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

Summary

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.

Abstract

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.

Introduction

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 e....

Protocol

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 tab....

Results

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.......

Discussion

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.......

Materials

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