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. 2023 Apr 13;4(2):102221.
doi: 10.1016/j.xpro.2023.102221. Online ahead of print.

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

Wenying Zhong  1 , Robert Oda  2 , Yasuyuki Ozeki  3 , Masato Yasui  1 , Mutsuo Nuriya  4
Affiliations

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

Wenying Zhong et al. STAR Protoc. .

Abstract

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.

Keywords: Cell Biology; Cell Culture; Microscopy; Molecular/Chemical Probes; Neuroscience.

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Conflict of interest statement

Declaration of interests R.O. is an employee of and holds stock options in Chordia Therapeutics Inc.

Figures

None
Graphical abstract
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.
Figure 2
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
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
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.
Figure 5
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.
Figure 6
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
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.
See this image and copyright information in PMC

References

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