The setup for SRS microscopy was similar to what was described earlier (Shen et al., 2014 ), which integrates picoEmerald laser system (Applied Physics and Electronics) and a laser-scanning microscope (FV1200MPE; Olympus). The images were taken through a 60× objective (water immersion, N.A. = 1.2, UPlanAPO/IR; Olympus). The lasers were tuned so that C-D bond vibration at 2101 cm−1 could be selectively imaged. Images were acquired by Fluoview Software and were later assigned pseudo colors in ImageJ. CH3 bond vibration signal was collected for use as protein content normalization.
Uplanapo ir
Manufactured by Olympus
The UPlanAPO/IR is a high-performance objective lens designed for infrared imaging applications. It provides excellent optical performance and is suitable for a variety of microscopy techniques.
Automatically generated - may contain errors
6 protocols using uplanapo ir
1
Stimulated Raman Scattering Microscopy
2
Correlative SRS and Confocal Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The setup for SRS microscopy was similar to what was described earlier (36 (link)). Correlative SRS and confocal fluorescence imaging was performed on the same microscope (FV1200; Olympus) and objective (60×, water immersion, N.A. = 1.2, UPlanAPO/IR; Olympus). Spontaneous Raman spectra were acquired using an upright confocal Raman spectrometer (Xplora; HORIBA Jobin Yvon).
3
Two-color SRS Imaging of Plant Cell Walls
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SRS chemical images were constructed based on a parallel two-color SRS microscope with the two orthogonal outputs of a dual-phase lock-in amplifier (LIA) as described previously (dual-phase SRS). The pulsed femtosecond laser beams from a commercial optical parametric oscillator (OPO) (Insight DS+, Newport, CA) were used as the excitation laser sources. A fundamental 1040 nm laser with a pulse duration of 150 fs was used as the Stokes beam, and the tunable OPO output (690–1300 nm, 120 fs) served as the pump beam. The two laser beams were combined using a dichroic mirror (DM), spatially and temporally overlapped, and then allowed to interact with the samples through a laser-scanning microscope (FV 1200, Olympus). To capture the Raman frequencies at 2900 cm−1 for cell wall polysaccharides and 1600 cm−1 for lignin in loquat flesh, the tunable outputs were turned to 802 and 892 nm, respectively. A 60× water immersion objective lens (NA = 1.2, UPlanApo/IR, Olympus) was used to focus the light into the sample, and an oil condenser (NA = 1.4, U-AAC, Olympus) was used to collect the signal. Both the reconstruction of large-scale images and the extraction of SRS spectra from the image stacks were performed with ImageJ software (National Institute of Health, Bethesda, MD, USA).
4
Stimulated Raman Scattering Microscopy Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SRS images were acquired using a lab‐built SRS microscope (Figure S1 , Supporting Information) previously reported.[22 ] A polygon scanner (Lincoln SA24, Cambridge Technology) scanned the Stokes beam onto a blazed grating (GR50‐0310, Thorlabs). As a reflective wedge, the grating introduces a continuous temporal delay between the pump and the retroreflected Stokes beam. Both the pump and Stokes beams were chirped with high‐dispersion glass (SF57, 90 cm in length for the Stokes beam and 75 cm in length for the pump beam). For all experiments, the power on the sample was 20 mW for 966 nm, 14 mW for 800 nm, and 75 mW for 1040 nm, if not otherwise noted. The microscope was equipped with a 60x water immersion objective (NA = 1.2, UPlan‐Apo/IR, Olympus). The SRS signal was then captured by a photodiode with a custom‐built resonant circuit and extracted by a lock‐in amplifier (UHFLI, Zurich Instrument). The same setup was used for two‐photon fluorescence imaging with 20 mW for 966 nm. After filtering the excitation beam following the interaction with the sample, a photomultiplier tube (H7422‐40, Hamamatsu) was used to measure the two‐photon fluorescence signal.
5
Quantifying Lipid Droplets in Touch Neurons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To quantify lipid droplet metrics in the touch neurons, 1-day adult worms were imaged using Stimulated Raman Scattering (SRS) microscopy. Worms were mounted onto 2% agarose pads with 0.5% NaN3 as anesthetic on glass microscope slides and imaged using a 60× water objective (UPlanAPO/IR; 1.2 N.A.; Olympus). A femtosecond-pulsed laser and picosecond-pulsed laser were used for simultaneously imaging label-free lipids (SRS channel) and GFP-labeled neurons (fluorescence channel). Images were analyzed using ImageJ software (NIH). Neuronal area was selected using fluorescent signal based on several Z-projected stacks to confirm the presence of lipid droplets within the cell and to exclude extracellular signal.
6
Femtosecond SRS Microscopy for Molecular Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SRS/pump-probe imaging was performed on a femtosecond SRS microscope. An ultrafast laser system with dual output at 80MHz (InSight DeepSee, Spectra-Physics) provided pump and Stokes beams. The pump and Stikes beams were set to 802 nm and 1045 nm, respectively, to be resonant with the C-H vibration band at 2,899 cm -1 . For the C-D vibration band at 2,168 cm - 1 , the pump beam was tuned to 852 nm. For the C≡C vibration band at 2,265 cm -1 , the pump beam was tuned to 846 nm. Stokes beam was modulated by an acoustooptic modulator (AOM, 1205-C, Isomet) at 2.2 MHz. Both beams were linearly polarized. A motorized translation stage was employed to scan the temporal delay between the two beams. Two beams were then sent into a home-built laser-scanning microscope. A 60x water immersion objective lens (NA = 1:2, UPlanApo/IR, Olympus) was used to focus the light into the sample, and an oil condenser (NA = 1:4, UAAC, Olympus) was used to collect the signal. The stimulated Raman loss and pumpprobe signals were detected by a photodiode, which was extracted with a digital lock-in amplifier (Zurich Instrument). The power of the pump beam (802nm, 846 nm and 852 nm) and the power of the Stokes beam at the specimen were maintained at ~30mW and 100mW, respectively. The images were acquired at 10 μs pixel dwell time. No cell damage was observed during the imaging procedure.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!