A Flexible Chamber for Time-Lapse Live-Cell Imaging with Stimulated Raman Scattering Microscopy

Podsumowanie

We report a stage-top, flexible environmental chamber for time-lapse imaging of live cells using upright stimulated Raman scattering microscopy with transmitted signal detection. Lipid droplets were imaged in SKOV3 cells treated with oleic acid for up to 24 h with a 3 min time interval.

Streszczenie

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many biological and biomedical applications. However, long-term time-lapse SRS imaging of live cells has not been widely adopted. SRS microscopy often uses a high numerical aperture (NA) water-immersion objective and a high NA oil-immersion condenser to achieve high-resolution imaging. In this case, the gap between the objective and the condenser is only a few millimeters. Therefore, most commercial stage-top environmental chambers cannot be used for SRS imaging because of their large thickness with a rigid glass cover. This paper describes the design and fabrication of a flexible chamber that can be used for time-lapse live-cell imaging with transmitted SRS signal detection on an upright microscope frame. The flexibility of the chamber is achieved by using a soft material - a thin natural rubber film. The new enclosure and chamber design can be easily added to an existing SRS imaging setup. The testing and preliminary results demonstrate that the flexible chamber system enables stable, long-term, time-lapse SRS imaging of live cells, which can be used for various bioimaging applications in the future.

Wprowadzenie

Optical microscopy is used to observe the microstructures of samples. Optical imaging is rapid, less invasive, and less destructive than other technologies¹. Live-cell imaging with optical microscopy is developed to capture the dynamics of cultured live cells over a long period². Different types of optical contrasts provide distinct information about biological samples. For instance, optical phase microscopy shows the subtle difference in the refractive indices across the sample³. Fluorescence microscopy is widely used to image specific biomolecules or cellular organelles. However, the broadband excitation and emission spectra of fluorescence usually result in spectral overlapping when multicolor imaging is performed⁴. Fluorescent molecules are light-sensitive and can be bleached after long-term, periodic light exposure. In addition, fluorescence labeling may change the biodistribution of the molecules in cells⁵. SRS microscopy is a label-free chemical imaging technology⁶. The contrast of SRS relies on the vibrational transition of specific chemical bonds. The vibrational frequency of a chemical bond often exhibits a narrow spectral bandwidth, making it feasible to image multiple Raman bands in the same samples⁷. SRS microscopy is a unique tool for live-cell imaging, providing multiple chemical contrasts in a label-free manner⁸.

While SRS imaging of unstained cells has been used for many studies, long-term time-lapse SRS imaging of live cells has not been widely adopted. One reason is that commercial open chambers cannot be directly used for SRS imaging because of their large thickness^9,10,11,12. These chambers with a glass lid are mostly designed for brightfield or fluorescence imaging using a single high NA objective with a backward detection scheme. However, SRS imaging prefers transmitted detection using both a high NA objective and a high NA condenser, which leaves only a very short gap (typically a few millimeters) between the objective and the condenser. To overcome this problem, we designed a flexible chamber using a soft material to enable time-lapse SRS imaging of live cells using an upright microscope frame. In this design, the water dipping objective was enclosed in the soft chamber and can freely move in three dimensions for focusing and imaging purposes.

The optimal temperature for culturing most mammalian cells is 37 °C, while the room temperature is always 10° lower than this. Temperature higher or lower than 37 °C has a dramatic effect on cell growth rate¹³. Therefore, temperature control of the cell culture environment is required in a live-cell imaging system. It is known that temperature instability will lead to defocusing issues during long-term imaging¹⁴. To achieve a stable 37 °C environment, we built a large enclosure chamber to cover the entire microscope frame, including a thermal insulation layer underneath the microscope (Figure 1). Within the sizeable temperature-control chamber, the small flexible chamber helps to accurately maintain the physiological humidity and pH via the regulated air flow supplemented with 5% CO₂ (Figure 2). The temperature and humidity of the chambers were measured to confirm that the double-chamber design provided the optimal cell culture condition for cell growth under long-term, periodic SRS imaging (Figure 3). We then demonstrated the application of the system for time-lapse imaging and tracking lipid droplets (LDs) in SKOV3 cancer cells (Figure 4, Figure 5, and Figure 6).

Protokół

1. Build the microscope environmental enclosure

NOTE: This large microscope environmental enclosure is used to control the temperature of the microscope body and the imaging environment to be stabilized at 37 °C (Figure 1A).

1. Mark the locations of the feet of the SRS microscope frame and the motorized stage using a marker pen on the optical table. Mount two Iris Diaphragms in front of the Galvanometer scanner of the microscope and adjust to make the pump and Stokes laser beams pass through the center of the Iris Diaphragms.
2. Remove the microscope frame and the stage from the optical table.
3. Lay the silicone rubber sheet (size: 31 x 29 inches, thickness: 1/8 inch) on the optical table (Figure 1B).
4. Cut the silicone rubber along the marks using a knife, remove small rubber pieces, and place the square MICA ceramic sheets (size: 6 x 6 inches, thickness: 1/4 inch) into the same locations.
   NOTE: MICA ceramic is an easy-to-machine material¹⁵. It is as hard as aluminum but is an excellent thermal insulator. MICA ceramic sheets were used to stop heat transfer from the metal microscope frame and stage to the stainless-steel optical table. A few through-holes should be drilled on the MICA sheets to allow the use of ¼-20 screws for mounting the feet of the frame and the stage.
5. Move the microscope frame and the stage back to the optical table and carefully align the feet onto the MICA ceramic sheets along the marker lines. Use ¼-20 screws to mount the frame and the stage on the table.
6. Realign the SRS optical path. Adjust the mirror mounts of Mirror 1 and Mirror 2 to make the laser beam pass through the center of the two premounted Iris Diaphragms (Figure 2).
   NOTE: Technical details of the lab-built SRS microscope used for the current live-cell imaging work are described previously¹⁶. The pulse width of the pump and Stokes beams are ~3-4 ps with glass rod dispersion. The system is controlled using the ScanImage¹⁷ software.
7. On top of this thermal insulation foundation, assemble the environmental enclosure to cover the entire microscope frame using five pieces of large polycarbonate sheets (size: 31 x 29 x 28 inches, thickness: 0.25 inch).
   NOTE: The size of the enclosure box is determined based on the dimensions of the microscope frame and the stage. The front panel of the microscope enclosure box will need to be removed temporarily to install the flexible chamber and load the cell culture dish.
   1. To assemble the enclosure, carry out simple machining work, including cutting, drilling, and tapping screw holes on each edge of the polycarbonate sheets. Cut two large holes with a diameter of 2.6 inches on the right and the left sheets of the enclosure to fit the inlet and outlet tubing, respectively. Cut a small hole with a diameter of 5 mm on the back sheet to allow the laser beams to enter the enclosure.
8. Seal the edges and interfaces of the box using aluminum foil tape.
9. Connect the flexible duct hose to the inlet and the outlet ports of the enclosure box to allow circulated warm airflow pumped and controlled by the heater module. Place the thermal sensor of the heater module in the flexible chamber where the cells are cultured and imaged. Set the targeted temperature at 37 °C.
   ​NOTE: A diffuser may be used to get a more uniform airflow distribution in the environmental enclosure.

2. Assemble the flexible chamber

1. Mount the machined hollow cylindrical aluminum piece 1 (material: aluminum 6061) to the nosepiece of the objective using three set screws (Figure 2).
2. Mount the machined hollow cylindrical aluminum piece 2 to the sample holder using four ¼-20 screws (Figure 2).
   NOTE: The sample holder must be modified to hold the 50 mm cover glass-bottom cell culture dish. Drill a through hole in the center of the sample holder using a 1-7/8 inches hole saw. Counterbore the hole using a 50 mm hole saw and keep the depth of the through hole ~1 mm.
3. Fit the sample holder with the aluminum piece 2 onto the motorized stage and mount it using screws.
4. Place the natural rubber film sleeve (thickness: 0.01 inch; glued with cyanoacrylate adhesive) between the two machined aluminum pieces and mount it using rubber bands at each end.
5. Connect the compressed CO₂ cylinder to the gas mixer module using proper tubing and connectors. Set the CO₂ input pressure at 20-25 psi. Use a built-in CO₂ sensor and a controller to ensure the air mixer module can regulate and mix 5% CO₂ into the airflow. Use inline filters to clean up the airflow.
6. Using proper tubing and connectors, guide the mixed air (with 5% CO₂) to the presterilized water bottle placed on the hot plate, and then guide the humidified air to the flexible chamber. Set the hot plate at 37 °C. Bubble the airflow in the warm water to increase the humidity of the airflow.

3. Preparation for time-lapse live-cell SRS imaging experiments

1. Wipe all parts of the flexible chamber with 70% ethanol, including the nosepiece, the water dipping objective, and the sample holder.
2. Decontaminate the entire enclosure system using a UV lamp placed in the enclosure for 20 to 30 min.
   NOTE: Do not stay in the lab room during the UV disinfection process for safety.
3. Culture SKOV3 cells in a 50-mm glass-bottom Petri dish for 12 h under normal physiological conditions in a regular incubator.
   NOTE: Start the SKOV3 cell culture¹⁸ in a standard biosafety cabinet.
4. Disconnect the machined aluminum piece 2 from the sample holder by removing the screws.
   NOTE: This is the way to open the flexible chamber to load the cell culture dish.
5. Load the cell culture dish. Remember to add immersion oil on the top of the condenser before placing the cell culture dish. Remove the cover of the dish and immobilize the dish using the clamps.
   NOTE: To avoid contamination, all the operations should be performed with gloves.
6. Lower the objective into the cell culture media for coarse focusing. Lower the aluminum piece 2 to enclose the flexible chamber and mount it to the sample holder using screws.
   NOTE: A 2 mm silicone rubber cushion pad is attached to the bottom of the aluminum piece 2 to seal the gap tightly.
7. Set the air supply with 5% CO₂ and 19% O₂ for normal cell culture with an airflow rate of 200 cc/min.
   ​NOTE: A lower airflow rate may be used. It depends on how well the flexible chamber is sealed.

4. Conduct time-lapse live-cell SRS imaging experiments

1. Tune the laser wavelength to 805 nm to target the 2854 cm^-1 Raman shift, which is attributed to the vibration of CH₂ chemical bonds. Use low laser power to reduce photodamage to the cells. To follow this protocol, use ~15 mW average power of the pump laser and ~7.5 mW of the Stokes laser (fixed at 1,045 nm) for long-term live-cell imaging.
   NOTE: Higher laser power will yield better SRS imaging quality. However, too high laser power will induce photodamage to live cells. There is a tradeoff between image quality and photodamage.
2. Adjust and focus the objective to achieve good SRS imaging of the cells using the FOCUS button on the MAIN CONTROLS panel of the software. To perform rapid focusing, set the pixel number to be 512 × 512 pixels with a pixel dwell time of 4.8 µs on the CONFIGURATION panel.
3. Set the lock-in amplifier input range (typically 5 mV) to be twice the maximal signal voltage. Set the low-pass filter to be the same as the pixel dwell time (4.8 µs).
   NOTE: Different lab-built SRS systems may use different data acquisition settings.
4. After achieving good focusing, set the imaging resolution to be 2,048 × 2,048 pixels for a 175 µm² field of view for the acquisition of high-quality images. Check the SAVE function on the MAIN CONTROLS panel and check the SRS channel on the CHANNELS panel. Set the interval time between two frames to be 180 s (3 min) on the MAIN CONTROLS channel. Set the acquisition number to 480 for time-lapse imaging over 24 h.
   NOTE: A lower imaging resolution may be used based on the purpose of the experiments.
5. Start automated imaging acquisition using the LOOP function on the MAIN CONTROLS panel.
   NOTE: Check whether there is focal drift due to temperature instability in the first hour of imaging. The first-hour imaging may not be stable. Check focusing every 2-3 h during the time-lapse imaging session.
6. Process the collected images using ImageJ¹⁹. Use the following two methods for LD quantification: (i) the LD/cell body area ratio, and (ii) the mean SRS intensity of total LDs. Measure the cell body area by thresholding and zeroing the non-cellular pixels in the SRS images at 2,854 cm^-1. Measure the LD area and intensity by thresholding and zeroing the non-LD pixels in the SRS images.
   NOTE: More details about SRS image processing were reported previously¹⁶.

Wyniki

We fabricated and assembled the flexible chamber system for time-lapse SRS imaging (Figure 1 and Figure 2), and then evaluated the performance of the system. The temperature inside the microscope environmental enclosure reached the expected 37 °C within 1 h, which did not significantly affect the room temperature (Figure 3A). The temperature in the flexible chamber reached 37 °C in 1.5 h, and it was stably maintained at 37...

Dyskusje

Time-lapse live-cell SRS microscopy is an alternative imaging technique for molecule tracking in a label-free manner. Compared to fluorescence labeling, SRS imaging is free from photobleaching, enabling long-term monitoring of molecules. However, to date, the live cell imaging system on an upright SRS microscopy is not commercially available. In this work, a live cell imaging system with a stable thermal-insulated microscope enclosure box and a flexible inner soft chamber was developed to enable transmitted SRS time-laps...

Materiały

Name	Company	Catalog Number	Comments
A lab-built SRS microscope			https://rdcu.be/cP6ve
HF2LI 50 MHz lock-in amplifer	Zurich Instruments	HF2LI
Iris diaphragm	Thorlabs Inc	SM1D12
Kinematic mirror mount	Thorlabs Inc	KM100
Microscope frame	Nikon Inc	FN-1
Motorized microscopy stage	Prior Scientific	Z-Deck
Oil-immersion condenser (C-AA Achromat/Aplanat, NA 1.4)	Nikon Inc	MBL71405
Water-immersion objective (CFI75 Apo 25XC W 1300)	Nikon Inc	MRD77225
Materials and parts for the microscope enclosure (31'' x 29'' x 28'' L x W x H)
Airtherm heater module	World Precision Instruments (WPI)	AIRTHERM-SAT-1W
Airtherm heater controller, CO2 and humidity monitor	World Precision Instruments (WPI)	AIRTHERM-SMT-1W
Air/CO2 mixer module	World Precision Instruments (WPI)	ECU-HOC-W
Flexible duct hose (2-1/2'' ID, 2-3/4'' OD)	McMaster-Carr	56675K71
High-temperature glass-mica ceramic, easy-to-machine (6'' x 6'', 1/4'' thickness)	McMaster-Carr	8489K62
Polycarbonate sheets (thickness 0.25'')	McMaster-Carr	8574K286
Silicone rubber sheets (36'' x 36'', thickness 1/8'')	McMaster-Carr	5827T43
Materials and parts for the Flexible chamber
Hot plate	McMaster-Carr	31745K11
High-purity inline filter, 1/4 NPT	McMaster-Carr	6645T18
Hole saw (cutting diameter 1-7/8 inch)	McMaster-Carr	4066A34
Hole saw (cutting diameter 50 mm)	McMaster-Carr	4556A19
High-temperature silicone rubber tubing, soft, 2 mm ID, 5 mm OD	McMaster-Carr	5054K313
Inline filter (1/4 NPT, 40 micron)	McMaster-Carr	98385K843
Multipurpose 6061 Aluminum round tube (1/8'' wall thickness, 4'' OD)	McMaster-Carr	9056K42
Multipurpose 6061 Aluminum round tube (3/4'' wall thickness, 3-3/4'' OD)	McMaster-Carr	9056K47
Multipurpose 6061 Aluminum bar (12'' x 12'', thickness 1/4'')	McMaster-Carr	8975K142
Multipurpose 6061 Aluminum bar (8'' x 8'', thickness 3/8'')	McMaster-Carr	9246K21
Objective nosepiece (single)	Nikon Inc	FN-MN-H
Sample holder (modified)	Prior Scientific	HZ202
Ultra-thin natural rubber film (thickness 0.01'')	McMaster-Carr	8611K13
Vacuum-sealable glass jar	McMaster-Carr	3231T44
Software
MATLAB	MathWorks
ImageJ (Fiji)		imagej.net
ScanImage	Vidrio Technologies, LLC		SRS imaging software
Materials for live-cell imaging
Cover glass bottom sterile culture dishes (Dia.x H, 50 x 7 mm)	Electron Microscopy Sciences (EMS)	70674-02
DMEM cell culture medium	ThermoFisher Scientific	11965092
Fetal bovine serum (FBS)	ThermoFisher Scientific	26140079
LysoSensor fluorescent dye DND-189	ThermoFisher Scientific	L7535 (Invitrogen)
Oleic acid	MilliporeSigma	364525
SKOV3 cell line	ATCC	HTB-77