional syringes to be driven.
    12. Light source (Newport, model: SOLA-SE-II ; Lumencorp)

  4. Cell loading and image acquisition
    1. Shaking incubator (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4333 )
    2. Spectrophotometer (Thermo Fisher Scientific, Thermo ScientificTM, model: NanoDropTM 2000c )
    3. Autoclave (Consolidated Sterilizer Systems, model: ADV-PLUS )
    4. Vacuum desiccator (5.8 L Pyrex glass; Corning, PYREX®, catalog number: 3121-200 )
    5. Vacuum pump (Welch Vacuum, catalog number: 2546 , B-01)
    6. Mini vortexer (Fisher Scientific, catalog number: 02-215-365 )
      Note: This product has been discontinued.
    7. Bunsen burner (accuFlame; Fisher Scientific, catalog number: 03-902Q )
    8. Environmental chamber/multFYLM microscope stage
      Chamber design file available at:
      https://github.com/finkelsteinlab/FYLM_mask_files/blob/master/FYLMChamber.scad

Software

  1. NIS-Elements Advanced Research (v4.30.02; Nikon Instruments)

Procedure

  1. Microfabrication
    multFYLM microfabrication follows conventional soft lithography methods. The first step is to generate a patterned mold, which can be used to cast devices in elastomeric silicone (PDMS). Such molds, or ‘master’ structures are created on silicon wafers, using UV lithography to deposit patterns on the surface in an epoxy resin (SU-8). The patterns are dictated by masks, which restrict the ability of a UV light source to cross-link the resin. Their alignment is critical to the proper patterning on the wafer, as features of the final master are contained on each of the two masks. A developer is used to remove unexposed resin, leaving a master that is now ready for use (Figure 1). A master can be used repeatedly for at least two years to make hundreds of multFYLM devices.
    Note: The procedures detailed below should be performed in a cleanroom. All instrument settings are unique to the equipment used and included as a guideline. These settings will need to be adjusted to match the instruments available in a user’s cleanroom. All microfabrication steps should be completed in a single day; although suitable stopping points may exist, they have not been tested.


    Figure 1. Overview of the multFYLM design. The multFYLM contains six independent paths. Media enters through each nanoport at the top of the device (Entry), and then follows the path indicated by blue arrows, before exiting through nanoports at the bottom of the device (Exit).

    1. Rinse the polished wafer surface with acetone, isopropanol, and then water.
    2. Air-dry the wafer while setting up the plasma cleaner.
    3. Set the hotplate to 200 °C.

    Plasma cleaning
    Plasma clean the wafer to yield an ultra-clean surface, so that resin patterns may be deposited on the surface with high resolution and adherence.
    1. Turn on the plasma cleaner and gas controller.
    2. Create a plasma cleaning program (Table 1) that will clean the wafer with a 30/70 ratio of O2 to N2. More time does not necessarily yield a better surface.

      Table 1. First plasma cleaning program


    3. Break the chamber vacuum, and load the wafer with the polished side up.
      Note: Manual operation works best for bleeding the vacuum.
    4. Run the cleaning program.
      1. Change the RF tuning switch to Auto, then start the program.
      2. Reverse power flow should be minimized during plasma flow, via adjustment of the C1 and C2 switches.
      3. Upon program completion, allow vacuum bleeding to finish, then stop the program.
    5. Remove wafer.
    6. Re-establish chamber vacuum to promote instrument longevity and cleanliness.
    7. Turn off components. 

    Prepare the mask aligner
    Turn on the mask aligner components, so they can equilibrate before use.
    1. Turn on gas and vacuum lines.
    2. Turn on the mask aligner.
    3. Start UV lamp–it requires a 10-min warm up period.

    Prepare wafer for first exposure
    Deposit the first layer of resin evenly on the wafer surface to yield a resin thickness of 5-6 µm. Alignment, spin parameters and resin application are all critical for proper deposition.
    1. Place the wafer directly on a hotplate with the polished surface face up for 20 min at 200 °C.
      Note: This step assures that the wafer is dry. The temperature of the wafer does not have to be maintained once removed from the hotplate, but one should proceed quickly to the next step. A hot-hand protector mitt may be used to transfer the wafer between instruments.
    2. Place a 100-mm carousel on the spin coater.
    3. Carefully transfer the wafer to the very center of the carousel, opening the vacuum line to firmly hold the wafer in place. An off-center wafer will not yield an even layer of SU-8 in Step A21.
    4. Turn on the spin coater, and set the spin coater program:
      1. 10 sec at 500 rpm, acceleration level 2 (266 rpm/sec).
      2. 35 sec at 1,500 rpm, acceleration level 4 (532 rpm/sec).
    5. Run the program, adding two drops of cyclopentanone to the wafer surface once the speed reaches 1,500 rpm.
    6. Add 6 ml of SU-8 2005 resin to the wafer surface as evenly as possible–avoid dripping SU-8 over the sides of the wafer.
    7. Wait 3 min while bubbles rise to the surface of the SU-8 on the wafer.
    8. Run the program from Step A17.
    9. The cover should be lifted slowly to avoid dripping SU-8 onto the freshly-spun wafer.
    10. Dampen a wipe with cyclopentanone and remove the SU-8 bead remaining on the edge of the wafer surface. Alternatively, an edge-bead removal protocol may be used if the spin coater is so equipped.
    11. Release vacuum pressure and remove the wafer from the carousel.
      Note: The resulting layer of SU-8 should be uniform. If not, the wafer must be cleaned with isopropanol and the procedure restarted from Step A1.
    12. Heat the wafer from room temperature to 95 °C on a hotplate that is initially off.
    13. Leave the wafer on the hotplate at 95 °C for 4 min.
    14. Turn off the hotplate and let the wafer cool down on it for 10 min. 

    Expose wafer with the first mask
    Install the first mask and the resin-covered wafer into the mask aligner. Expose the wafer to UV light long enough to produce patterns in the resin at sufficient resolution. Under-exposure results in incomplete patterning or diminished features, while over-exposure results in enlarged features and low resolution.
    1. Adjust the mask aligner parameters (Table 2). The parameters here should only be used as a guideline.

      Table 2. Mask aligner parameters for the first layer


    2. Set mask 1 into the mask holder.
      1. Remove and install the correct mask holder for 100-mm wafers.
      2. Set the mask in the holder chrome-side face up, using vacuum to hold the mask in.
    3. Position the mask in the approximate center of the viewable region.
    4. Load wafer into the wafer holder.
    5. Align wafer with the mask, using alignment marks as a guide.
    6. Expose the wafer to UV light, and wait for the exposure to complete.
    7. Remove the wafer. 

    Remove unexposed photoresist from the wafer
    Use developer to remove the unexposed resin from the wafer surface; this process reveals the deposited features. Excessive developing will cause the deposited features to be washed off.
    1. Place wafer back on a cooled hotplate.
    2. Heat up to 95 °C, then incubate at that temperature for one minute.
    3. Place wafer in a 1 L flask photoresist-side up.
    4. Pour developer over wafer to cover it completely.
    5. Allow developing to proceed for 30 sec with agitation.
    6. Remove wafer.
    7. Rinse wafer surface with fresh developer.
    8. Rinse wafer surface with isopropanol.
    9. Dry the wafer using pressurized N2.

    Prepare the wafer for second exposure
    Deposit the second layer of resin evenly on the wafer surface to yield a resin thickness of 20-30 µm. Both the resin and spin parameters have been optimized for depositing a resin layer with the proper characteristics for the second exposure.
    1. Clean the wafer surface in the plasma cleaner following Steps A4-A10 but with the following program (Table 3).

      Table 3. Second plasma cleaning program


    2. Place the wafer directly on a hotplate with the polished surface face up for 20 min at 200 °C.
      Note: This step assures that the wafer is dry. The temperature of the wafer does not have to be maintained once removed from the hotplate, but one should proceed quickly to the next step. A hot-hand protector mitt may be used to transfer the wafer between instruments.
    3. Place a 100-mm carousel on the spin coater.
    4. Carefully transfer the wafer to the very center of the carousel, opening the vacuum line to firmly hold the wafer in place. An off-center wafer will not yield an even layer of SU-8 in Step A21.
    5. Set the spin coater program:
      a. 14 sec at 500 rpm, acceleration level 2 (266 rpm/sec).
      b. 37 sec at 3000 rpm, acceleration level 4 (532 rpm/sec).
    6. Add 6 ml of SU-8 2010 to the wafer surface as evenly as possible–avoid dripping SU-8 over the sides of the wafer.
    7. Wait 9 min while bubbles rise to the surface of the SU-8 on the wafer.
    8. Close cover and run the program from Step A48.
    9. The cover should be lifted slowly to avoid dripping SU-8 onto the freshly-spun wafer.
    10. Dampen a wipe with cyclopentanone and remove the SU-8 bead remaining on the edge of the wafer surface. Alternatively, an edge-bead removal protocol may be used if the spin coater is so equipped.
    11. Release vacuum pressure and remove the wafer from the carousel.
    12. Heat the wafer from room temperature to 85 °C on a hotplate that is initially off.
    13. Leave the wafer on the hotplate at 85 °C for 15 min.
    14. Turn off the hotplate and let the wafer cool down on it for 10 min.

    Expose wafer with the second mask
    Install the second mask and the resin-covered wafer into the mask aligner. Expose the wafer to UV light long enough to produce patterns in the resin at sufficient resolution. Alignment at this step is critical, as it ensures that features produced using the second mask will overlay properly with those already on the wafer surface.
    1. Adjust the mask aligner parameters (Table 4). The parameters here should only be used as a guideline.

      Table 4. Mask aligner parameters for the second layer


    2. Set mask 2 into the mask holder.
      1. Remove and install the correct mask holder for 100-mm wafers.
      2. Set the mask in the holder chrome-side face up, using vacuum to hold the mask in.
    3. Position the mask in the approximate center of the viewable region.
    4. Load wafer into the wafer holder.
    5. Adjust the position of the wafer such that it is aligned with the mask, using the alignment marks on the second mask and the wafer (from the first exposure).
    6. Expose the wafer to UV light, and wait for the exposure to complete.
    7. Remove the wafer. 

    Remove unexposed photoresist from the wafer
    Use developer to remove the unexposed resin from the wafer surface. This process reveals the deposited features. Excessive developing will cause the deposited features to be washed off.
    1. Place wafer back on a cooled hotplate.
    2. Heat up to 85 °C, then incubate at that temperature for ten minutes.
    3. During the incubation, remove the second mask from the mask aligner, and then turn off the mask aligner and UV lamp.
    4. Place wafer in a 1 L flask photoresist-side up.
    5. Pour developer over wafer to cover it completely.
    6. Allow developing to proceed for 3 min with agitation.
    7. Remove wafer.
    8. Rinse wafer surface with fresh developer.
    9. Rinse wafer surface with isopropanol.
    10. Repeat Steps A72-A73.
    11. Dry the wafer using pressurized N2.

  2. multFYLM assembly
    Assembly of the multFYLM via soft-lithography proceeds once the master structure is complete. The master structure is used as a mold for PDMS. Before the PDMS hardens, ports are added to allow media flow into the microfluidic structures. Once the silicone has set, it is cleaned and adhered to a large cover glass. The thin, transparent cover glass forms the base of the multFYLM and allows imaging of cells that are captured within the individual arms of the device.

    Cast the multFYLM in polydimethylsiloxane (PDMS)
    Prepare a PDMS solution according to the manufacturer’s protocol (Figure 2). Wrap the master structure with tape to create a vertical barrier for the PDMS. Pour half of the solution onto the master structure. Soft-bake the first layer until it is tacky, then place a clean port over each conduit passage present on the master structure. Pour the remaining PDMS onto the first layer, then bake it until all the PDMS has fully hardened.


    Figure 2. Soft lithography. A. Paper tape surrounds the wafer containing the SU-8 master to keep the PDMS in place while it sets. B. First layer of PDMS. C. Layer one is semi-hardened. D. Nanoports are placed on the first layer. E. The second PDMS layer is poured around the nanoports. F. The fully-cured multFYLM, removed from the master structure.

    1. Preheat oven to 75 °C.
    2. Mix 30 g of PDMS with 3 g of the hardening agent in a 50 ml conical tube. Install the cap.
    3. Mix the PDMS solution on a lab roller for 45 min at room temperature.
    4. Clean 12 nanoports in 2% Hellmanex in a bath sonicator for 20 min on the sonication setting (nanoports can also be cleaned ahead of time).
    5. Rinse the nanoports thoroughly in filtered DI water.
    6. Place the nanoports in 70% EtOH in the bath sonicator for 20 min on the sonication setting.
    7. Create a barrier around the circumference of the patterned wafer, using standard lab tape.
      a. At least 2 mm of the tape should extend evenly below the circumference of the wafer.
      b. Tape adhesion is critical in order to avoid PDMS leaking from the wafer surface.
    8. Set the wafer inside a large (150 mm) Petri dish.
    9. Centrifuge the mixed PDMS solution at room temperature at > 400 x g for 90 sec to remove large bubbles.
    10. Pour 13 g PDMS onto the wafer, allowing it to wet the entire surface evenly.
    11. Dry the nanoports at 70 °C on a hotplate for 30 min.
    12. Remove residual air bubbles by placing the wafer in a vacuum desiccator for 15 min at 60-70 cmHg.
    13. Remove the vacuum rapidly to remove bubbles still trapped in the PDMS. Repeat if necessary.
    14. Place the wafer with PDMS in the oven at 70 °C for 15 min.
    15. Test the PDMS on the wafer for proper hardness.
      1. PDMS should be semi-solid and very tacky, making a small peak when probed with a 200 µl pipette tip.
      2. If not, place it back in the oven, checking every few minutes for proper hardness.
    16. Using a dissection scope, delicately place each of the twelve nanoports over the end of each media conduit of the PDMS as seen in the wafer’s pattern.
      Avoid placing a nanoport down more than once on the PDMS surface. Multiple placements can damage multiple fluid passages, and a misaligned port may prevent fluid flow to the corresponding passage.
    17. Pour 14 g PDMS onto the wafer, allowing it to wet the entire surface evenly.
    18. Place the wafer in a vacuum desiccator for 15 min at 60-70 cmHg. This removes any air that may be trapped under the nanoports, ensuring a good seal between the nanoports and the PDMS.
    19. Return the wafer to the oven (70 °C) for 3 h, or until fully cured. 

    Cut, clean and assemble the multFYLM
    Remove the multFYLM from the master structure, then use a razor blade to trim away excess PDMS. Use a biopsy punch to make a direct path from each molded conduit to the nanoport on the opposite side of the multFYLM. Ultraclean the multFYLM and a large cover glass, then adhere them to one another.  This completes assembly of the multFYLM.
    1. Place a cover glass in a Petri dish containing a 2% Helmannex solution for one hour with agitation on a rocker.
    2. Rinse the cover glass twice with diH2O.
    3. Rinse the cover glass twice with isopropanol.
    4. Place the cover glass in a large Petri dish containing a single layer of aluminum foil.
    5. Set the dish on a hotplate at 70 °C for at least two hours to dry.
    6. Carefully remove the tape from the circumference of the patterned wafer and multFYLM.
    7. Carefully peel the cast multFYLM from the wafer surface.
      1. Peel gently as to avoid splitting the polymerized PDMS.
      2. Do not touch the surface that was in contact with the patterned wafer. 
    8. Invert the multFYLM in the Petri dish, ports-side down.
    9. Cut away excess PDMS from the patterned/ported region of the multFYLM using a new, sharp razor blade.
    10. Position the multFYLM under a dissection microscope.
    11. Using a 1 mm biopsy punch, gently punch a hole through the center of each nanoport from the bottom surface through to the top surface.
      Note: The punched region should include the conduit that the nanoport was placed over.
    12. Remove the ‘punched-out’ core of PDMS from the biopsy punch using fine tweezers before withdrawing the biopsy punch from the multFYLM.
    13. Remove the biopsy punch from the multFYLM with light pressure and a slight rotating motion to avoid separating the nanoport from the PDMS.
    14. Inspect the nanoports and remove any remaining PDMS particles with the fine tweezers.
    15. Submerge the multFYLM in a beaker containing 100% isopropanol and place the beaker in the bath sonicator for 30 min on the sonication setting.
    16. Remove the multFYLM from the beaker and place it ports-side down in a large Petri dish containing a single layer of aluminum foil.
    17. Set the dish on a hotplate at 70 °C for two hours to dry.
    18. Place the recently-dried multFYLM and cover glass in the plasma cleaner, with the surfaces that will contact the cover glass facing up.
    19. Turn on the plasma cleaner.
    20. Turn on the vacuum to evacuate the chamber for at least one minute.
    21. Turn the RF setting to ‘high’ for 20 sec.
    22. Immediately remove the components from the plasma cleaner.
    23. Adhere the cover glass and multFYLM by carefully setting the cleanest side of the cover glass onto the center of multFYLM.
    24. Apply light pressure to the multFYLM to assure that it has fully-adhered to the cover glass.
      1. For best results, the multFYLM should be used within several hours of assembly. Alternatively, the multFYLM may be stored in 70% ethanol for extended, sterile storage.
      2. The completed multFYLM should be stored in a container to avoid contamination.

    Microscope and microfluidics setup
    Whole-lifespan imaging adds additional technical challenges to operating any microfluidic device. First, the microfluidic system must provide fresh media to the captured cells while also removing waste. Imperfections in the flow path can cause air bubbles that dislodge cells, potentially disrupting a multi-day experiment. Moreover, additional precautions must be taken to remove cells that are trapped upstream of the multFYLM. This is because these cells may grow into microcolonies during multFYLM operation, ultimately obstructing the flow of fresh media to the device. Second, the microscope should be equipped with stable optical and mechanical components for up to a week of continuous imaging. An active feedback focus-finding system ensures that the multFYLM can be imaged for several days without requiring any user intervention. Similarly, a light source (i.e., LED lamp) that does not change in output intensity or spectrum during a week of continuous operation is recommended. Finally, we recommend that the entire device is enclosed in an incubator jacket that maintains optimal growth conditions for the desired cells (see Equipment D8).

    Prepare microfluidic tubing
    Clean all the microfluidic fittings (Figure 3) that will be used for attaching to the multFYLM, then fit them onto microfluidic tubing. It is necessary to put a right angle in the tubing immediately after the fittings that will attach to the nanoports, otherwise the tubing will not clear the environmental chamber and microscope components.


    Figure 3. Microfluidic fittings

    1. Submerge all microfluidic fittings in a beaker containing 2% Hellmannex detergent and sonicate in a bath sonicator for 20 min on the sonication setting.
    2. Rinse all fittings with diH2O three times.
    3. Submerge all microfluidic fittings in a beaker containing 100% ethanol and sonicate in a bath sonicator for 20 min on the sonication setting.
    4. Rinse all fittings with 100% ethanol.
    5. Dry all the fittings in a Petri dish on a hotplate at 70 °C for 30 min or longer.
    6. Cut twelve sections of tube to the length of 60 cm. Cut ends to be as square as possible.
    7. Using a Bunsen burner as an aid, permanently bend a 95° angle into one end of each tube, approximately 17 mm from the end.
    8. For six tubes that will become the waste lines, attach the following fittings at the bent end:
      1. F-333N coned nut, threads away from the bend.
      2. F-142N ferrule, blunt end towards the bend. The tubing should extend beyond the ferrule by 1-2 mm.
    9. For six tubes that will become the media lines, attach the following fittings:
      1. F-333N coned nut, threads away from the bend.
      2. F-142N ferrule, blunt end towards the bend. The tubing should extend beyond the ferrule by 1-2 mm.
      3. P-215 flangeless nut, threads toward the straight end.
      4. P-272 ferrule, blunt end away from the flangeless nut.
      5. P-658 Luer adapter, screwed onto the flangeless nut, sandwiching the ferrule.
    10. Connect each media line to a waste line using P-235 connectors.
      Note: Tubing should be prepared ahead of time, and can be stored in ethanol or sterile water until use.

    Prepare the microscope for imaging
    Turn on the microscope and peripherals, so that they can warm up before the experiment begins. The NIS Elements software (or other control software) should also be opened, as some peripherals may not turn on completely without a signal from the correctly-configured software.
    1. Turn on the following components:
      1. Microscope
      2. Camera
      3. Shutter controller
      4. Stage
      5. Objective heater–set to achieve 30 °C within the multFYLM. The heater should be installed on the 60x air objective. The temperature setting should be determined empirically, as a higher programmed temperature will likely be required to account for heat loss.
      6. Stage heater–set to achieve 30 °C within the multFYLM. The temperature setting should be determined empirically, as a higher programmed temperature will likely be required to account for heat loss.
      7. LED light source
      8. White light source
    2. Start the NIS Elements software suite.
      1. Select ‘Neo/Zyla’ as the image grabber if prompted.
      2. Move the 10x objective into position.

  1. Cell loading and image acquisition
    Below, we describe a protocol to maximize the number of cells that are captured in the multFYLM. Since the multFYLM contains many fine passages, it can become clogged with cell clumps or other debris. Care must be taken while preparing and loading the media and cells to avoid any particles or cell clumps. Further, air can easily dislodge captured cells, and so it should be purged from any upstream components in the fluid path. Use sterile techniques to prevent other microbes from contaminating cells in the multFYLM.
    Image acquisition of cells in the multFYLM requires image collection at dozens of locations, regular time intervals, multiple Z planes, and filters corresponding to the range of fluorophores present. While an in-focus Z plane is used for the majority of imaging, the out-focus Z plane allows for greater certainty in defining the cell boundaries. Care should be taken when selecting fluorophores and filters, as spectral separation allows for unambiguous attribution of fluorescence to individual fluorophores.

    Prepare media and cells
    Make a liter of degassed, filtered YES 225 media, and culture the yeast strains so that they will be in exponential growth-phase on the first day of the experiment.
    1. Prepare one liter of YES 225 agar media (Recipe 1).
    2. Prepare one liter of YES 225 liquid media (Recipe 2).
    3. Prepare 1 ml of sterile 20% BSA solution in a conical tube.
    4. Streak cells from glycerol stocks onto the agar plates four days prior to the start of the experiment. Plates should be incubated at 30 °C until colonies are well-formed, then left at room temperature.
    5. Select a 2-3 day-old colony and inoculate 10 ml of YES 225 media in a test tube.
    6. Incubate the cell culture overnight in a shaking incubator at 30 °C.
    7. When the optical density at 595 nm (OD595) of the cell culture reaches 0.1, inoculate a fresh test tube containing 10 ml of YES 225 media.
    8. Incubate the new cell culture in a shaking incubator at 30 °C until the OD595 is 0.5 to 1.0 (4-6 h).
    9. Degas the YES 225 media by placing it in a vacuum desiccator with the bottle cap loose for 15 min. This should be done just prior to loading the media into syringes.

    Connect and clean media/waste lines
    Load the prepared media into syringes large enough to hold enough media for the entire experimental time course. Clean the media and waste lines using ethanol and sterile water, as sterility is essential to experimental success. Install the multFYLM in the environmental chamber, then connect the waste lines (Figure 4).


    Figure 4. Epifluorescent microscope prepared for imaging of the multFYLM. A. The complete multFYLM microfluidic path. B. Microfluidic fittings connect lines to the multFYLM.

    1. Turn on the syringe pump.
    2. Determine how many flowpaths within the multFYLM will be used.
      Only three or four of the available six flowpaths are typically used due to spatial constraints and image collection rates. All six flowpaths can be used if the image collection rate is infrequent enough, the lines do not over-torque the multFYLM, and all areas can be observed by the microscope.
    3. Fill N 10 ml syringes (‘N’ equal to as many flowpaths that will be used) with 70% ethanol.
    4. Load these syringes into the syringe holder on the syringe pump.
    5. Connect N media/waste line sets to each ethanol syringe.
    6. Set the syringe pump parameters and run:
      1. Syringe: B-D Plastipak 10 ml syringe
      2. 5 min
      3. 1 ml/min
    7. Fill N 10 ml syringes with diH2O.
    8. Replace the ethanol syringes with the water syringes.
    9. Rerun the pump according to Step C15.
    10. Load N syringes with the degassed YES media.
      1. Attach a large syringe needle to the syringe to aid in loading the syringe without introducing any air bubbles.
      2. Any air in the syringes should be removed immediately.
    11. Replace the water syringes with the YES media syringes.
    12. Set the syringe pump parameters and run it to replace the water in the lines with YES media:
      1. Custom syringe–diameter 31.75 mm
      2. 1 min
      3. 1 ml/min 
    13. Retrieve the multFYLM and attach it to the heated stage insert using spring metal clips or lab tape.
      By convention, the multFYLM is oriented as parallel to the imaging area as possible, with entrance ports oriented closest to the user. The entrance ports lead to the end of the microfluidic pattern that is not directly accessible to the waste trenches at the periphery of the channels intended to hold the cells.
    14. Detach the waste lines from media lines and attach them to the exit channels of the multFYLM.
      1. Take care to avoid placing lines over regions that will be imaged during the experiment.
      2. Connecting lines to all six paths concurrently is difficult. It is generally advisable to run no more than three or four flowpaths in parallel.
      3. Be sure to perform this task in as sterile a manner as possible.
      4. Media lines should be kept sterile until connected. Storing them in an open conical tube is typically sufficient to prevent contamination.

    Load cells into the multFYLM
    Carefully vortex and load cells into each entry port, then attach the media lines while avoiding introduction of any air. Establish a program for the syringe pump that typically provides a consistent flow rate, with an occasional, increased flow rate; this will help dislodge any debris that might otherwise clog the passages of the multFYLM.
    1. Transfer 400 µl of each cell culture into separate microfuge tubes.
    2. Add 100 µl of sterile 20% BSA solution to each tube.
    3. Vortex each tube for one minute.
    4. Using a micropipette, transfer 40 µl of cell solution to each appropriate entry port.
      1. Take care to introduce as little air as possible. This volume assures that enough liquid is present to allow a drop-to-drop connection with the media line without over-filling the nanoport during setup.
      2. The pipette tip should be held just above the base of the port to avoid introducing air to the flowpath.
    5. The cells may be observed using white light and the 10x objective. They should begin to flow into the multFYLM due to surface tension.
    6. Set the syringe pump at a rate of 40 µl/min and run.
    7. As YES media begins to exit each media line, gently attach it to an entry port.
      Be very careful while attaching: use a drop-to-drop connection strategy to avoid introducing air to the flowpaths, and do not torque on the multFYLM. Ports can easily separate, or the cover glass can crack.
    8. Observe the cells using white light and the 10x objective. They should be filling the channels of the microfluidic flowpath, starting near the entry ports first (Figure 5A).
    9. Create a program for the pump with the following parameters:
      1. One minute at a flow rate of 55 µl/min
      2. Fourteen minutes at 5 µl/min.
      3. Repeat 725 times.
    10. Once cells have filled most of the channels to be observed, start the above program.


      Figure 5. Schizosaccharomyces pombe cells loaded into the multFYLM. A. 10x image of cells within a single flowpath immediately following the loading process. B. 60x image of cells viewable within the defined region of interest (ROI).

    Begin image acquisition
    Using the NIS Elements software, set up a multi-dimensional acquisition strategy that will capture images of cells in each compartment of the multFYLM at regular time intervals, an in-focus and out-of-focus Z plane, and all filter sets necessary for the emission of the fluorophores in use (Table 5). Other software suites may be used, though the following directions are specific to NIS Elements.
    1. Move the 60x air objective into place.
    2. Obtain focus, then turn on the Perfect Focus System (PFS) using the PFS button on the front of the microscope.
    3. Using the stage controller, bring the left-most flowpath in use into view.
    4. Change the Region of Interest (ROI) to the same size as the viewable area of cell channels.
      1. In the Zyla settings menu in NIS Elements, use the ‘Commands’ > ‘ROI’ > ‘Load ROI’ drop-down menu and then select the *.CAMROI file downloaded from below.
        Camera ROI file:
        https://github.com/finkelsteinlab/FYLM_mask_files/blob/master/FYLM_ROI_2X2_new.camroi
      2. In the same sub-menu, select ‘Use current ROI’ (Figure 5B).
    5. Under the ND Acquisition menu, set the folder and file names.
      1. Path: Location where the generated files will be stored.
      2. Filename: Name of the files to be generated. A three-digit number will be appended to the end automatically.
    6. Under the ND Acquisition menu, set Time:
      1. The interval and total duration of the image collection. Frequency is dependent on the number of channels that will be collected, but for white light-only images, a 2 min interval is reasonable.
      2. It is recommended that the duration be 36 h or less, to balance file size with image collection restart frequency.
    7. Under the ND Acquisition menu, set Z:
      1. In-focus (as determined using PFS)
      2. 4 µM offset (Step: 4, 2 steps, Below: -4, Above: 0)
    8. Under the ND Acquisition menu, set λ:
      1. Optical configurations should be set up for each fluorescent image filter set. Exposure times should be determined experimentally.
      2. Select all optical configurations that will be used during the experiment.
      3. Fluorescent images do not need to be collected at every time period (reduces the likelihood of photo-toxicity), and frequency can be set using T Pos.
      4. Z-depth is selectable. It is recommended that fluorescent images only be collected at the ‘Home’ Z Pos. 
    9. Under the ND Acquisition menu, set XY:
      1. X-Y positions should be tiled across the observable cells.
      2. It is recommended that positions be defined in a loop pattern to avoid large changes in the focal plane, which can lead to loss of focus mid-experiment. 
    10. Once all parameters have been set (Table 5), press ‘Run’.

      Table 5. Example parameters for multi-dimensional image acquisition


    11. Observe the first few rounds of imaging to assure that everything remains as set.
    12. The experiment should be observed at least once a day to check for errors and to collect a new image file. Downstream analysis is optimized for files containing 24 h of data.
    13. After 24 h, press ‘Finish’ to complete one day’s collection.
      1. This will also save the file, though saving can be assured by accessing ’Save’ in the ‘File’ menu.
      2. If prompted, it is not necessary to complete the current loop before finishing.
    14. Image analysis software may now be used to create videos and analyze the collected data.

Data analysis

Information on how data collected using this methodology is analyzed can be found in these references (Greenstein et al., 2017; Spivey et al., 2017).

Notes

  1. During microfabrication, the type and volume of photoresist, and the spin parameters can be varied to alter the height of the deposited features. Similarly, the type of photoresist and exposure time and intensity can be varied to alter the resolution and width of the deposited features. This can be particularly useful for capturing cells with slightly larger dimensions.
  2. A common failure point during multFYLM assembly is punching out the PDMS from the center of the nanoports. Often, removal of the punch results in the nanoport lifting away from the PDMS, creating a small pocket of air. With care, such pockets of air can later be expelled when the coverglass is pressed to the PDMS. If pockets remain, they can become a reservoir for air that will dislodge cells while passing through the multFYLM, or for other cells that can clump and block the passageways as the experiment proceeds.
  3. Loading the multFYLM with cells often works best with a freshly-assembled device, as the interior is still quite dehydrated, thus media and cells readily flow into it in order to rehydrate the surfaces. If the multFYLM has been stored for a length of time, it is advisable to run one ml of 70% ethanol, then one ml of water through the device backwards, so that air is not trapped in the exit channels. Otherwise, trapped air will not be displaced from the exit pathways, and adjacent channels will not yield the required pressure differential necessary for subsequent cell loading.

Recipes

  1. YES 225 agar media (1 L)
    1. 36.13 g YES 225 powder, 20 g agar; add diH2O up to 1 L total volume
    2. Autoclave, then pour 25 ml into individual Petri plates using sterile technique
  2. YES 225 liquid media (1 L)
    1. 36.13 g of the YES 225 powder; add diH2O up to 1 L total volume.
    2. Filter sterilize the solution–this will also remove small particulates that can lead to clogged passages. Autoclave treatment is not sufficient, as it will sterilize the solution but will not remove particulates

Acknowledgments

We would like to thank members of the Finkelstein laboratory for their input and advice during the development and preparation of this method. This work was generously supported by the following grants and fellowships: the American Federation for Aging Research (AFAR-020 to I.J.F.), the Welch Foundation (F-1808 to I.J.F.), and the NIH (F32 AG053051 to S.K.J.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Science Foundation. This protocol was adapted from prior designs (Spivey et al., 2014; Spivey et al., 2017).

References

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简介

整个寿命的单细胞分析极大地增加了我们对细胞老化等基本细胞过程的理解。为了观察整个寿命期间的个体细胞,必须从生长培养基中移除所有后代,通常通过手动显微切割。然而,手动显微切割费力,低通量,并且与荧光显微镜不兼容。在这里,我们描述了多路复用裂变酵母寿命显微解剖器(multFYLM)的组装和操作,这是一种用于快速获取单细胞全寿命成像的高通量微流体装置。 multFYLM从多达六种不同的遗传背景或治疗方案中捕获约一千个杆状裂殖酵母细胞。将固定的细胞荧光成像超过一周,而将子代细胞从装置中取出。得到的数据集产生记录每个细胞复制寿命的高分辨率多通道图像。我们预计multFYLM将广泛适用于裂殖酵母(Schizosaccharomyces pombe)和其他对称分裂的单细胞生物的单细胞整个寿命研究。

【背景】细胞衰老导致细胞功能的累积下降,最终导致死亡。大多数关于细胞衰老的研究侧重于模型单细胞生物的复制寿命,例如出芽酵母酿酒酵母(Nyström和Liu,2014; Wasko和Kaeberlein,2014; Wierman和Smith,2014; Ruetenik和Barrientos ,2015)。细胞的复制寿命(RLS)被定义为母细胞在其生命过程中产生的女儿的数量(Henderson和Gottschling,2008; Sutphin等人,2014)。 RLS研究极大地扩展了我们对有丝分裂活性细胞中细胞衰老的理解。例如,在出芽酵母中,老母亲优先保留来自萌芽子细胞的错误折叠的蛋白和其他细胞衰老因子(Aguilani等人,2003; Hughes和Gottschling,2012; Liu等人。2010; Saka et al。,2013; Zhou et al。,2014; Paoletti et al。,2016) 。这项壮举是通过限制这些“衰老因子”在芽隔中的流动来实现的,以防止它们在复原的女儿中积累(Shcheprova et al。,2008; Higuchi-Sanabria et al。 ,2014)。衰老因子是否也在对称分裂细胞中分离尚不清楚(Wang等人,2010; Coelho等人,2013; Nakaoka和Wakamoto,2017)。事实上,对于细胞对称分裂老化的机制和原因知之甚少。

整个寿命期细胞衰老研究需要将衰老细胞与其后代分开。开创性的早期研究在出芽酵母中通过手动显微切割从母体中移出子细胞(Mortimer和Johnston,1959)。自从1959年第一次这样的研究以来,人工显微切割仍然是一种流行的研究大多数单细胞生物复制老化的方法(Mortimer和Johnston,1959; Kennedy等人,1994; Barker和Walmsley ,1999; Fu等人,2008)。然而,该测定的低通量和费力的性质限制了我们对复制老化的当前理解。最近,在捕获并保留个体衰老细胞的微流体装置中自动去除后代细胞(Wang等人,2010; Lee等人,2012; Xie 2012; Zhang等人,2012; Tian等人,2013; Crane等人, 2014; Nobs和Maerkl,2014; Jo等人,2015; Liu等人,2015; Nakaoka和Wakamoto,2017; Spivey< em>等,2017)。使用这样的设备,可以相对独立地跟踪相对较大的单个细胞群(100s至1000s的细胞)。然而,这些方法大多集中在原核细胞或不对称分裂芽殖酵母上(Spivey和Finkelstein,2014; Chen等人,2017)。

在这里,我们描述了微流控装置的制造和装配,用于在整个复制寿命期间捕获和成像数千个裂殖酵母细胞。多路复用裂殖酵母寿命显微解剖器(multFYLM)使实验者能够追踪1000多个裂殖酵母细胞的寿命(Spivey等人,2017年)。细胞可连续成像多达六个独立群体超过一周,在每个细胞的复制寿命期间产生高分辨率成像。 multFYLM由使用通过UV光刻制造的模板的有机硅弹性体构成。此处包含的协议详细介绍了multFYLM的构建,装载分裂酵母细胞以及使用荧光显微镜进行图像采集。我们预计该协议将广泛用于棒状真核细胞的长期成像,并将阐明多种生物学过程,包括细胞周期调控,染色质动力学,蛋白质体内稳态和细胞衰老。

关键字:细胞衰老, 生命期, 显微解剖, 微流体, 光刻, 制造

材料和试剂

  1. 微细加工
    1. SU-8 2005光刻胶(Microchem)
    2. SU-8 2010光刻胶(Microchem)
    3. P掺杂硅片(大学晶片,目录号:452;直径100毫米;测试级)
    4. 定制石英光掩膜(Compugraphics)
      Photomask设计文件可在:
      https://github.com/finkelsteinlab/FYLM_mask_files/raw/master/l1 -151030.gds
      https://github.com/finkelsteinlab/FYLM_mask_files/raw/master/l2 -151030.gds
    5. SU-8开发商(Microchem)
    6. 丙酮(Pharmco-Aaper,Midland Scientific,目录号:329000000CSGF)
    7. 异丙醇(Fisher Chemical,目录号:BP26184)
    8. 环戊酮(Sigma-Aldrich,目录号:W391018-1KG-K)

  2. multFYLM组件
    1. 50ml锥形管(Genesee Scientific,Olympus Plastics,目录号:21-108)
    2. 大培养皿(150毫米; Fisher Scientific,目录号:FB0875714)
    3. 200μl移液枪头(Genesee Scientific,目录号:23-150RL)
    4. 活检穿孔器(P125; 1毫米Acu-punch; Acuderm)
    5. 玻璃盖玻片(48 x 65 mm#1; Gold Seal,Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:3335)
    6. 铝箔(Fisher Scientific,目录号:01-213-102)
    7. 剃刀刀片(duridium风格,单边;宝石/星)
    8. 实验室胶带(Fisher Scientific,目录号:15-901-10R)
    9. Nanoports(IDEX Health& Science,产品目录号:N-333; 12×; 10-32线;无头滚花头)
    10. 聚二甲基硅氧烷(PDMS; Dow Corning,Sylgard 184,Fisher Scientific,目录号:50-366-794)
      制造商:Electron Microscopy Sciences,目录号:2423610。
    11. Hellmanex III(Hellma Analytics)
    12. 乙醇(Pharmco-Aaper,目录号:111000200CSPP)

  3. 显微镜和微流体装置
    1. PFA Tubing(IDEX Health& Science,目录号:1512L; 1/16“OD)
    2. 锥形螺母和套圈(IDEX Health& Science,目录号:F-333N; 12×; 10-32螺纹;无头滚花头)
    3. 在线过滤器(IDEX Health& Science,目录号:P-272; 6×)
    4. Luer适配器(IDEX Health& Science,产品目录号:P-658; 6 x;¼-28螺纹; Luer-Lok螺纹)
    5. 无凸缘螺母(IDEX Health& Science,目录编号:P-215; 6 x;¼-28螺纹)
    6. Union(IDEX Health& Science,目录号:P-704-01; 6次; 10-32次)

  4. 细胞加载和图像采集
    1. 试管(14毫升; Corning,目录号:352051)
    2. 大规格注射器针头(16 G 1.5“; BD,目录号:305198)
    3. 大注射器(100毫升; Veterinary Concepts,目录号:60271)
    4. 10毫升注射器(Luer-Lok tip; BD,目录号:309604)
    5. Steriflip真空过滤管(50ml;20μM尼龙网; Millipore Sigma,目录号:SCNY00020)
    6. 培养皿(100mm; Fisher Scientific,目录号:FB0875713)
    7. Stericup-GP过滤灭菌模块(500ml;0.22μmPES; Millipore Sigma,目录号:SCGPU05RE)
    8. 酵母菌株
    9. 牛血清白蛋白(Sigma-Aldrich,目录号:A2153)
    10. 琼脂粉(Sigma-Aldrich,目录号:A1296)
    11. 是225粉(250克;日出科学,目录号:2011-250)
    12. 是的225琼脂媒体(配方1)
    13. 是225液体介质(配方2)

设备

  1. 微细加工
    1. 1L烧瓶(No.1000; Corning,Pyrex,目录号:1000-1L)
    2. Suss MA-6掩模对准器(Suss MicroTec Lithography GmbH)
    3. 旋涂机(Laurell Technologies)
    4. 热板(Cimarec +; Thermo Fisher Scientific,Thermo Scientific,目录号:HP88857100)
    5. 各向异性RIE等离子蚀刻机(Nordson March,目录号:CS170IF)
    6. 热手保护器米特(SP Scienceware - Bel-Art Products - H-B Instrument,产品目录号:F38000-0001)

  2. multFYLM组件
    1. 迷你labroller(Labnet)
    2. 等离子清洁器(哈里克等离子,产品目录号:PDC-32G)
    3. 实验室烤箱(Ecotherm,Precision)
    4. 解剖显微镜(AmScope,目录号:SM-1T-PL)
    5. 精密镊子(Fisher Scientific,目录号:16-100-103)
    6. Sonicator(Bransonic,目录号:2510R-DTH)
    7. 摇摆/鼓动者(肚皮舞者; Stovall生命科学)
    8. 本生灯(accuFlame; Fisher Scientific,目录号:03-902Q)
    9. 离心机(Beckman Coulter,型号:Avanti J-26XP)
    10. 离心机转子(Beckman Coulter,型号:JLA-16.250)

  3. 显微镜和微流体装置
    1. 落射荧光成像显微镜(Eclipse Ti;尼康)
    2. 焦点维护系统(尼康Perfect Focus,尼康)
    3. CMOS相机(安道尔,型号:Zyla 5.5 sCMOS)
    4. 10倍,0.3 NA目标(福陆计划;尼康)
    5. 60x,0.95 NA目标(Plan Apo Lambda; Nikon)
    6. 计算机控制的显微镜舞台(Proscan III机动舞台;事先)
    7. 目标加热器(Bioptechs,目录号:150819-19)
    8. 荧光成像的适当过滤器
      1. eGFP(Chroma,目录号:49002)
      2. mKO(Chroma,目录号:49010)
      3. E2Crimson(Chroma,目录号:49015)
    9. 光源快门(SmartShutter;萨特仪器)
    10. 快门控制器(Lambda SC; Sutter Instrument)
    11. 计算机控制的注射器泵(KD Scientific,型号:LEGATO 210)
      注意:该泵配置为两个注射器。如果需要两个以上的注射器,可以使用多个泵,或者可以制造适配器(图4)以允许驱动额外的注射器。
    12. 光源(纽波特,型号:SOLA-SE-II; Lumencorp)

  4. 细胞加载和图像采集
    1. 摇动培养箱(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:4333)
    2. 分光光度计(Thermo Fisher Scientific,Thermo Scientific TM,型号:NanoDrop TM 2000c)
    3. 高压灭菌器(统一灭菌器系统,型号:ADV-PLUS)
    4. 真空干燥器(5.8L Pyrex玻璃; Corning,PYREX®,目录号:3121-200)
    5. 真空泵(Welch Vacuum,目录号:2546,B-01)
    6. 迷你涡旋器(Fisher Scientific,目录号:02-215-365)
      注意:此产品已停产。
    7. 本生灯(accuFlame; Fisher Scientific,目录号:03-902Q)
    8. 环境室/ multFYLM显微镜舞台
      分庭设计文件可在:
      https://github.com/finkelsteinlab/FYLM_mask_files/blob/master/FYLMChamber.scad

软件

  1. NIS-Elements高级研究(v4.30.02; Nikon Instruments)

程序

  1. 微细加工
    multFYLM微制造遵循常规的软光刻方法。第一步是生成图案化的模具,该模具可用于在弹性硅胶(PDMS)中浇铸器件。这种模具或“主”结构是在硅晶片上创建的,使用紫外光刻技术在环氧树脂(SU-8)表面沉积图案。图案由掩模决定,这限制了UV光源交联树脂的能力。它们的对齐对于晶圆上正确的图案化至关重要,因为最终主控的特征包含在两个掩模中的每一个上。使用显影剂去除未曝光的树脂,留下一个现在可以使用的主体(图1)。主人可以重复使用至少两年,以制造数百个multFYLM设备。
    注意:下面详细介绍的程序应在洁净室内进行。所有仪器设置对于所用设备都是独一无二的,并作为指导。这些设置将需要进行调整以匹配用户洁净室中可用的仪器。所有微细加工步骤应在一天内完成;尽管可能存在合适的停止点,但它们尚未经过测试。


    图1. multFYLM设计概述。multFYLM包含六个独立的路径。介质通过设备顶部的每个纳米孔进入(入口),然后沿着蓝色箭头指示的路径行进,然后通过设备底部的出口(出口)退出。


    1. 用丙酮,异丙醇和水冲洗抛光的晶圆表面。
    2. 在安装等离子清洁器的同时空气干燥晶圆。

    3. 将电炉设置为200°C
    等离子清洗
    等离子清洁晶圆以产生超洁净的表面,以便树脂图案可以以高分辨率和附着力沉积在表面上。
    1. 打开等离子清洁器和气体控制器。
    2. 创建一个等离子体清洁程序(表1),该程序将以30/70比率的O 2至N 2/2清洁晶圆。更多的时间不一定会产生更好的表面。

      表1.第一个等离子清洗程序


    3. 打破室真空,并加载晶圆的抛光面朝上。
      注意:手动操作最适合放空真空。
    4. 运行清洁程序。
      1. 将射频调整开关更改为自动,然后启动程序。

      2. 通过调整C1和C2开关,在等离子体流动过程中,应使反向功率流量最小化
      3. 程序完成后,让真空渗血完成,然后停止程序。
    5. 去除晶圆。
    6. 重新建立真空室,以提高仪器的使用寿命和清洁度。
    7. 关闭组件。 

    准备面具对齐器
    打开面罩对齐器组件,使其在使用前平衡。
    1. 打开燃气和真空管路。
    2. 打开面具对齐器。
    3. 启动紫外灯 - 它需要10分钟的预热时间。

    为首次曝光准备晶圆
    将第一层树脂均匀地沉积在晶片表面上以产生5-6μm的树脂厚度。对齐,旋转参数和树脂应用对于正确的沉积都是至关重要的。
    1. 将晶圆直接放在电炉上,抛光表面朝上,在200°C下保持20分钟。
      注:此步骤可确保晶圆干燥。晶片温度一旦从热板上取下就不必保持,但应快速进行下一步。可以使用热手保护手套在仪器之间传送晶片。

    2. 在旋转涂布机上放置一个100毫米的旋转木马
    3. 小心地将晶圆转移到转盘的中心,打开真空线,将晶圆牢固地固定到位。步骤A21中偏离中心的晶圆不会产生均匀的SU-8层。
    4. 打开旋转涂布机,并设置旋转涂布机程序:
      1. 在500转/分,10秒加速等级2(266转/秒)。
      2. 1,500 rpm时为35秒,加速度为4(532 rpm / sec)。
    5. 运行该程序,一旦速度达到1,500 rpm,向晶片表面添加两滴环戊酮。
    6. 尽可能均匀地在晶圆表面添加6毫升SU-8 2005树脂 - 避免SU-8滴落在晶圆两侧。
    7. 等待3分钟,同时气泡上升到晶圆上SU-8的表面。
    8. 从步骤A17运行程序。
    9. 应该缓慢提起盖子,以避免SU-8滴到新纺出的硅片上。
    10. 用环戊酮湿润擦拭并去除残留在晶片表面边缘上的SU-8珠粒。或者,如果旋转涂布机如此装备,则可以使用边缘珠去除协议。
    11. 释放真空压力并从传送带上取下晶圆。
      注意:SU-8的最终层应该是均匀的。如果不是,晶圆必须用异丙醇清洗,并从步骤A1重新开始。

    12. 在最初关闭的热板上将晶圆从室温加热到95°C。

    13. 在95°C的热板上放置晶片4分钟。


    14. 关掉电炉,让晶片在其上冷却10分钟。 

    使用第一个掩模曝光晶圆
    将第一个掩模和树脂覆盖的晶圆安装到掩模对准器中。将晶片暴露于紫外线足够长的时间,以足够的分辨率在树脂中产生图案。曝光不足导致图案不完整或功能减弱,而过度曝光会导致放大功能和低分辨率。
    1. 调整掩模对齐器参数(表2)。这里的参数只能作为指导。

      表2.第一层的遮罩对齐参数


    2. 将面罩1放入面罩支架。

      1. 移除并安装适用于100毫米晶圆的正确面罩

      2. 使用真空将面罩固定在支架镀铬面朝上。
    3. 将遮罩置于可视区域的大致中央。
    4. 将晶圆装入晶圆支架。

    5. 使用对齐标记作为指导,将晶圆与面罩对齐。
    6. 将晶圆暴露在紫外线下,等待曝光完成。
    7. 去除晶圆。 

    从晶圆上去除未曝光的光刻胶
    使用显影剂从晶圆表面去除未曝光的树脂;该过程揭示了沉积的特征。过度的显影会导致沉积的特征被冲走。
    1. 将晶圆放回冷却的热板上。
    2. 加热至95°C,然后在此温度下孵育1分钟。
    3. 将晶圆置于1升烧瓶中光刻胶面朝上。
    4. 将开发人员倒在晶圆上以完全覆盖它。

    5. 允许开发者在搅拌的情况下继续进行30秒。
    6. 去除晶圆。
    7. 用新鲜的显影剂清洗晶圆表面。

    8. 用异丙醇清洗晶圆表面。

    9. 使用加压氮气干燥晶片。

    准备二次曝光晶圆
    将第二层树脂均匀地沉积在晶片表面上以产生20-30μm的树脂厚度。树脂和旋转参数都进行了优化,以沉积具有用于第二次曝光的适当特性的树脂层。
    1. 按照步骤A4-A10清洁等离子清洁器中的晶圆表面,但使用以下程序(表3)。

      表3.第二次等离子清洁程序


    2. 将晶圆直接放在电炉上,抛光表面朝上,在200°C下保持20分钟。
      注:此步骤可确保晶圆干燥。晶片温度一旦从热板上取下就不必保持,但应快速进行下一步。可以使用热手保护手套在仪器之间传送晶片。

    3. 在旋转涂布机上放置一个100毫米的旋转木马
    4. 小心地将晶圆转移到转盘的中心,打开真空线,将晶圆牢固地固定到位。步骤A21中偏离中心的晶圆不会产生均匀的SU-8层。
    5. 设置旋涂机程序:
      一个。
      在500 rpm时为14秒,加速等级2(266 rpm / sec)。

      在3000 rpm下为37秒,加速度为4(532 rpm / sec)。
    6. 尽可能均匀地在晶片表面添加6毫升SU-8 2010 - 避免SU-8滴落在晶圆两侧。
    7. 等待9分钟,同时气泡上升到晶圆上SU-8的表面。
    8. 关闭封面并从步骤A48运行程序。
    9. 应该缓慢提起盖子,以避免SU-8滴到新纺出的硅片上。
    10. 用环戊酮湿润擦拭并去除残留在晶片表面边缘上的SU-8珠粒。或者,如果旋转涂布机如此装备,则可以使用边缘珠去除协议。
    11. 释放真空压力并从传送带上取下晶片。

    12. 在最初关闭的热板上将晶圆从室温加热到85°C。

    13. 在85°C的热板上放置晶片15分钟。

    14. 关掉电炉,让晶片冷却10分钟。

    用第二个掩膜曝光晶圆
    将第二个掩模和树脂覆盖的晶圆安装到掩模对准器中。将晶片暴露于紫外线足够长的时间,以足够的分辨率在树脂中产生图案。此步骤的对齐非常重要,因为它确保使用第二个掩膜产生的特征能够与晶圆表面上已有的特征正确叠加。
    1. 调整掩模对齐器参数(表4)。这里的参数只能作为指导。

      表4.第二层的遮罩对准器参数


    2. 将面罩2放入面罩支架。

      1. 移除并安装适用于100毫米晶圆的正确面罩

      2. 使用真空将面罩固定在支架镀铬面朝上。
    3. 将遮罩置于可视区域的大致中央。
    4. 将晶圆装入晶圆支架。
    5. 使用第二个掩模和晶圆上的对准标记(从第一次曝光开始),调整晶圆的位置,使其与掩模对齐。
    6. 将晶圆暴露在紫外线下,等待曝光完成。
    7. 去除晶圆。 

    从晶圆上去除未曝光的光刻胶
    使用显影剂从晶圆表面去除未曝光的树脂。这个过程揭示了沉积的特征。过度的显影会导致沉积的特征被冲走。
    1. 将晶圆放回冷却的热板上。
    2. 加热到85°C,然后在该温度下孵育10分钟。
    3. 在孵化期间,从掩模对准器上取下第二个掩模,然后关闭掩模对准器和UV灯。
    4. 将晶圆置于1升烧瓶中光刻胶面朝上。
    5. 将开发人员倒在晶圆上以完全覆盖它。

    6. 允许开发者在搅拌的情况下进行3分钟。
    7. 去除晶圆。
    8. 用新鲜的显影剂清洗晶圆表面。

    9. 用异丙醇清洗晶圆表面。
    10. 重复步骤A72-A73。

    11. 使用加压氮气干燥晶片。

  2. multFYLM组装
    一旦主结构完成后,通过软光刻技术组装multFYLM。主结构被用作PDMS的模具。在PDMS硬化之前,添加端口以允许介质流入微流体结构。一旦硅树脂凝固后,它就会被清洗并粘附在一块大玻璃罩上。薄而透明的盖玻片构成了multFYLM的基础,并允许在设备的单个臂内捕获细胞的成像。
    将聚甲基硅氧烷(PDMS)中的multFYLM浇铸
    根据制造商的协议准备PDMS解决方案(图2)。用胶带包裹主结构为PDMS创建垂直屏障。将一半溶液倒入主结构。软烤第一层,直到它变粘,然后在主结构上的每个导管通道上放置一个干净的端口。将剩余的PDMS倒入第一层,然后烘烤,直到所有PDMS完全硬化。


    图2.软光刻。 :一种。纸带围绕包含SU-8主盘的晶圆,以便在设置PDMS时保持原位。 B.第一层PDMS。 C.第一层是半硬化的。 D.纳米orts被放置在第一层。 E.将第二个PDMS层倒在纳米管周围。 F.完全固化的multFYLM,从主结构中移除。

    1. 将烤箱预热至75°C。
    2. 将30克PDMS与3克硬化剂混合在50毫升锥形管中。安装帽子。

    3. 在室温下将PDMS溶液在实验室滚筒上混合45分钟。
    4. 在超声波处理设备中,在浴室超声波仪中用2%Hellmanex清洗12个nanoports 20分钟(nanoports也可以提前清洗)。
    5. 用过滤去离子水彻底冲洗nanoorts。

    6. 在超声波处理设备中,将纳米棒放置在70%的乙醇中,超声波清洗20分钟

    7. 使用标准的实验室胶带在图案化的圆片周围创建一个屏障。
      一个。
      至少2毫米的胶带应均匀地延伸到晶圆的圆周下方 湾
      为避免PDMS从晶圆表面泄漏,胶带附着力至关重要
    8. 将晶圆放入大型(150毫米)培养皿中。
    9. 在室温下在> 50℃离心混合的PDMS溶液。 400克x克 90秒,去除大气泡。
    10. 将13克PDMS倒入晶圆上,使其均匀地浸湿整个表面。

    11. 在70°C的热板上干燥30分钟
    12. 将晶圆置于60-70cmHg的真空干燥器中15min,除去残留的气泡。
    13. 快速移除真空以去除仍然困在PDMS中的气泡。如有必要重复。

    14. 将PDMS晶圆放入70°C烘箱中15分钟。
    15. 测试晶圆上的PDMS以获得适当的硬度。
      1. PDMS应该是半固体且非常粘性的,当用200μl移液枪头探查时会形成一个小峰。
      2. 如果没有,请将它放回烤箱,每隔几分钟检查一次硬度。
    16. 使用解剖范围,精确地将12个纳米口中的每一个放置在PDMS的每个介质导管的末端,如晶片的图案所示。
      避免在PDMS表面上多次放置纳米柱。多次放置会损坏多个流体通道,并且未对准的端口可能会阻止流体流向相应的通道。
    17. 将14克PDMS倒入晶圆,使其均匀地浸湿整个表面。
    18. 将晶片在60-70cmHg的真空干燥器中放置15分钟。这可以去除纳米端口下可能存在的空气,确保纳米端口与PDMS之间的良好密封。

    19. 。将晶圆放回烤箱(70°C)3小时,或直至完全固化。 

    剪切,清理并组装multFYLM
    从主结构中移除multFYLM,然后使用剃刀刀片修剪掉多余的PDMS。使用活检穿孔器从每个模制导管到multFYLM另一侧上的纳米孔进行直接路径。超清洁multFYLM和一个大玻璃盖,然后将它们粘在一起。   完成 multFYLM 的组装。

    1. 在摇杆上搅动一个小时,将盖玻片置于含有2%Helmannex溶液的培养皿中一小时。

    2. 用diH 2 O冲洗护盖玻璃两次

    3. 用异丙醇冲洗护盖玻璃两次
    4. 将盖玻片放入含有单层铝箔的大培养皿中。

    5. 在70°C的电炉上放置盘子至少两个小时晾干。

    6. 小心地从图案化晶圆和multFYLM的圆周上取下胶带
    7. 小心地从晶圆表面上剥离铸造的multFYLM。
      1. 柔和地去皮以避免分裂聚合的PDMS。背景颜色:透明;框尺寸:边框;颜色:#666666; font-family:Arial,Helvetica,sans-serif; font-size: 13.33px;字型样式:正常;字体变量:正常;字体重量:400;字母间距:正常;孤儿:2;文本对齐:对齐;文字修饰:无;文本缩进:0像素;文本-transform:无; -webkit文本行程宽度:0像素;空白:正常;字间距:0像素;” />
      2. 不要触摸与图案化晶圆接触的表面。 

    8. 翻转培养皿中的multFYLM,端口朝下。

    9. 使用新型锋利的剃刀刀片,从multFYLM的图案化/移植区域切掉多余的PDMS。
    10. 将multFYLM放置在解剖显微镜下。
    11. 使用1毫米活检穿孔器,从底面到顶面轻轻地穿过每个纳米孔的中心。
      注意:冲孔区域应包括nanoort放置在其上的导管。

    12. 在从multFYLM取出活检穿孔器之前,使用精密镊子从活检穿孔器中取出PDMS'穿孔'核心。
    13. 用轻微的压力和轻微的旋转运动从multFYLM上取下活检穿孔器,以避免将纳米孔与PDMS分开。
    14. 检查nanoorts并用精密镊子清除任何残留的PDMS颗粒。
    15. 将multFYLM浸没在含有100%异丙醇的烧杯中,并在超声处理设置中将烧杯置于浴超声波仪中30分钟。
    16. 从烧杯中取出multFYLM,并将其端口朝下放入含有单层铝箔的大培养皿中。

    17. 在70°C的电炉上将盘子放置两个小时晾干。
    18. 将最近干燥的multFYLM和玻璃罩放入等离子清洁器中,使与玻璃罩接触的表面朝上。
    19. 打开等离子清洁器。
    20. 打开真空抽气室至少一分钟。


    21. 将射频设置设置为“高”,持续20秒
    22. 立即从等离子清洗机中取出组件。

    23. 通过小心地将保护盖玻璃的最干净的一面放在multFYLM的中心位置,粘贴保护玻璃和multFYLM。
    24. 施加轻微的压力到multFYLM,以确保它完全粘附在玻璃罩上。
      1. 为获得最佳效果,multFYLM应在组装数小时内使用。或者,multFYLM可以储存在70%的乙醇中以延长无菌储存。 -serif;字体大小:13.33px;字型样式:正常;字体变量:正常;字体重量:400;字母间距:正常;孤儿:2;文本对齐:对齐;文字修饰:无;文本缩进:0像素;文字变换:无; -webkit文本行程宽度:0像素;空白:正常;字间距:0像素;” />
      2. 完成的multFYLM应存放在容器中以避免污染。

    显微镜和微流体装置
    整个寿命成像增加了操作任何微流体装置的额外技术难题。首先,微流体系统必须向捕获的细胞提供新鲜的培养基,同时还要清除废物。流路中的缺陷会导致气泡脱落细胞,从而可能会中断多天的实验。此外,必须采取额外的预防措施来清除multFYLM上游的细胞。这是因为这些细胞可能会在multFYLM操作过程中长成微菌落,最终阻碍新鲜培养基流向装置。其次,显微镜应配备稳定的光学和机械部件,长达一周的连续成像。主动反馈聚焦寻找系统确保multFYLM可以成像数天,无需任何用户干预。类似地,推荐在连续操作的一周内不改变输出强度或光谱的光源(即,,LED灯)。最后,我们建议将整个装置封装在一个培养箱内,以保持所需细胞的最佳生长条件(参见设备D8)。

    准备微流体管
    清洁将用于连接到multFYLM的所有微流体接头(图3),然后将它们安装到微流体管道上。有必要在连接到纳米管的接头之后立即在管道中放一个直角,否则管道不会清除环境室和显微镜组件。


    图3.微流体接头

    1. 将所有微流体接头浸入含有2%Hellmannex洗涤剂的烧杯中,并在超声波处理设备中在超声浴器中超声20分钟。

    2. 用diH 2 O 3冲洗所有配件。
    3. 将所有的微流体接头浸入含有100%乙醇的烧杯中,并在超声波处理设备中在超声浴器中超声20分钟。
    4. 用100%乙醇冲洗所有接头。

    5. 在70°C的热板上将培养皿中的所有接头干燥30分钟或更长时间。
    6. 将十二段管切成60厘米的长度。剪切尽可能平方。
    7. 使用本生灯作为辅助工具,将95°角永久弯曲到每个管的一端,距离端约17 mm。
    8. 对于将成为废物管线的六根管子,请在弯曲端安装以下配件:
      1. font-family
      2. F-142N套圈,朝向弯头的钝端。
        管道应延伸超出套圈1-2毫米。
    9. 对于将成为媒体线路的六个管道,请连接以下配件:

      1. F-333N锥形螺母远离弯道
      2. F-142N套圈,朝向弯头的钝端。
        管道应延伸超出套圈1-2毫米。

      3. P-215无法兰螺母朝直线端螺纹
      4. P-272套圈,钝端远离无法兰螺母。

      5. P-658 Luer适配器,拧在无法兰螺母上,夹住套圈。

    10. 使用P-235连接器将每条介质线连接到废物管线。
      注意:管道应提前准备好,并可存放在乙醇或无菌水中直至使用。

    准备用于成像的显微镜
    打开显微镜和外围设备,以便在实验开始前他们可以预热。 NIS Elements软件(或其他控制软件)也应该打开,因为某些外设在没有正确配置软件的信号的情况下可能无法完全打开。
    1. 打开以下组件:
      1. 显微镜
      2. 相机
      3. 快门控制器
      4. 阶段
      5. 目标加热器设置为在multFYLM内达到30°C。加热器应安装在60x空气物镜上。温度设置应该凭经验确定,因为可能需要更高的程序设计温度来解决热损失。
      6. 舞台加热器设置为在multFYLM内达到30°C。温度设置应该凭经验确定,因为可能需要更高的程序设计温度来解决热损失。
      7. LED光源
      8. 白光源
    2. 启动NIS Elements软件套件。

      1. 如果出现提示,请选择'Neo / Zyla'作为图像采集卡
      2. 将10倍的目标移动到位置。

  1. 细胞加载和图像采集
    下面,我们描述一个协议,以最大化在multFYLM中捕获的单元的数量。由于multFYLM包含许多细小的段落,因此可能会堵塞细胞团或其他碎片。在准备和装载培养基和细胞时要小心,以避免任何颗粒或细胞团块。此外,空气可以很容易地移除捕获的细胞,因此它应该从流体路径中的任何上游组件清除。使用无菌技术来防止其他微生物污染multFYLM中的细胞。
    在multFYLM中对细胞进行图像采集需要在数十个位置,固定时间间隔,多个Z平面和与存在的荧光团范围对应的滤光片进行图像采集。尽管大多数成像都使用了对焦Z平面,但外平焦Z平面允许更加确定地定义单元边界。选择荧光基团和滤光片时应该小心,因为光谱分离可以明确地将荧光归因于单个荧光基团。

    准备媒体和单元格
    制作一升脱气,过滤的YES 225培养基,培养酵母菌株,使其在实验的第一天处于指数生长期。
    1. 准备一升YES 225琼脂培养基(配方1)。
    2. 准备一升YES 225液体介质(配方2)。

    3. 在锥形管中准备1毫升无菌20%BSA溶液
    4. 在实验开始前四天将来自甘油储液的细胞接种到琼脂平板上。培养皿应在30°C下培养至菌落形成良好,然后置于室温下。
    5. 选择2-3天龄的菌落,并在试管中接种10 ml YES 225培养基。

    6. 在30°C摇动培养箱中孵育细胞培养过夜。
    7. 当细胞培养物的595nm(OD 595)光密度达到0.1时,接种含有10ml YES 225培养基的新鲜试管。
    8. 将新细胞培养物在30℃振荡培养箱中孵育直至OD 595为0.5至1.0(4-6小时)。
    9. 将YES 225培养基放入真空干燥器中,使瓶盖松动15分钟。这应该在将介质装入注射器之前完成。

    连接并清洁媒体/废品行
    将准备好的介质加载到足够大的注射器中,以便在整个实验时间过程中容纳足够的介质。使用乙醇和无菌水清洁介质和废物管线,因为无菌对实验成功至关重要。将multFYLM安装在环境室中,然后连接废物管线(图4)。


    图4.准备用于成像multFYLM的荧光显微镜。 :一种。完整的multFYLM微流体路径。 B.微流体接头将管线连接到multFYLM。

    1. 打开注射泵。
    2. 确定multFYLM中将使用多少个流路。
      由于空间限制和图像采集速率,通常仅使用三条或四条可用六条流路。如果图像采集频率不够频繁,线条不会过度扭转multFYLM,并且所有区域都可以通过显微镜观察,则可以使用全部六条流路。

    3. 用70%乙醇填充N 10毫升注射器('N'等于将要使用的许多流路)。
    4. 将这些注射器装入注射器泵的注射器支架。
    5. 将N个培养基/废液管线组连接到每个乙醇注射器。
    6. 设置注射泵参数并运行:
      1. 注射器:B-D Plastipak 10毫升注射器
      2. 5分钟
      3. 1毫升/分钟

    7. 填充N 10毫升注射器diH sub 2 O.

    8. 用水注射器替换乙醇注射器。
    9. 根据步骤C15重新运行泵。
    10. 用脱气的YES介质装载N注射器。

      1. 。将一个大注射器针头连接到注射器上,以帮助加载注射器而不引入任何气泡。

      2. 注射器中的空气应立即清除

    11. 用YES媒体注射器替换注射器。
    12. 设置注射泵参数并运行以用YES介质替换管线中的水:
      1. 定制注射器直径31.75毫米
      2. 1分钟
      3. 1 ml / min 

    13. 使用弹簧金属夹或实验室胶带取回multFYLM并将其连接到加热的舞台插入物上。
      按照惯例,multFYLM被定向为尽可能平行于成像区域,入口端口最靠近用户。入口端口通向微流体模式的末端,而微流体模式不能直接进入用于容纳细胞的通道外围的废弃沟槽。
    14. 分离媒体线路中的废弃线路并将它们连接到multFYLM的出口通道。
      1. 注意避免将线条放在实验过程中要成像的区域上。
      2. 将线路同时连接到所有六个路径是困难的。通常建议并行运行不超过三条或四条流路。

      3. 尽量以无菌方式执行此任务。
      4. 媒体线路应保持无菌,直到连接。将它们存放在开放的锥形管中通常足以防止污染。

    将单元格加载到multFYLM
    中 小心地将漩涡和称重传感器放入每个入口端口,然后连接介质线,同时避免引入任何空气。建立注射泵的程序,通常提供一致的流速,偶尔增加流速;这将有助于消除任何可能阻塞multFYLM通道的碎片。
    1. 将每个细胞培养物400μl转移到单独的微量离心管中。

    2. 加入100μl无菌20%BSA溶液
    3. 将每个管涡旋一分钟。
    4. 使用微量移液器,将40μl细胞溶液转移到每个相应的进样口。
      1. 小心引入尽可能少的空气。该体积确保有足够的液体存在以允许与介质管线的滴落连接,而不会在安装期间过度填充纳米孔。

      2. 移液器吸头应保持在端口底部的上方,以避免将空气引入流道。
    5. 可以使用白光和10倍物镜观察细胞。
      由于表面张力,他们应该开始流入multFYLM。

    6. 以40μl/ min的速率设置注射泵并运行。
    7. 当YES媒体开始退出每条媒体线时,请轻轻将其连接到输入端口。
      附着时要非常小心:使用点对点连接策略以避免向流动路径引入空气,并且不要在multFYLM上施加扭矩。
      端口可以很容易分开,否则盖玻片可能会破裂。
    8. 使用白光和10倍物镜观察细胞。他们应该填充微流体流路的通道,从入口附近开始(图5A)。
    9. 用以下参数为泵创建一个程序:
      1. 一分钟,流速为55μl/ min

      2. 5μl/ min,14分钟
      3. 重复725次。
    10. 一旦细胞填充了大部分要观察的通道,请启动上述程序。


      图5.载入到multFYLM中的Schizosaccharomyces pombe 细胞。 :一种。在加载过程之后立即在单个流路内形成10倍的细胞图像。 B.在所定义的感兴趣区域(ROI)内可见的细胞的60x图像。

    开始图像采集
    使用NIS Elements软件,建立一个多维采集策略,以定期的时间间隔捕获multFYLM每个隔室中的细胞图像,聚焦和离焦Z平面,以及所有必要的过滤器组在使用中荧光团的排放(表5)。可以使用其他软件套件,但以下说明仅适用于NIS Elements。
    1. 将60x空气物镜移动到位。
    2. 获得焦点,然后使用显微镜正面的PFS按钮打开Perfect Focus System(PFS)。
    3. 使用舞台控制器,将最左侧的流动路径投入使用。
    4. 将感兴趣区域(ROI)更改为与小区信道的可视区域相同的大小。
      1. 在NIS Elements的Zyla settings菜单中,使用'Commands'> 'ROI'> '加载投资回报率'下拉菜单,然后选择从下面下载的* .CAMROI文件。
        相机ROI文件:
        https://github.com/finkelsteinlab/FYLM_mask_files/blob/master/FYLM_ROI_2X2_new.camroi
      2. 在相同的子菜单中,选择“使用当前ROI”(图5B)。
    5. 在ND采集菜单下,设置文件夹和文件名称。
      1. 路径:生成的文件将被存储的位置。
      2. 文件名:要生成的文件的名称。一个三位数字将自动附加到结尾。
    6. 在ND采集菜单下,设置时间:
      1. 图像采集的时间间隔和总持续时间。频率取决于要采集的通道数量,但对于纯白光图像,2分钟的间隔是合理的。
      2. 建议持续时间为36小时或更短,以平衡文件大小与图像采集重启频率。
    7. 在ND采集菜单下,将Z:
      1. 聚焦(如使用PFS确定)
      2. 4μM偏移(步骤:4,2步,以下:-4,以上:0)
    8. 在ND采集菜单下,设置λ:
      1. 应为每个荧光图像滤光片组设置光学配置。曝光时间应通过实验确定。
      2. 选择实验过程中将使用的所有光学配置。
      3. 荧光图像不需要在每个时间段收集(减少光毒性的可能性),频率可以使用T Pos设置。
      4. Z深度是可选的。建议仅在'Home'Z Pos收集荧光图像。 
    9. 在ND采集菜单下,设置XY:
      1. X-Y位置应平铺在可观察细胞上。
      2. 建议将位置定义为环形图案以避免焦平面发生较大变化,这可能会导致焦点在实验中丢失。 
    10. 一旦所有参数设置好(表5),按下'运行'。

      表5.多维图像采集的示例参数


    11. 观察前几轮成像,以确保一切都保持不变。
    12. 应该每天至少观察一次实验以检查错误并收集新的图像文件。下游分析针对包含24小时数据的文件进行了优化。
    13. 24小时后,按'完成'完成一天的收集。
      1. 这也将保存文件,但通过访问“文件”菜单中的“保存”可以保证保存。
      2. 如果出现提示,则不必在完成前完成当前循环。
    14. 现在可以使用图像分析软件来创建视频并分析收集的数据。

数据分析

关于如何使用这种方法收集数据的信息可以在这些参考文献中找到(Greenstein等人,2017; Spivey等人,2017)。

笔记

  1. 在微制造期间,光致抗蚀剂的类型和体积以及自旋参数可以变化以改变沉积特征的高度。类似地,可以改变光致抗蚀剂的类型和曝光时间和强度以改变沉积特征的分辨率和宽度。这对于捕捉尺寸稍大的细胞特别有用。
  2. multFYLM组装过程中的一个常见故障点是从纳米管的中心冲出PDMS。通常,去除冲头会导致纳米棒脱离PDMS,形成一小块空气。小心的是,当盖玻璃被压到PDMS上时,这样的空气袋可以稍后排出。如果口袋仍然存在,它们可以成为空气的储存器,当通过multFYLM时,它会移出细胞,或者随着实验的进行,其他细胞会堵塞和堵塞通道。
  3. 用细胞加载multFYLM通常对于新装配的设备来说效果最好,因为内部仍然非常脱水,因此介质和细胞容易流入其中以使表面再水化。如果multFYLM已经储存了一段时间,建议使用1毫升70%乙醇,然后将1毫升水倒流通过设备,以便空气不会被困在出口通道中。否则,截留的空气不会从出口通道排出,并且相邻的通道不会产生所需的压力差,以便进行后续的电池装载。

食谱

  1. 是225琼脂培养基(1 L)
    1. 36.13克是225粉末,20克琼脂;加diH 2 0 O总量高达1 L
    2. 高压灭菌器,然后用无菌技术将25毫升倒入各个培养皿中。
  2. 是225液体培养基(1 L)
    1. 36.13克的YES 225粉末;将diH 2 0添加至总体积1 L。
    2. 过滤器对溶液进行消毒 - 这也会去除可能导致通道堵塞的小颗粒。高压灭菌器处理是不够的,因为它会消毒的解决方案,但不会去除颗粒

致谢

我们要感谢芬克尔斯坦实验室的成员在开发和准备这种方法的过程中提供的意见和建议。这项工作得到以下赠款和研究金的慷慨支持:美国老龄研究联合会(AFAR-020至I.J.F.),韦尔奇基金会(F-1808至I.J.F.)和NIH(F32 AG053051至S.K.J.)。内容完全是作者的责任,并不一定代表国家科学基金会的官方观点。该协议是根据以前的设计进行改编的(Spivey等人,2014年; Spivey等人,2017年)。

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Copyright Jones Jr et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Jones Jr, S. K., Spivey, E. C., Rybarski, J. R. and Finkelstein, I. J. (2018). A Microfluidic Device for Massively Parallel, Whole-lifespan Imaging of Single Fission Yeast Cells. Bio-protocol 8(7): e2783. DOI: 10.21769/BioProtoc.2783.
  2. Spivey, E. C., Jones, S. K., Rybarski, J. R., Saifuddin, F. A. and Finkelstein, I. J. (2017). An aging-independent replicative lifespan in a symmetrically dividing eukaryote. Elife 6.
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