LP emission during flow was collected by pumping LPs using a 10 ns pulse width at 1064 nm with 2 MHz repetition rate (CNILaser FL-1064-Nano-LAB). Excitation light was focused using a 10× 0.3 NA objective (Leica HC PL FLUOTAR). Emission light was collected and dispersed using a custom-built grating spectrometer equipped with a linescan camera (Sensors Unlimited 2048 L). This camera was connected to an FPGA (National Instruments PCIe-1473R) that determined whether sorting criteria were met. Sorting accuracy was assessed by comparing the flow device inputs and outputs using a confocal microscope (Olympus Fluoview 3000). This microscope was able to attain brightfield images of cells and LPs and was equipped with a cell incubator (Tokai Hit). Furthermore, the microscope was modified to measure LPs using a 1060–1070 pump laser operating at 2 MHz with 10 ns pulse width (Spectra Physics VGEN-ISP-POD). Spectra were collected by a 20× 0.45NA objective (Olympus IMS LCPLN20XIR) and sent to a spectrometer (Andor Kymera 328i) equipped with a linescan camera (Sensors Unlimited 2048 L). All camera data was saved and analyzed using custom code based on MATLAB and Python.
Hc pl fluotar
Manufactured by Leica camera
The HC PL FLUOTAR is a series of high-performance microscope objectives manufactured by Leica. These objectives are designed for fluorescence microscopy applications, providing high-quality optical performance and accurate color reproduction. The core function of the HC PL FLUOTAR objectives is to deliver sharp, high-contrast images with excellent chromatic correction and uniform illumination across the field of view.
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Lab products found in correlation
Fluoview 3000, by Olympus (1 mentions)
Pcie 1473r, by National Instruments (1 mentions)
Vgen isp pod, by Spectra-Physics (1 mentions)
Stellaris 5, by Leica camera (1 mentions)
Leica application suite software, by Leica camera (1 mentions)
Uv 2550 spectrophotometer, by Hitachi (1 mentions)
Labram hr evolution, by Horiba (1 mentions)
F 4500 fluorescence spectrometer, by Hitachi (1 mentions)
Tcs sp5 aobs confocal microscope, by Leica camera (1 mentions)
Leica application suite advanced fluorescence las af software, by Leica Microsystems (1 mentions)
Raman spectrometer, by Renishaw (1 mentions)
7 protocols using hc pl fluotar
1
Laser-based Single Particle Characterization
2
Confocal Microscopy for Fluorescent Proteins
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Fluorescence microscopy was performed with a confocal microscope (SP5, Leica) using a 10× objective lens (HC PL FLUOTAR, Leica) under the differential interference contrast polarizing setting. Image resolutions were set at 512 × 512 pixel2, and four frame averages were taken per image. The overall laser power was set to 20%, the smart offset was set to −1%, and the pinhole was held at 100 μm throughout.
The following settings were used for each fluorophore: mVenus: 514-nm laser at 30%, PMT detector between 525 and 575 nm, and smart gain at 800; mCherry: 543-nm laser at 40%, PMT detector between 600 and 700 nm, and smart gain at 1100; emGFP: 488-nm laser at 25%, PMT detector between 500 and 600 nm, and smart gain at 900; and TAMRA: 543-nm laser at 15%, PMT detector between 560 and 625 nm, and smart gain at 725.
All fluorescence images were analyzed using Fiji (ImageJ). Line profiles from images are shown in the same figures. Any brightness/contrast changes made to the images were the same for both +UV and −UV image sets.
The following settings were used for each fluorophore: mVenus: 514-nm laser at 30%, PMT detector between 525 and 575 nm, and smart gain at 800; mCherry: 543-nm laser at 40%, PMT detector between 600 and 700 nm, and smart gain at 1100; emGFP: 488-nm laser at 25%, PMT detector between 500 and 600 nm, and smart gain at 900; and TAMRA: 543-nm laser at 15%, PMT detector between 560 and 625 nm, and smart gain at 725.
All fluorescence images were analyzed using Fiji (ImageJ). Line profiles from images are shown in the same figures. Any brightness/contrast changes made to the images were the same for both +UV and −UV image sets.
3
Automated Fluorescence Measurement on Metasurface Biosensors
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An optical unit to conduct FL measurement on the metasurface biosensors was incorporated in the automated MF system in Section 2.4 . A 10× objective lens of numerical aperture (NA) 0.28 (M Plan Apo, Mitsutoyo, Kawasaki, Japan) focused the illumination green LED (M530F2, Thorlabs, Newton, NJ, USA) light and collected the FL emitted on the metasurface. An uncooled CCD camera (Infinity-3S, Teledyne-Lumenera, Ottawa, ON, Canada) was used in the automated MF system; in the FL and background measurement, exposure time was set to 2 s and gain of signal was set to 10. Without any active cooling mechanism, the background was not low. When evaluating the measured FL intensities, we subtracted the background level in data analysis.
To conduct low-background FL measurement for the extremely diluted samples, confocal FL microscope (Stellaris 5, Leica, Wetzlar, Germany) was used. In the photon counting mode, the background coming from the instrument was suppressed to almost zero. A 10× objective lens of NA 0.32 (HC PL FLUOTAR, Leica) was used. In accordance with the FL-molecule HEX, excitation wavelength was set to 521 nm and detection wavelengths were set to 570–700 nm. An FL image was acquired through 10-frame accumulation.
To conduct low-background FL measurement for the extremely diluted samples, confocal FL microscope (Stellaris 5, Leica, Wetzlar, Germany) was used. In the photon counting mode, the background coming from the instrument was suppressed to almost zero. A 10× objective lens of NA 0.32 (HC PL FLUOTAR, Leica) was used. In accordance with the FL-molecule HEX, excitation wavelength was set to 521 nm and detection wavelengths were set to 570–700 nm. An FL image was acquired through 10-frame accumulation.
4
Live-cell imaging of tube formation
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Using the LX AF6000 microscope and the Leica Application Suite software (Leica) cells were imaged at regular intervals over an extended period of time (The Mark and Find Figure S2D ). function allowed the center of the wells to be defined and saved, and recalled with high precision accuracy to allow images of the same position to be imaged over time. Care was taken to ensure that no air bubbles were present in the Matrigel or the media as this interfered with analysis (Figure S2E ). Images were captured with a 5x objective (Leica, HC PL FLUOTAR, dry immersion lens, aperture 0.15, working distance 12,000) positioned in the center of the well and imaged every 2 min 30 s over 6–7 h or every 5 min beyond 6 h. Images were opened as a group in FIJI 17 and exported as an .AVI movie at a frame rate that would result in a video of ~4–10 s to allow visualization of tube forming kinetics.
For the hBEC-MSC co-culture experiment, the microscope was set up as above with minor adjustments. A second channel containing the 488 laser was activated and images were captured sequentially in a bright field-FITC manner. The resulting file when opened for analysis displayed all bright field images stacked, and all fluorescent images stacked separately.
For the hBEC-MSC co-culture experiment, the microscope was set up as above with minor adjustments. A second channel containing the 488 laser was activated and images were captured sequentially in a bright field-FITC manner. The resulting file when opened for analysis displayed all bright field images stacked, and all fluorescent images stacked separately.
5
Spectroscopic Characterization of DTE Powder
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Spontaneous Raman spectra were collected with a confocal Raman microscope (LabRAM HR Evolution, HORIBA) in dark room to avoid ambient light. The DTE powder samples were excited through a 50× air NIR objective (HC PL Fluotar, 0.55 NA, Leica) by a 1064 nm laser (75 mW after the objective). The acquisition time was 10 s for each spectrum. UV-Vis spectra and photoluminescence spectra were measured on Shimadzu UV-2550 spectrophotometer and Hitachi F-4500 fluorescence spectrometer with DMSO solution (1×10−4 M), respectively.
6
Confocal Imaging of Spinal Cord and Cerebral Cortex
7
Raman Mapping of Atherosclerotic Plaques
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The spontaneous Raman measurements were recorded in mapping mode using a commercially available Raman spectrometer, Renishaw inVia, with an excitation wavelength of 532 nm. The attached Leica microscope was equipped with a ×50 objective (Leica HC PL FLUOTAR) with a NA of 0.8. The following settings were kept constant for each measured plaque. The laser power was set to 5% corresponding to 3 mW at the sample plane with a spot size of less than 1 µm. Per pixel, two or three accumulations were recorded with an exposure time of 1 s. The tissue sustained no detectable damage during the measurement. We recorded the fingerprint region from around 850–1850 cm−1 with a resolution of approximately 4 cm−1 FWHM. The total Raman spectra acquisition time per sample was up to 23 h, depending on the mapped area. Based on the prior acquired auto-fluorescence images, we mapped an area around the plaque location in 1 µm steps. To analyze and compare the resonance enhancement, additional measurements were done with an excitation source of 785 nm and the laser power set to 100%, measured as 82 mW at the sample plane.
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