Protocol to image deuterated propofol in living rat neurons using multimodal stimulated Raman scattering microscopy

Summary

Propofol is a widely used anesthetic important in clinics, but like many other bioactive molecules, it is too small to be tagged and visualized by fluorescent dyes. Here, we present a protocol to visualize deuterated propofol in living rat neurons using stimulated Raman scattering (SRS) microscopy with carbon-deuterium bonds serving as a Raman tag. We describe the preparation and culture of rat neurons, followed by optimization of the SRS system. We then detail neuron loading and real-time imaging of anesthesia dynamics.

For complete details on the use and execution of this protocol, please refer to Oda et al.¹

Graphical abstract

Highlights

• •Protocol for real-time cellular imaging using multimodal SRS microscopy
• •High-resolution imaging of deuterated propofol in living rat neurons
• •Real-time monitoring of anesthesia dynamics in living neurons
• •Has potential to be applied to other small-sized drugs

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Propofol is a widely used anesthetic important in clinics, but like many other bioactive molecules, it is too small to be tagged and visualized by fluorescent dyes. Here, we present a protocol to visualize deuterated propofol in living rat neurons using stimulated Raman scattering (SRS) microscopy with carbon-deuterium bonds serving as a Raman-tag. We describe the preparation and culture of rat neurons, followed by optimization of the SRS system. We then detail neuron loading and real-time imaging of anesthesia dynamics.

Before you begin

Background

Dynamics of bioactive molecules is a key determinant of their biological activities. For drugs, their pharmacological actions are determined by their distribution inside cells and tissues. Therefore, imaging the localization and dynamics of drugs of interests is a crucial step in understanding the mechanisms of their pharmacological actions. However, this is not straightforward since a large number of drugs have a molecular weight smaller than 500 Da.² Commonly used fluorescent tags themselves have molecular weights of 500 Da or more; conjugation of these fluorescent tags can severely affect the original properties of drugs due to their size. This difficulty in tagging and imaging have made many drugs “invisible” with conventional imaging strategies.

Recent progress in Raman scattering imaging opens new possibilities to overcome this limitation. The first technological breakthrough was the development of coherent Raman scattering microscopy, including stimulated Raman scattering (SRS) and coherent anti-Stokes Raman scattering (CARS).^{3,4,5,6,7,8,9} These microscopy techniques dramatically increased the sensitivity of Raman imaging which was the key limitation of traditional spontaneous Raman scattering microscopy. The second breakthrough was the invention of small-sized and bioorthogonal “Raman-tags” that emit specific Raman signals that do not exist in biological samples in the so called “silent region” of Raman spectrum where signals from biological materials are very low.^10,11 The combination of these two inventions have made it possible to image small bioactive molecules that could not have been visualized otherwise.¹² Although it has not been actively applied to pharmacological research yet, this strategy has great potential in visualizing fluorescence-incompatible small-sized drugs, and thereby allowing for detailed characterizations of the spatiotemporal dynamics of those drugs at cellular and/or subcellular resolution.¹³

Here in this protocol, we describe steps to perform bioorthogonal Raman imaging of a Raman-tagged general anesthetic, propofol, using SRS microscopy in living neurons.¹ After optimizing the laboratory-built SRS microscope system using Raman-tagged standard samples, living neurons incubated with deuterated propofol are exposed to laser lights. Aided by simultaneously acquired two-photon excitation fluorescence signals, the distribution and dynamics of deuterated propofol are characterized at subcellular resolution. This imaging method is non-invasive, highly sensitive, and possesses high molecular specificity and therefore should find broad applications in live-cell imaging.

Preparation of stock solutions

Timing: 1 h

• 1.
  100 mM propofol-d17 in dimethylsulfoxide (DMSO)

  • a.
    Prepare 100 mM stock solution of deuterated propofol by dissolving 5 mg of a deuterated propofol in 256 μL of DMSO.
  • b.
    For preservation, the stock solution should be aliquoted and stored at −80°C up to one year.

Note: Propofol-d17 is actually visualized at a concentration of 100 μM, diluted with HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid) ACSF (artificial cerebral spinal fluid) before each use. The volume of propofol-d17 solution depends on the size and number of dishes used in the observation. When using a 35 mm dish with 3 mL solution, 3 μL of 100 mM of propofol-d17 solution is needed each time.

• 2.
  100 mM propofol in DMSO

  • a.
    Prepare 100 mM Stock solution of normal propofol by diluting 5.4M original propofol stock solution with DMSO by 54 times.
  • b.
    For preservation, the stock solution should be aliquoted and stored at −80°C up to one year.

Note: The amount of propofol solution may be the same as that of propofol-d17 solution.

• 3.
  Stock of calcein AM (acetoxymethyl ester)

  • a.
    Before opening, allow the vial to come to room temperature (20°C–25˚C).
  • b.
    Dissolve 1 mg dye in 201 μL high-quality, anhydrous DMSO. This makes a final stock solution concentration 5 mM.
  • c.
    For preservation, the stock solution should be aliquoted and stored at −80°C up to one year.

CRITICAL: Calcein-AM should be dissolved in high quality DMSO. Once dissolved, it should be stored at −20°C or −80°C and used as soon as possible while avoiding multiple freeze and thaw cycles.

Preparation of SRS standard sample with alkyne estrogen

Timing: 1 h (for steps 4 to 8)

Alkyne tagged estrogen (17α-ethinylestradiol) on a slide glass was used as an SRS standard sample because of its strong Raman signal at ∼2,100 cm⁻¹ which is similar to other deuterated compounds.¹⁴ In addition, to easily identify the focal plane, fluorescent beads with a wide range of fluorescence signals (TetraSpeck) are included in this standard sample.

• 4.Apply 2.5 μL of 4.0 μm-diameter TetraSpeck bead suspension and suitable amounts (∼ 1 mg) of 17α-ethinylestradiol powder to the surface of a clean glass microscope slide (Matsunami, 0.9–1.2 mm thickness) and spread with a pipette tip.

Note: 17α-ethinylestradiol is not soluble in aqueous solution and therefore will remain as a solid; this works well as a standard sample similar to fluorescent beads (see below). Before placing on coverglass, tap the tube of TetraSpeck to ensure that beads are uniformly suspended.

• 5.Apply a drop of ProLong antifade reagent to the mixture as a mounting solution.
• 6.Mount the specimens with a coverslip (Matsunami, 0.13–0.17 mm thickness).
• 7.Incubate overnight (12–24 h) at 37°C.
• 8.Following the curing time, seal the edges of the coverslip with nail polish and stored at room temperature.

Note: Sealing the edges slows oxidation and extends the sample's life by several months. We keep the sample slides in dark as a precaution to avoid potential bleaching.

Preparation of primary cultured neurons for live imaging

Timing: 2 weeks

• 9.
  Coating and preparation of media

  • a.
    Coat collagen-coated glass-coverslips (12 mm, IWAKI) in 35 mm cell culture dishes each containing four coverslips (total 32 coverslips in 8 dishes) with 30 μg/mL poly-D-lysine solution in 100 mM borate buffer overnight in a moisture cell culture incubator set to 37˚C and 5% CO₂.
  • b.
    Prepare primary neuron growth media (PNGM, Lonza) according to the manufacturer’s instructions as follows:

    • i.
      1 x PNBM Basal Medium (CC-3256), 200 mL.
    • ii.
      1 x PNGM SingleQuots Growth Supplement Pack (CC-4462).
    • iii.
      Keep PNGM media at 4°C up to 1 month.
• 10.
  Plating neuron on coverslip

  • a.
    Before starting neuron seeding, remove poly-D-lysine solution from coverslip-containing dishes and replace with 1.5 mL of PNGM.
  • b.
    Gently press coverslips down onto the bottom of the cell culture dish with a pipet to prevent them from moving.
  • c.
    Place these dishes back into the incubator while preparing for neurons.
  • d.
    Primary neurons (Lonza R-Hi-501) are stored in liquid nitrogen for long term storage. Retrieve one tube of frozen neuron stock and thaw for 90 s in a 37°C water bath.
  • e.
    After thawing, carefully transfer neurons into a new sterile 50 mL conical tube.
  • f.
    Carefully resuspend cells by adding 16 mL of PNGM drop by drop.

    CRITICAL: This process should be very slow and careful to prevent osmotic shock of neurons.

  • g.
    While pipetting, gently shake the 50 mL conical tube to mix cells in media.
  • h.
    After pipetting all media, retrieve dishes with coverslips from the incubator and remove PNGM from the dishes.
  • i.
    Add 2 mL of cell suspension to each dish and gently shake the cell culture dish to distribute cells evenly.
  • j.
    Place cells in the incubator at 37˚C and 5% CO₂ and let cells settle for 4 h.
  • k.
    After 4 h, confirm that cells have adhered to the coverslips with a microscope.
  • l.
    Gently remove 1.5 mL of media and add 2 mL of fresh PNGM and return cells to the incubator.
• 11.
  Maintenance

  • a.
    Replace PNGM every 2–3 days by removing 0.75 mL and adding 1 mL of fresh media.

Note: Neurons will be ready for imaging after a minimum of 7 days of culture, with optimal use after two weeks, and can be used for up to one month (Figure 1).

Figure 1.

Phase contrast images of primary cultured hippocampal neurons at 1 and 10 days in vitro

Neurites extending from somata can be readily observed.

Optimization of SRS microscope system

Timing: 30 min

• 12.
  Initial SRS System Setup

  Note: SRS imaging is performed using an Emerald Engine laser source and OPO (Optical Parametric Oscillator) (APE, Angewandte Physik & Elektronik GmbH) (Figure 2). The OPO is fed with a 516 nm second harmonic light generated from a 1032 nm fundamental laser. The fundamental laser output and OPO output were used as Stokes and pump pulses, respectively. Stokes pulses are intensity-modulated with an electro-optic modulator, and then combined with pump pulses. Then the pulses are sent to a custom-made laser-scanning microscope. Further details can be found in ref.¹

  Note: Pay special attention when using high power near infrared lasers. Use appropriate eye protection (goggles) when working with exposed light sources and avoid wearing reflective surfaces.

  • a.
    Adjust the wavelength of the pump laser using the OPO to set the desired wavelength for imaging (with Stokes laser at 1,031.7 nm, wavelengths of pump laser to visualize 2,055 cm⁻¹ and 2,100 cm⁻¹ are 851.2 nm and 848.0 nm, respectively).
  • b.
    Set the power of pump and Stokes pulses to ∼200 mW after shutter.

    Note: The power loss in the optical path varies system to system and should be checked for each microscope; it is reduced to 50% of these values when measured at the objective lens in our system (i.e., ∼100 mW at the sample). Also note that the power should be optimized for each sample preparation to maximize the signal intensity while avoiding photobleaching of fluorophores and phototoxicity.

  • c.
    Align pump and Stokes lasers carefully to ensure spatial synchronization.
  • d.
    Adjust pulses in time through a delay line to maximize temporal overlap.
• 13.
  Image calibration

  • a.
    Use alkyne-estrogen having a Raman signal at ∼2,100 cm⁻¹ as a standard sample.

Note: Inclusion of fluorescent beads allow for easy identification of the focal plane, making it simple to confirm the Raman signal's position (Figure 3).

• 14.
  Adjustment settings for propofol-d17

  • a.
    Change the wavelength of the pump laser to 851.2 nm to visualize Raman signals at 2,055 cm⁻¹.

Optional: As an option in setting up the SRS system, signals of deuterated palmitic acid inside cells can be used as a cell-based positive control. For this purpose, incubate cells overnight in palmitic acid-d31 which is diluted with cell culture medium to a concentration of 10 μM.

• 15.
  Setting up the perfusion system (Figure 4)

  • a.
    Connect two lines of tubing containing both an inlet and an outlet line from the peristaltic pump to the imaging chamber.
  • b.
    Adjust the motor speed and ensure there is sufficient starting liquid in both the tube and the imaging dish before the experiment begins.
  • c.
    Set the system for 1000-frame (1.07 frames/second) acquisition of HEPES ACSF solution with 100 μM of propofol-d17.

Note: Motor speed depends on fluid flow rate in washout experiments. To assess the speed of solution exchange, influx of 1 μM Alexa 594 in HEPES ACSF may be monitored.

Figure 2.

Custom-made SRS system

Optical components were aligned to merge pump laser emitted from OPO (blue box on top) and Stokes laser from Emerald Engine (under black sheet on top right). The combined lasers are introduced to the scanner (red box at bottom right) for laser scanning.

Figure 3.

Representative images of the standard sample

SRS signal of alkyne-estrogen (A) and two-photon excited fluorescence signal of TetraSpeck beads (B) can be acquired simultaneously from the same field of view. Scale bars represent 100 μm.

Figure 4.

Setup of perfusion system

(A) Full view of perfusion system.

(B) Close up view of the objective lens and dish.

(C) A multi-channel peristaltic pump connected with the imaging chamber.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Biological samples
Rat hippocampal neurons	Lonza	R-Hi-501
Chemicals, peptides, and recombinant proteins
Calcein AM	Thermo Fisher	C1430
Alexa Fluor 594	Thermo Fisher	A10438
TetraSpeck Microspheres, 4.0 μm	Thermo Fisher	T7283
ProLong Glass Antifade Mountant	Thermo Fisher	P36980
Propofol (2,6-diisopropylphenol)-d17	Toronto Research Chemicals	P829752
Dimethyl sulfoxide	SIGMA	67-68-5
17α-Ethinylestradiol	Sigma-Aldrich	E4876
Propofol (2,6-diisopropylphenol)	Wako	321-98212
PNBM Primary Neuron Basal Medium	Lonza	CC-3256
PNGM Primary Neuron Growth Medium SingleQuots Supplements and Growth Factors	Lonza	CC-4462
Palmitic acid-d31	Toronto Research Chemicals	P144501
Poly-D-lysine hydrobromide	Sigma-Aldrich	P7886
Boric acid	Sigma-Aldrich	B0252
Di-sodium tetraborate decahydrate	Sigma-Aldrich	B9876
Dextrose	Wako	049-31165
HEPES	Nacalai Tesque	17514-15
Software and algorithms
ImageJ (Fiji)	Schindelin et al.15	https://imagej.net/Fiji
ScanImage	Pologruto et al.16	http://scanimage.vidriotechnologies.com/
MATLAB	Mathworks	R2021b
Other
Emerald Engine Yb-fiber-based Pump Laser	Angewandte Physik & Elektronik GmbH	N/A
Levante IR ps Optical Parametric Oscillator	Angewandte Physik & Elektronik GmbH	N/A
Custom-made laser-scanning microscope	Oda et al.1	N/A
Rainin Dynamax RP-1 Peristaltic Pump	Rainin	RD-RP1
COLI-coated cover glass (12 mm)	IWAKI	N/A
Micro slide glass	Matsunami	MAS-01
Micro cover glass	Matsunami	C015001

Materials and equipment

PNGM media

Reagent	Final concentration	Amount
PNBM Primary Neuron Basal Medium	1×	200 mL
NSF-1	1×	4 mL
L-Glutamine	1×	2 mL
GA (gentamicin)	1X	0.2 mL

Store in dark at 4°C for up to one month.

CRITICAL: Because NSF-1 degrades rapidly, it is strongly advised that it be aliquoted in 500 μL units (amount per 25 ml of culture media), frozen, and then added to the media as needed immediately before use.

HEPES ACSF Buffer

Reagent	Final concentration (mM)	Amount
Sodium Chloride	150	8.77 g
Potassium Chloride	3	0.22 g
Dextrose	10	1.80 g
HEPES	10	2.38 g
H2O	N/A	To 1L
Total	N/A	1L

Add final 1 mM Mg²⁺ and 3 mM Ca²⁺ and adjust the pH to 7.3 and the osmolarity to 305–310 mOsm.

The solution should be stored at 4°C and can be used for one year. Because neurons are particularly sensitive to temperature, HEPES ACSF is heated in a 37°C water bath before each usage.

Borate Buffer

Reagent	Final concentration (mM)	Amount
Boric Acid	50.1	3.1 g
di-Sodium tetraborate decahydrate	12.5	4.75 g
H2O	N/A	To 1L
Total	N/A	1L

Borate buffer solution should be sterile filtered before use and can be stored at room temperature for up to one year.

SRS microscope system

The SRS microscope consists of a pump and a Stokes picosecond laser, custom-made microscope and detectors. For full details of the system, please see Oda et al.¹

Computer software

MATLAB ScanImage (formerly by Vidrio Technologies), an open-source software application is applied as laser scanning microscopy controller. Of note, ScanImage—which is designed in MATLAB—is entirely scriptable and customizable.

Step-by-step method details

Loading neurons

Timing: 45 min

Optional: For better identification of cells, neurons may be loaded with fluorescent dyes. Calcein AM was chosen in this protocol to identify the cells' cytoplasm and thereby their morphologies.

• 1.
  Staining neurons with fluorescent dyes

  • a.
    Dilute 5 mM calcein AM stock to a final working concentration of 100 nM in HEPES ACSF.
  • b.
    Load cells with 100 nM calcein AM in HEPES ACSF for 30 min at 37°C in 5% CO₂ incubator.
  • c.
    After loading, wash cells in fresh HEPES ACSF and transfer to 35 mm glass base dish (IWAKI) with 3 mL of HEPES ACSF.

CRITICAL: Calcein AM is sensitive to hydrolysis. Calcein AM-containing aqueous working solutions should be made shortly before use and used within one day.

CRITICAL: The concentration of calcein AM and the incubation time are strictly controlled since relatively high concentrations of calcein AM can be toxic to cells, especially neurons; low concentrations of calcein AM can also lead to difficulties in observation.

• 2.
  Check cells with conventional microscope

  • a.
    After staining is complete, use fluorescence microscope to assess staining of neurons.

Note: Wait 15–60 minutes to allow for AM ester removal if fluorescence color presentation is insufficient for cell identification.

Observation of propofol in living neurons

Timing: 1 h

• 3.
  Detection of propofol-d17 signal

  • a.
    Align the two lasers to maximize the SRS signals using alkyne-containing standard sample (alkyne-estrogen).
  • b.
    Just before imaging, add propofol-d17 to the cell culture dish to make a final concentration of 100 μM.
  • c.
    Place the dish on the sample stage and prepare for observation.

    Optional: If the system is equipped with single photon confocal microscopy, turn it on, illuminate cells with 488 nm laser, locate the cells with the fluorescence of calcein AM.

  • d.
    After checking the safety of the optical line, open a shutter for pump and Stokes lasers, run program to start the experiment.
  • e.
    Referring to the two-photon microscope channel, adjust the bottom objective lens to position the cell again.
  • f.
    Check the signal of the SRS channel.
  • g.
    If the objective lens is equipped with correction collar, adjust it to maximize the SRS signal.

    Note: Although SRS signals do not bleach, fluorescence signals may be susceptible to bleaching with high intensity lasers used for SRS imaging.

  • h.
    Adjust the number of frame and average according to the strength of the signal and acquire the image (Figure 5).

    Note: Frame averaging result in acquisition of images with better signal to noise ratio but reduces temporal resolution.

• 4.
  Confirmation of the signal of propofol-d17

  • a.
    Set the pump wavelength to make the wavenumber below 2,000 cm⁻¹, off-resonance wavenumber of propofol-d17.
  • b.
    Obtain the image in the same frame averaging setting.
  • c.
    Compare images under on- and off-resonance pulses to confirm the signal of propofol-d17.
• 5.Data analysis

Figure 5.

Representative images of multimodal multiphoton imaging of primary cultured neurons

SRS signals of propofol-d17 at 2055 cm⁻¹ (A) and two-photon excited fluorescence of calcein AM (B) can be acquired simultaneously.

Fiji was used to analyze the intensity distribution of the SRS signals, which can further confirm the location of propofol-d17 on neuron.

Measuring dynamics of propofol by washout experiment

Timing: 3 h (for steps 6 and 7)

High sensitivity and low toxicity of multimodal multiphoton system allows characterization of the spatiotemporal dynamics of propofol. For this purpose, a perfusion system was used to wash away propofol-d17 on the neuronal culture while monitoring SRS signals.

• 6.
  Washout experiments

  • a.
    Using the loaded neuronal cells described above, choose a target cell for timelapse imaging.
  • b.
    Perform a timelapse imaging for a total 1,000 frames while perfusing of 3 mL 100 μM propofol-d17 solution with fresh HEPES ACSF solution (Figure 6).

Optional: Use ImageJ (Fiji) to process the data, make a composite image, and group every 20 images to get a time-lapse image set and perform mathematical analysis.

Optional: Alternatively, a set of 1000 frames can be obtained during the washout of 3 mL 100 μM propofol-d17 with fresh 100 μM of regular (non-deuterated) propofol on the same kind of neuronal cells.

• 7.
  Data Analysis

  Note: The microscope system can acquire data from four channels at the same time as photodetectors can be equipped with a bandpass filter to detect various signals. In this experiment, two main signal channels are used i.e., SRS signal and two-photon excited fluorescence signal are acquired simultaneously, in which the two-photon excited fluorescence image is used as a guide to identify the location of cells.

  • a.
    Choose the region of interest for analysis (cell body in the example shown in Figure 7).
  • b.
    Use appropriate software (e.g., Fiji) to analyze the SRS channels' signal changes.
  • c.
    Plot intensity of propofol-d17 SRS signal intensity against time to visualize timecourse of SRS signal changes upon washout (Figure 7).

Figure 6.

SRS image before (left) and after (right) exchange of propofol-d17 with fresh HEPES ACSF on hippocampal neuron

Reduction in the SRS signal intensity of propofol-d17 at 2055 cm⁻¹ at somatic regions (blue and red boxes) after washout can be seen.

Figure 7.

Changes of SRS signals upon washout of propofol-d17

Intensity profiles of SRS signals from two different neurons after washout of propofol-d17.

Expected outcomes

Using this protocol, deuterated propofol can be directly visualized in living neurons and its dynamics can be monitored together with other parameters such as cell morphology. Specificity of the Raman signal from deuterated propofol can be confirmed by imaging the same field of view at on and off resonance. In cell observation experiments, we can obtain high-resolution images of propofol-d17 in living neurons using 2,055 cm⁻¹ as the on-resonance peak and 2,000 cm⁻¹ as the off-resonance peak, and the signal proved to be high at the cellular membrane of the neuronal cell body and neurites, although the propofol-d17 was still present in the extracellular solution.

Time-lapse images of propofol were taken in the dynamic analysis utilizing our SRS system. Analysis of the intensity of propofol signals in the cells in washout experiments revealed a drop in SRS intensity, implying that propofol-d17 was quickly replaced by new incoming propofol. An exponential decay curve can be well fit to SRS signal of propofol-d17.

Limitations

Although SRS is far superior to spontaneous Raman scattering, the Raman signals obtained with an SRS microscope is still low compared to fluorescence microscopy. Further improvements in sensitivity are needed for wider applications of this technique to biology. The linear relationship between the SRS signal and molecular concentration in SRS imaging could be affected by background noises arising from the unintended optical processes (e.g., cross-phase modulation, and two-photon absorption).⁸

Availability of SRS microscope systems is still limited compared to other imaging systems. As such, to see the drugs of interests by this technique, collaboration with laboratories having SRS microscope running may be needed. Similarly, availability of Raman-tagged drugs is critical for this protocol. In this respect, fortunately, a wide range of deuterated compounds are commercially available.

Troubleshooting

Problem 1

Phototoxicity during imaging (related to steps 3 and 6).

Potential solution

Phototoxicity during multimodal, multiphoton microscopy often results from excessive absorption of photons by fluorophores. In this protocol, excess fluorescence from calcein AM may be an issue. To avoid such phototoxicity, optimization of calcein AM loading to achieve just enough fluorescence signals is suggested.

Problem 2

The signal of the propofol-d17 SRS signal is low (related to steps 3 and 6).

Potential solution

Optimize the SRS microscope system and acquisition settings. Increasing the laser powers helps to increase SRS signals, but care must be taken to avoid increased phototoxicity. Also, frame averaging to obtain images will help to improve the signal to noise ratio.

Problem 3

Perfusion system has many instabilities, which will affect the results of the experiment (related to step 6).

Potential solution

Since the perfusion system is constructed manually, it is important to constantly monitor the fluid in the tube. Use of fluorescence dyes as a marker of solution exchange helps for characterization.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Mutsuo Nuriya (mnuriya@keio.jp).

Materials availability

This study did not generate new unique reagents.

Data and code availability

The published article includes all datasets generated or analyzed during this study. Original images are available from the corresponding author on request.