We want to thank the 2019 Undergraduate Senior Design Team (Suk Chul Yoon, Ian Foxton, Louis Mazza, and James Walsh) at Binghamton University for the design, fabrication, and testing of the microscope enclosure box. We thank Scott Hancock, Olga Petrova, and Fabiola Moreno Olivas at Binghamton University&#160;for helpful discussions. This research was supported by the National Institutes of Health under Award Number R15GM140444.

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 &#176;C (Figure 1A).
<ol>
	<li>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.</li>
	<li>Remove the microscope frame and the stage from the optical table.</li>
	<li>Lay the silicone rubber sheet (size: 31 x 29 inches, thickness: 1/8 inch) on the optical table (Figure 1B).</li>
	<li>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 &#188;-20 screws for mounting the feet of the frame and the stage.</li>
	<li>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 &#188;-20 screws to mount the frame and the stage on the table.</li>
	<li>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.</li>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Seal the edges and interfaces of the box using aluminum foil tape.</li>
	<li>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 &#176;C. 
	&#8203;NOTE: A diffuser may be used to get a more uniform airflow distribution in the environmental enclosure.</li>
</ol>
2. Assemble the flexible chamber
<ol>
	<li>Mount the machined hollow cylindrical aluminum piece 1 (material: aluminum 6061) to the nosepiece of the objective using three set screws (Figure 2).</li>
	<li>Mount the machined hollow cylindrical aluminum piece 2 to the sample holder using four &#188;-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.</li>
	<li>Fit the sample holder with the aluminum piece 2 onto the motorized stage and mount it using screws.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C. Bubble the airflow in the warm water to increase the humidity of the airflow.</li>
</ol>
3. Preparation for time-lapse live-cell SRS imaging experiments
<ol>
	<li>Wipe all parts of the flexible chamber with 70% ethanol, including the nosepiece, the water dipping objective, and the sample holder.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Set the air supply with 5% CO2 and 19% O2 for normal cell culture with an airflow rate of 200 cc/min. 
	&#8203;NOTE: A lower airflow rate may be used. It depends on how well the flexible chamber is sealed.</li>
</ol>
4. Conduct time-lapse live-cell SRS imaging experiments
<ol>
	<li>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.</li>
	<li>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 &#215; 512 pixels with a pixel dwell time of 4.8 &#181;s on the CONFIGURATION panel.</li>
	<li>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 &#181;s). 
	NOTE: Different lab-built SRS systems may use different data acquisition settings.</li>
	<li>After achieving good focusing, set the imaging resolution to be 2,048 &#215; 2,048 pixels for a 175 &#181;m2 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&#160;resolution may be used based on the purpose of the experiments.</li>
	<li>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.</li>
	<li>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.</li>
</ol>

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The authors have no conflicts of interest to disclose.

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...

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 thickness9,10,11,12. 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 &#176;C, while the room temperature is always 10&#176; lower than this. Temperature higher or lower than 37 &#176;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 &#176;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).

<table><tbody><tr><td>&lt;strong&gt;A lab-built SRS microscope&lt;/strong&gt;</td><td></td><td></td><td>https://rdcu.be/cP6ve</td></tr><tr><td>HF2LI 50 MHz lock-in amplifer</td><td>Zurich Instruments</td><td>HF2LI</td><td></td></tr><tr><td>Iris diaphragm</td><td>Thorlabs Inc</td><td>SM1D12</td><td></td></tr><tr><td>Kinematic mirror mount</td><td>Thorlabs Inc</td><td>KM100</td><td></td></tr><tr><td>Microscope frame</td><td>Nikon Inc</td><td>FN-1</td><td></td></tr><tr><td>Motorized microscopy stage</td><td>Prior Scientific</td><td>Z-Deck</td><td></td></tr><tr><td>Oil-immersion condenser (C-AA Achromat/Aplanat, NA 1.4)</td><td>Nikon Inc</td><td>MBL71405</td><td></td></tr><tr><td>Water-immersion objective (CFI75 Apo 25XC W 1300)</td><td>Nikon Inc</td><td>MRD77225</td><td></td></tr><tr><td>&lt;strong&gt;Materials and parts for the microscope enclosure (31&#039;&#039; x 29&#039;&#039; x 28&#039;&#039; L x W x H)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Airtherm heater module</td><td>World Precision Instruments (WPI)</td><td>AIRTHERM-SAT-1W</td><td></td></tr><tr><td>Airtherm heater controller, CO&lt;sub&gt;2&lt;/sub&gt; and humidity monitor</td><td>World Precision Instruments (WPI)</td><td>AIRTHERM-SMT-1W</td><td></td></tr><tr><td>Air/CO&lt;sub&gt;2&lt;/sub&gt; mixer module</td><td>World Precision Instruments (WPI)</td><td>ECU-HOC-W</td><td></td></tr><tr><td>Flexible duct hose (2-1/2&#039;&#039; ID, 2-3/4&#039;&#039; OD)</td><td>McMaster-Carr</td><td>56675K71</td><td></td></tr><tr><td>High-temperature glass-mica ceramic, easy-to-machine (6&#039;&#039; x 6&#039;&#039;, 1/4&#039;&#039; thickness)</td><td>McMaster-Carr</td><td>8489K62</td><td></td></tr><tr><td>Polycarbonate sheets (thickness 0.25&#039;&#039;)</td><td>McMaster-Carr</td><td>8574K286</td><td></td></tr><tr><td>Silicone rubber sheets (36&#039;&#039; x 36&#039;&#039;, thickness 1/8&#039;&#039;)</td><td>McMaster-Carr</td><td>5827T43</td><td></td></tr><tr><td>&lt;strong&gt;Materials and parts for the Flexible chamber&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hot plate</td><td>McMaster-Carr</td><td>31745K11</td><td></td></tr><tr><td>High-purity inline filter, 1/4 NPT</td><td>McMaster-Carr</td><td>6645T18</td><td></td></tr><tr><td>Hole saw (cutting diameter 1-7/8 inch)</td><td>McMaster-Carr</td><td>4066A34</td><td></td></tr><tr><td>Hole saw (cutting diameter 50 mm)</td><td>McMaster-Carr</td><td>4556A19</td><td></td></tr><tr><td>High-temperature silicone rubber tubing, soft, 2 mm ID, 5 mm OD</td><td>McMaster-Carr</td><td>5054K313</td><td></td></tr><tr><td>Inline filter (1/4 NPT, 40 micron)</td><td>McMaster-Carr</td><td>98385K843</td><td></td></tr><tr><td>Multipurpose 6061 Aluminum round tube (1/8&#039;&#039; wall thickness, 4&#039;&#039; OD)</td><td>McMaster-Carr</td><td>9056K42</td><td></td></tr><tr><td>Multipurpose 6061 Aluminum round tube (3/4&#039;&#039; wall thickness, 3-3/4&#039;&#039; OD)</td><td>McMaster-Carr</td><td>9056K47</td><td></td></tr><tr><td>Multipurpose 6061 Aluminum bar (12&#039;&#039; x 12&#039;&#039;, thickness 1/4&#039;&#039;)</td><td>McMaster-Carr</td><td>8975K142</td><td></td></tr><tr><td>Multipurpose 6061 Aluminum bar (8&#039;&#039; x 8&#039;&#039;, thickness 3/8&#039;&#039;)</td><td>McMaster-Carr</td><td>9246K21</td><td></td></tr><tr><td>Objective nosepiece (single)</td><td>Nikon Inc</td><td>FN-MN-H</td><td></td></tr><tr><td>Sample holder (modified)</td><td>Prior Scientific</td><td>HZ202</td><td></td></tr><tr><td>Ultra-thin natural rubber film (thickness 0.01&#039;&#039;)</td><td>McMaster-Carr</td><td>8611K13</td><td></td></tr><tr><td>Vacuum-sealable glass jar</td><td>McMaster-Carr</td><td>3231T44</td><td></td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MATLAB</td><td>MathWorks</td><td></td><td></td></tr><tr><td>ImageJ (Fiji)</td><td></td><td>imagej.net</td><td></td></tr><tr><td>ScanImage</td><td>Vidrio Technologies, LLC</td><td></td><td>SRS imaging software</td></tr><tr><td>&lt;strong&gt;Materials for live-cell imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Cover glass bottom sterile culture dishes (Dia.x H, 50 x 7 mm)</td><td>Electron Microscopy Sciences (EMS)</td><td>70674-02</td><td></td></tr><tr><td>DMEM cell culture medium</td><td>ThermoFisher Scientific</td><td>11965092</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>ThermoFisher Scientific</td><td>26140079</td><td></td></tr><tr><td>LysoSensor fluorescent dye DND-189</td><td>ThermoFisher Scientific</td><td>L7535 (Invitrogen)</td><td></td></tr><tr><td>Oleic acid</td><td>MilliporeSigma</td><td>364525</td><td></td></tr><tr><td>SKOV3 cell line</td><td>ATCC</td><td>HTB-77</td><td></td></tr></tbody></table>

a flexible chamber for time-lapse live-cell imaging with stimulated raman scattering microscopy

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.

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 &#176;C within 1 h, which did not significantly affect the room temperature (Figure 3A). The temperature in the flexible chamber reached 37 &#176;C in 1.5 h, and it was stably maintained at 37...

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

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.

Stimulated Raman scattering (SRS) microscopy is a label-free chemical imaging technology. Live-cell imaging with SRS has been demonstrated for many ...

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Research

JoVE Journal

Bioengineering

1.2K Views.  State University of New York. 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.

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

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. SRS imaging modality can be obtained using commercial laser-scanning microscopes by equipping with a non-descanned forward detector with proper bandpass filters and lock-in amplifier (LIA) detection scheme. A schematic layout of a typical SRS microscope includes the following: two pulsed laser beams, (i.e., the pump and probe directed in a scanning microscope), which must be overlapped in both space and time at the image plane, then focused by a microscope objective into the sample through two scanning mirrors (SMs), which raster the focal spot across an x-y plane. After interaction with the sample, transmitted output pulses are collected by an upper objective and measured by a forward detection system inserted in an inverted microscope. Pump pulses are removed by a stack of optical filters, whereas the probe pulses that are the result of the SRS process occurring in the focal volume of the specimen are measured by a photodiode (PD). The readout of the PD is demodulated by the LIA to extract the modulation depth. A two-dimensional (2D) image is obtained by synchronizing the forward detection unit with the microscope scanning unit. In this paper, the implementation of an SRS microscope is described and successfully demonstrated, as well as the reporting of label-free images of polystyrene beads with diameters of 3 &#181;m. It is worth noting that SRS microscopes are not commercially available, so in order to take advantage of these characteristics, the homemade construction is the only option. Since SRS microscopy is becoming popular in many fields, it is believed that this careful description of the SRS microscope implementation can be very useful for the scientific community.

In this manuscript, the implementation of a stimulated Raman scattering (SRS) microscope, obtained by the integration of an SRS experimental set-up with a laser scanning microscope, is described. The SRS microscope is based on two femtosecond (fs) laser sources, a Ti-Sapphire (Ti:Sa) and synchronized optical parametric oscillator (OPO).

Stimulated Raman scattering (SRS) microscopy uses near-infrared excitation light; therefore, it shares many multi-photon microscopic imaging properties. ...

Engineering

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular level in the chemical and biomedical sciences. Although commercial SIM systems are available, systems that are custom designed in the laboratory can outperform commercial systems, the latter typically designed for ease of use and general purpose applications, both in terms of imaging fidelity and speed. This article presents an in-depth guide to building a SIM system that uses total internal reflection (TIR) illumination and is capable of imaging at up to 10 Hz in three colors at a resolution reaching 100 nm. Due to the combination of SIM and TIRF, the system provides better image contrast than rival technologies. To achieve these specifications, several optical elements are used to enable automated control over the polarization state and spatial structure of the illumination light for all available excitation wavelengths. Full details on hardware implementation and control are given to achieve synchronization between excitation light pattern generation, wavelength, polarization state, and camera control with an emphasis on achieving maximum acquisition frame rate. A step-by-step protocol for system alignment and calibration is presented and the achievable resolution improvement is validated on ideal test samples. The capability for video-rate super-resolution imaging is demonstrated with living cells.

This article provides an in depth guide for the assembly and operation of a structured illumination microscope operating with total internal reflection fluorescence illumination (TIRF-SIM) to image dynamic biological processes with optical super-resolution in multiple colors.

Optical super-resolution imaging with structured illumination microscopy (SIM) is a key technology for the visualization of processes at the molecular ...

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps at high speed, and high spatial resolution of thin samples by directly interrogating their molecular vibrations. In its standard implementation, SRS microscopy is narrowband and forms images with only a single vibrational frequency at a time. However, this approach not only hinders the chemical specificity of SRS but also neglects the wealth of information encoded within vibrational spectra. 
These limitations can be overcome by broadband SRS, an implementation capable of extracting a vibrational spectrum per pixel of the image in parallel. This delivers hyperspectral data that, when coupled with chemometric analysis, maximizes the amount of information retrieved from the specimen. Thus, broadband SRS improves the chemical specificity of the system, allowing the quantitative determination of the concentration of the different constituents of a sample. Here, we report a protocol for chemical imaging with broadband SRS microscopy, based on a home-built SRS microscope operating with a custom differential multichannel-lock-in amplifier detection. It discusses the sample preparation, alignment of the SRS apparatus, and chemometric analysis. By acquiring vibrational Raman spectra, the protocol illustrates how to identify different chemical species within a mixture, determining their relative concentrations.

We present a protocol to acquire chemical images with broadband stimulated Raman scattering (SRS) microscopy. Based on an SRS microscope that operates with differential multichannel-lock-in detection, the protocol describes the sample preparation, adjustment of the SRS apparatus, and chemometrics to disentangle different constituents of chemically heterogeneous samples.

Stimulated Raman scattering (SRS) microscopy is a nonlinear optical technique for label-free chemical imaging. This analytical tool delivers chemical maps ...

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to conventional epi-fluorescence microscopy, various imaging techniques have been developed to achieve specific experimental goals. Some of the widely used techniques include single-molecule fluorescence resonance energy transfer (smFRET), which can report conformational changes and molecular interactions with angstrom resolution, and single-molecule detection-based super-resolution (SR) imaging, which can enhance the spatial resolution approximately ten&#160;to twentyfold compared to diffraction-limited microscopy. Here we present a customer-designed integrated system, which merges multiple imaging methods in one microscope, including conventional epi-fluorescent imaging, single-molecule detection-based SR imaging, and multi-color single-molecule detection, including smFRET imaging. Different imaging methods can be achieved easily and reproducibly by switching optical elements. This set-up is easy to adopt by any research laboratory in biological sciences with a need for routine and diverse imaging experiments at a reduced cost and space relative to building separate microscopes for individual purposes.

Here we present a practical guide of building an integrated microscopy system, which merges conventional epi-fluorescent imaging, single-molecule detection-based super-resolution imaging, and multi-color single-molecule detection, including single-molecule fluorescence resonance energy transfer imaging, into one set-up in a cost-efficient way.

Fluorescence microscopy is a powerful tool to detect biological molecules in situ and monitor their dynamics and interactions in real-time. In addition to ...

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to visualize these highly dynamic events, two complementary light microscopy techniques that allow fast imaging of live samples were combined: spinning disk microscopy (SD) for fast and high-resolution volume recording and total internal reflection fluorescence (TIRF) microscopy for precise localization and visualization of the plasma membrane. A comprehensive and complete imaging protocol will be shown for guiding through sample preparation, microscope calibration, image formation and acquisition, resulting in multi-color SD-TIRF live imaging series with high spatio-temporal resolution. All necessary image post-processing steps to generate multi-dimensional live imaging datasets, i.e. registration and combination of the individual channels, are provided in a self-written macro for the open source software ImageJ. The imaging of fluorescent proteins during initiation and maturation of adhesion complexes, as well as the formation of the actin cytoskeletal network, was used as a proof of principle for this novel approach. The combination of high resolution 3D microscopy and TIRF provided a detailed description of these complex processes within the cellular environment and, at the same time, precise localization of the membrane-associated molecules detected with a high signal-to-background ratio.

An advanced microscope that permit fast and high-resolution imaging of both, the isolated plasma membrane and the surrounding intracellular volume, will be presented. The integration of spinning disk and total internal reflection fluorescence microscopy in one setup allows live imaging experiments at high acquisition rates up to 3.5 s per image stack.

In living cells, processes such as adhesion formation involve extensive structural changes in the plasma membrane and the cell interior. In order to ...

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze membrane fusion within eukaryotic cells. The SNARE protein family consists of about 36 different members. Specific intracellular transport routes are catalyzed by specific sets of 3 or 4 SNARE proteins that thereby contribute to the specificity and fidelity of membrane trafficking. However, studying the precise function of SNARE proteins is technically challenging, because SNAREs are highly abundant and functionally redundant, with most SNAREs having multiple and overlapping functions. In this protocol, a new method for the visualization of SNARE complex formation in live cells is described. This method is based on expressing SNARE proteins C-terminally fused to fluorescent proteins and measuring their interaction by F&#246;rster resonance energy transfer (FRET) employing fluorescence lifetime imaging microscopy (FLIM). By fitting the fluorescence lifetime histograms with a multicomponent decay model, FRET-FLIM allows (semi-)quantitative estimation of the fraction of the SNARE complex formation at different vesicles. This protocol has been successfully applied to visualize SNARE complex formation at the plasma membrane and at endosomal compartments in mammalian cell lines and primary immune cells, and can be readily extended to study SNARE functions at other organelles in animal, plant, and fungal cells.

This protocol describes a new method allowing for the quantitative visualization of complex formation of SNARE proteins, based on F&#246;rster resonance energy transfer, and fluorescence lifetime imaging microscopy.

Soluble N-ethylmaleimide sensitive fusion protein (NSF) attachment protein receptor (SNARE) proteins are key for membrane trafficking, as they catalyze ...

Immunology and Infection

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic molecular vibration, thus providing a perfect tool for in vivo study of biological processes at subcellular resolution. By integrating two-photon excited fluorescence (TPEF) imaging into the SRS microscope, the dual-modal in vivo imaging of tissues can acquire critical biochemical and biophysical information from multiple perspectives which helps understand the dynamic processes involved in cellular metabolism, immune response and tissue remodeling, etc. In this video protocol, the setup of a TPEF-SRS microscope system as well as the in vivo imaging method of the animal spinal cord is introduced. The spinal cord, as part of the central nervous system, plays a critical role in the communication between the brain and peripheral nervous system. Myelin sheath, abundant in phospholipids, surrounds and insulates the axon to permit saltatory conduction of action potentials. In vivo imaging of myelin sheaths in the spinal cord is important to study the progression of neurodegenerative diseases and spinal cord injury. The protocol also describes animal preparation and in vivo TPEF-SRS imaging methods to acquire high-resolution biological images.

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Stimulated Raman scattering (SRS) microscopy allows label-free imaging of biomolecules based on their intrinsic vibration of specific chemical bonds. In this protocol, the instrumental setup of an integrated SRS and two-photon fluorescence microscope is described to visualize cellular structures in the spinal cord of live mice.

Stimulated Raman scattering (SRS) microscopy enables label-free imaging of the biological tissues in its natural microenvironment based on intrinsic ...

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has been shown to be able to provide hematoxylin and eosin (H&amp;E)-equivalent images that allow fast and reliable diagnosis of brain cancer. Such capability has enabled exciting intraoperative cancer diagnosis applications. Two-color SRS imaging of tissue can be done with either a picosecond or femtosecond laser source. Femtosecond lasers have the advantage of enabling flexible imaging modes, including fast hyperspectral imaging and real-time, two-color SRS imaging. A spectral-focusing approach with chirped laser pulses is typically used with femtosecond lasers to achieve high spectral resolution. 
Two-color SRS acquisition can be realized with orthogonal modulation and lock-in detection. The complexity of pulse chirping, modulation, and characterization is a bottleneck for the widespread adoption of this method. This article provides a detailed protocol to demonstrate the implementation and optimization of spectral-focusing SRS and real-time, two-color imaging of mouse brain tissue in the epi-mode. This protocol can be used for a broad range of SRS imaging applications that leverage the high speed and spectroscopic imaging capability of SRS.

Stimulated Raman scattering (SRS) microscopy is a powerful, nondestructive, and label-free imaging technique. One emerging application is stimulated Raman histology, where two-color SRS imaging at the protein and lipid Raman transitions are used to generate pseudo-hematoxylin and eosin images. Here, we demonstrate a protocol for real-time, two-color SRS imaging for tissue diagnosis.

Stimulated Raman scattering (SRS) microscopy has emerged as a powerful optical imaging technique for tissue diagnosis. In recent years, two-color SRS has ...

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating nonlinear optical signals such as stimulated Raman scattering (SRS), two-photon excited fluorescence (TPEF), and transient absorption (TA) into an imaging platform, it can be used to reveal biomolecular contrast of cellular structures and AuNPs in a multimodal manner. This article presents a multimodal nonlinear optical microscopy and applies it to perform chemically specific imaging of AuNPs in cancer cells. This imaging platform provides a novel approach for developing more efficient functionalized AuNPs and determining whether they are within vasculatures surrounding the tumor, pericellular, or cellular spaces.

This article presents the integration of a spectral-focusing module and a dual-output pulse laser, enabling rapid hyperspectral imaging of gold nanoparticles and cancer cells. This work aims to demonstrate the details of multimodal nonlinear optical techniques on a standard laser scanning microscope.

Probing gold nanoparticles (AuNPs) in living systems is essential to reveal the interaction between AuNPs and biological tissues. Moreover, by integrating ...

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

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Rozdziały w tym wideo

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Implementation of a Nonlinear Microscope Based on Stimulated Raman Scattering

A Guide to Structured Illumination TIRF Microscopy at High Speed with Multiple Colors

Multiplex Chemical Imaging Based on Broadband Stimulated Raman Scattering Microscopy

Conducting Multiple Imaging Modes with One Fluorescence Microscope

Visualizing Adhesion Formation in Cells by Means of Advanced Spinning Disk-Total Internal Reflection Fluorescence Microscopy

Visualizing Intracellular SNARE Trafficking by Fluorescence Lifetime Imaging Microscopy

In vivo Imaging of Biological Tissues with Combined Two-Photon Fluorescence and Stimulated Raman Scattering Microscopy

Real-Time, Two-Color Stimulated Raman Scattering Imaging of Mouse Brain for Tissue Diagnosis

Biomolecular Imaging of Cellular Uptake of Nanoparticles using Multimodal Nonlinear Optical Microscopy