Three-dimensional label-free imaging throughout adipocyte differentiation by stimulated Raman microscopy

Ferrara, Maria Antonietta; Filograna, Angela; Ranjan, Rajeev; Corda, Daniela; Valente, Carmen; Sirleto, L.

doi:10.1371/journal.pone.0216811

Cited by 30 publications

(18 citation statements)

References 62 publications

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“…This SRS microscope is used to take advantage of two laser combinations. Ti:Sa and OPO laser combination cover the C-H region in SRG modality [17][18][19][20][21][22]. The latter Ti:Sa and SHG laser combination provide the extension of the microscope to the silent region (<1800 cm−1) and fingerprint region (1800-2800 cm−1) in the SRL modality [23].…”

Section: Methods and Resultsmentioning

confidence: 99%

“…We note that typical and widespread commercial laser sources combinations, such as Chameleon compact OPO (Coherent, Inc.) and Ti:Sa, although tailored for multimodal imaging, their minimum Raman shift is 2500 cm -1 . This means that by adopting this laser combination, only the CH-OH region of Raman spectra can be explored, while the and fingerprint regions are out of emission range [17][18][19][20][21][22]. Therefore, they cannot accomplish the demand of biorthogonal platforms based on femtosecond stimulated Raman microscopy.…”

Section: Introductionmentioning

confidence: 99%

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Stimulated Raman Microscopy Implemented by Three Femtosecond Laser Sources

2021

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In this work, the implementation of a femtosecond Stimulated Raman Scattering microscope, equipped with three femtosecond laser sources: a Titanium-Sapphire (Ti:Sa), an optical parametric oscillator (OPO), and a second harmonic generator (SHG); is presented. Our microscope is designed so that it can cover all the regions of Raman spectra, taking advantage of two possible laser combinations. The first, Ti:Sa and OPO laser beams, which cover the C-H region (>2800 cm-1 ) in stimulated Raman gain (SRG) modality, whereas the second, Ti:Sa and SHG laser beams, covering the C-H region and the fingerprint region in stimulated Raman losses (SRL) modality. The successful realization of the microscope is demonstrated, reporting images of polystyrene beads using both SRL and SRG modalities.

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Section: Methods and Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Stimulated Raman Microscopy Implemented by Three Femtosecond Laser Sources

2021

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“…The minimum of these widths was found by fitting a parabola. The effective SRS beam width was determined as described by equation (8). Figure 1b shows the smallest widths from knife edge measurements of the carrier and the probe with a Gaussian fit.…”

Section: Focal Spot Size and Optical Spatial Resolutionmentioning

confidence: 99%

“…Upon calibration of CTN and CSN, the noise contributions can be simulated as well for each TC and filter order with equation 6and (7) and the NEPBW expression in equation 4. For the optical point spread function (PSF), the required input is the 1/e 2 radius of the probe and carrier beams and equation (8). A detailed description of the input parameters and the setup parameters is given in Figure S3.…”

Section: Optimal Settings Simulationmentioning

confidence: 99%

Stimulated Raman Scattering Simulation for Imaging Optimization

Zada

Fokker

Leslie

et al. 2020

Preprint

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Two simulation programs of a stimulated Raman scattering microscopy (SRS) imaging system with lock-in amplifier (LIA) detection were developed. SRS is an imaging technique based on the vibrational Raman cross-section as the contrast mechanism and enables fast, label-free imaging. Most SRS implementations are based on a LIA detection of a modulated signal. However, building and operating such SRS set-ups still poses a challenge when selecting the LIA parameter settings for optimized acquisition speed or image quality. Moreover, the type of sample, e.g. a sparse sample vs. a densely packed sample, the required resolution as well as the Raman cross-section and the laser powers affect the parameter choice. A simulation program was used to find these optimal parameters. The focal spot diameters of the individual lasers (pump and Stokes) were used to estimate the effective SRS signal focal spot and the (optical) spatial resolution. By calibrating the signal and noise propagation through an SRS system for a known molecule, we estimated the signal and noise input to the LIA. We used a low pass filter model to simulate the LIA behavior in order to find the optimal parameters (i.e. filter order and time constant). Optimization was done for either image quality (expressed as contrast to noise ratio) or acquisition time. The targeted object size was first determined as a measure for the required resolution. The simulation output consisted of the LIA parameters, pixel dwell time and contrast to noise ratio. In a second simulation we evaluated SRS imaging based on the same principles as the optimal setting simulation, i.e. the signals were propagated through an imaging system and LIA detection. The simulated images were compared to experimental SRS images of polystyrene beads. Finally, the same software was used to simulate multiplexed SRS imaging. In this study we modeled a six-channel frequency-encoded multiplexed SRS system demodulated with six LIA channels. We evaluated the inter-channel crosstalk as a function of chosen LIA parameters, which in multiplex SRS imaging also needs to be considered. These programs to optimize the contrast to noise ratio, acquisition speed, resolution and crosstalk will be useful for operating stimulated Raman scattering imaging setup, as well as for designing novel setups.

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“…Specifically for the LIA in the context of SRS, to the best of our knowledge no in-depth analysis has been reported for the input parameters of the filter bandwidth. While most SRS studies mention the time constant (TC), almost none [9] mention the equally important filter-order (n), which makes it impossible to calculate the bandwidth or compare modalities reported in the literature. Since the conventional imaging manner of non-linear modalities, such as SRS, is scanning the focal spot across the sample, the scanning speed, the filter response, the focal spot size, and the spatial resolution in the scanning direction are related.…”

Section: Introductionmentioning

confidence: 99%

Stimulated Raman scattering simulation for imaging optimization

Zada

Fokker

Leslie

et al. 2021

J. Eur. Opt. Soc.-Rapid Publ.

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Two simulation programs of a stimulated Raman scattering microscopy (SRS) imaging system with lock-in amplifier (LIA) detection were developed. SRS is an imaging technique based on the vibrational Raman cross-section as the contrast mechanism and enables fast, label-free imaging. Most SRS implementations are based on LIA detection of a modulated signal. However, building and operating such SRS set-ups still poses a challenge when selecting the LIA parameter settings for optimized acquisition speed or image quality. Moreover, the type of sample, e.g. a sparse sample vs. a densely packed sample, the required resolution as well as the Raman cross-section and the laser powers affect the parameter choice.A simulation program was used to find these optimal parameters. The focal spot diameters of the individual lasers (pump and Stokes) were used to estimate the effective SRS signal focal spot and the (optical) spatial resolution. By calibrating the signal and noise propagation through an SRS system for a known molecule, we estimated the signal and noise input to the LIA. We used a low pass filter model to simulate the LIA behavior in order to find the optimal parameters (i.e. filter order and time constant).Optimization was done for either image quality (expressed as contrast to noise ratio) or acquisition time. The targeted object size was first determined as a measure for the required resolution. The simulation output consisted of the LIA parameters, pixel dwell time and contrast to noise ratio.In a second simulation we evaluated SRS imaging based on the same principles as the optimal setting simulation, i.e. the signals were propagated through an imaging system and LIA detection. The simulated images were compared to experimental SRS images of polystyrene beads.Finally, the same software was used to simulate multiplexed SRS imaging. In this study we modeled a six-channel frequency-encoded multiplexed SRS system demodulated with six LIA channels. We evaluated the inter-channel crosstalk as a function of chosen LIA parameters, which in multiplex SRS imaging also needs to be considered.These programs to optimize the contrast to noise ratio, acquisition speed, resolution and crosstalk will be useful for operating stimulated Raman scattering imaging setup, as well as for designing novel setups.

show abstract

Three-dimensional label-free imaging throughout adipocyte differentiation by stimulated Raman microscopy

Cited by 30 publications

References 62 publications

Stimulated Raman Microscopy Implemented by Three Femtosecond Laser Sources

Stimulated Raman Microscopy Implemented by Three Femtosecond Laser Sources

Stimulated Raman Scattering Simulation for Imaging Optimization

Stimulated Raman scattering simulation for imaging optimization

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