ld plan neofluar, by Zeiss - Product details

1

Targeted Chloroplast Ablation in Moss Cells

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Leaves of 4- to 5-week-old gametophores were excised and divided into 6 segments with a carbon knife. All cells not targeted for reprogramming were ablated by irradiating just one chloroplast with a solid-state ultraviolet laser (355 nm) through a 20X objective lens (LD Plan-NEOFLUAR, NA 0.40; Zeiss) at a laser focus diameter of less than 1 µm using the laser pressure catapulting function of the PALM microdissection system (Zeiss). The analysis was performed in targeted cells which survived for 72 h after isolation. A chloroplast to irradiate was chosen at a position distantly located from the targeted cell to minimize the irradiation effect.

Sato Y., Sugimoto N., Hirai T., Imai A., Kubo M., Hiwatashi Y., Nishiyama T, & Hasebe M. (2017). Cells reprogramming to stem cells inhibit the reprogramming of adjacent cells in the moss Physcomitrella patens. Scientific Reports, 7, 1909.

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2

Imaging Granulomas Using Epifluorescence and Confocal Microscopy

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Epifluorescent imaging of granulomas was conducted on an inverted Zeiss AxioObserverZ.1 using a 20× long working distance/0.4 NA lens (Zeiss, LD Plan NeoFluar), a Zeiss MRm camera and either an Xcite 120Q or an Xcite 120LED (Lumen Dynamics) light source. Images were acquired using the Zen Blue 2012 imaging suite (Zeiss).
Confocal images were acquired on an Andor XD spinning disk confocal, consisting of an Olympus IX81 inverted microscope equipped with a Yokogawa CSU-X1 spinning disk using a 20×/0.5 UplanFl lens (Olympus) and detected with a Andor Ixon3 897 EMCCD. Images were acquired using Metamorph.
Confocal images of CLARITY cleared granulomas and granulomas from (cdh1-tdtomato)^xt18 animals were obtained on a Zeiss AxioObserverZ.1 equipped with an XLIGHT V2 spinning disk unit (Crest Optics, Rome, Italy) using a 20×/0.5 NA lens, an ORCA Flash4.0 V3 (Hamamatsu, Bridgewater, NJ) and an LDI multiline laser (89 North, Williston VT).
Lightsheet microscopy was performed on a Zeiss Lightsheet Z1 using a Plan-Apochromat 20×/1.0 NA Aqueous immersion objective and a PCO.edge camera. Individual channels were acquired sequentially using dual-side illumination.
For presentation, images were prepared and adjusted using Zen software and FIJI for contrast adjustments and maximum projections.

Cronan M.R., Matty M.A., Rosenberg A.F., Blanc L., Pyle C.J., Espenschied S.T., Rawls J.F., Dartois V, & Tobin D.M. (2018). An Explant Technique for High-Resolution Imaging and Manipulation of Mycobacterial Granulomas. Nature methods, 15(12), 1098-1107.

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3

Three-Dimensional Dark-Field Microscopy Imaging

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Three-dimensional dark-field imaging was performed using a commercial inverted dark-field microscope (Axiovert 200 M, Zeiss, Jena, Germany) coupled with a color CCD camera (8051C-CL, Thorlabs Inc., Newton, NJ, USA). The system employs an oil immersion ultradark-field condenser (NA of 1.2–1.4, Zeiss), a 63× objective (0.75 NA LD Plan-Neofluar, Zeiss) mounted on a piezoelectric objective scanner (P-725, PI, Hannover, Germany), and a 530 nm longpass filter (530LP RapidEdge, Omega Optical, VT, USA) for z-stack acquisition. All three-dimensional DFM images were acquired with a 28.5 μm depth z-stack with a 0.7125 μm step size with custom-written image acquisition software that synchronizes camera acquisition and piezoelectric position. Each focal plane was imaged with a 10 ms acquisition time. The image dimensions are 0.1 μm/pixel in the X and Y directions and 0.75 μm/pixel in the Z direction. Illumination was provided by an integrated 100 W halogen source.

Jeong S., González G., Ho A., Nowell N., Austin L.A., Hoballah J., Mubarak F., Kapur A., Patankar M.S., Cramer D.W., Krauledat P., Hansen W.P, & Evans C.L. (2020). Plasmonic Nanoparticle-Based Digital Cytometry to Quantify MUC16 Binding on the Surface of Leukocytes in Ovarian Cancer. ACS sensors, 5(9), 2772-2782.

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4

Immunostaining of Fixed Brain Slices

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Slices were fixed in 4% paraformaldehyde in PBS for 30 min at 4°C before staining. Free-floating slices were washed in PBS and permeabilized overnight at 4°C with 1% triton X-100 dissolved in PBS. The slices were blocked for 3 h in blocking buffer (4% milk, 0.3% Triton X-100/PBS), stained with primary antibodies diluted in blocking buffer (2–3 days at 4°C) and washed in PBS. Immunoreactivity was detected using Alexa dye-conjugated secondary antibodies diluted in blocking buffer. The slices were incubated with the secondary antibodies for 2 h and then washed in 0.1% triton X-100 in PBS. Finally, slices were washed 2–3 times in PBS and mounted with ProLong Gold antifade reagent (Life Technologies) on glass microscope slides. Imaging was performed with Zeiss LSM 780 confocal system equipped with a 20× (LD Plan Neofluar, NA 0.8) and a 63× (Plan Apochromat, NA 1.4) oil immersion objective with a pinhole size of one and pixel format of 1024 × 1024. Line averaging was performed to reduce noise. Images were transferred to ImageJ/FIJI.

Petersen A.V., Jensen C.S., Crépel V., Falkerslev M, & Perrier J.F. (2017). Serotonin Regulates the Firing of Principal Cells of the Subiculum by Inhibiting a T-type Ca2+ Current. Frontiers in Cellular Neuroscience, 11, 60.

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5

3D Imaging of Mouse Cortical Layers

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Cleared, flat-mounted cortices were imaged with a confocal laser scanning microscope (LSM710, Zeiss) equipped with a long-working-distance 20× objective (LD-Plan Neofluar, NA = 0.40, working distance = 8.4 mm, Zeiss) and GaAsP detectors. EGFP was excited with an Argon laser (488 nm), and the laser power was manually adjusted (2–80%) for consistent fluorescent intensities at different depths. Z-stack and tiled images were acquired using a motorized stage and ZEN software (ZEN 2011 SP7 FP1 (black), Zeiss) at a resolution of 512 × 512 pixels for the x- and y-axes (0.84 μm per pixel) and at a 5 μm pitch for the z-axis. The imaging area was determined to include S1 (for P3) and both S1 and M1 (for P5 and P7) from the pial surface to the bottom of the white matter.

Oka Y., Doi M., Taniguchi M., Tiong S.Y., Akiyama H., Yamamoto T., Iguchi T, & Sato M. (2021). Interstitial Axon Collaterals of Callosal Neurons Form Association Projections from the Primary Somatosensory to Motor Cortex in Mice. Cerebral Cortex (New York, NY), 31(11), 5225-5238.

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6

Rapid Cell Deformability Characterization

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Real-time deformability cytometry (RT-DC) is used for high-throughput mechanical cell and spheroid characterization. The system is built on a commercial solution (AcCellerator, Zellmechanik Dresden) consisting of an inverted microscope (Axio Observer A.1, Zeiss), a CMOS camera (MC1362, Mikrotron), a microsecond-pulsed LED illumination (L1, Zellmechanik Dresden), and a dedicated syringe pump (NemeSys, Cetoni).
Either a microfluidic chip or a glass cuvette is assembled on the xy-stage of the microscope. Cells or spheroids translocate through a constriction and deform by hydrodynamic shear and normal stress^{26 (link),27 (link)}. Image acquisition and analysis are performed in real-time with a throughput exceeding 1000 cells or 10 spheroids per second (depending on initial concentration) and deformation is quantified using the circularity of each particle:

\begin{matrix} Deformation = 1 - Circularity = 1 - \frac{2 \sqrt{π Area}}{Perimeter} . \end{matrix}

Measurements on single cells are performed using a 40x objective (PDMS chip: Apochromat, Zeiss; glass cuvette: LD Plan Neofluar, Zeiss) with an optical resolution of 0.34 µm per pixel, and on spheroids using a 20x objective (LDC, Zeiss) with 0.68 µm per pixel.

Panhwar M.H., Czerwinski F., Dabbiru V.A., Komaragiri Y., Fregin B., Biedenweg D., Nestler P., Pires R.H, & Otto O. (2020). High-throughput cell and spheroid mechanics in virtual fluidic channels. Nature Communications, 11, 2190.

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7

Imaging Granulomas Using Epifluorescence and Confocal Microscopy

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Epifluorescent imaging of granulomas was conducted on an inverted Zeiss AxioObserverZ.1 using a 20× long working distance/0.4 NA lens (Zeiss, LD Plan NeoFluar), a Zeiss MRm camera and either an Xcite 120Q or an Xcite 120LED (Lumen Dynamics) light source. Images were acquired using the Zen Blue 2012 imaging suite (Zeiss).
Confocal images were acquired on an Andor XD spinning disk confocal, consisting of an Olympus IX81 inverted microscope equipped with a Yokogawa CSU-X1 spinning disk using a 20×/0.5 UplanFl lens (Olympus) and detected with a Andor Ixon3 897 EMCCD. Images were acquired using Metamorph.
Confocal images of CLARITY cleared granulomas and granulomas from (cdh1-tdtomato)^xt18 animals were obtained on a Zeiss AxioObserverZ.1 equipped with an XLIGHT V2 spinning disk unit (Crest Optics, Rome, Italy) using a 20×/0.5 NA lens, an ORCA Flash4.0 V3 (Hamamatsu, Bridgewater, NJ) and an LDI multiline laser (89 North, Williston VT).
Lightsheet microscopy was performed on a Zeiss Lightsheet Z1 using a Plan-Apochromat 20×/1.0 NA Aqueous immersion objective and a PCO.edge camera. Individual channels were acquired sequentially using dual-side illumination.
For presentation, images were prepared and adjusted using Zen software and FIJI for contrast adjustments and maximum projections.

Cronan M.R., Matty M.A., Rosenberg A.F., Blanc L., Pyle C.J., Espenschied S.T., Rawls J.F., Dartois V, & Tobin D.M. (2018). An Explant Technique for High-Resolution Imaging and Manipulation of Mycobacterial Granulomas. Nature methods, 15(12), 1098-1107.

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8

Femtosecond Laser Tissue Ablation

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The laser beam is focused on the fixed tissue (rat liver dissection). Thereon, a Galvano scanner (IntelliScan 14, SCANLAB GmbH, Germany) steers the laser beam along a straight line of 400 μm length with a speed of 50 mm/s, repeating this process five hundred times. The laser-affected tissue is imaged using a confocal microscope (Zeiss LSM 880, Carl Zeiss AG, Germany) with a 20× microscope objective (LD Plan-Neofluar, Carl Zeiss AG, Germany). The average power of the femtosecond pulsed laser is characterized by the laser power percentage (LPP), which is modulated by a beam attenuator (Ultrafast, Altechna, Lithuania). A photodiode power sensor (S130VC, Thorlabs, USA) measures the average laser power at the focal plane of a 10× microscope objective (M Plan Apo, Mitutoyo, Japan).

Huaroto J.J., Capuano L., Kaya M., Hlukhau I., Assayag F., Mohanty S., Römer G.W, & Misra S. (2023). Two-photon microscopy for microrobotics: Visualization of micro-agents below fixed tissue. PLOS ONE, 18(8), e0289725.

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9

In Vivo Speckle and Fluorescence Imaging

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The experiment setup is presented in Fig. 1. The wide-field illumination was provided by a mercury lamp passed through a single band filter set (ET470/24x, 510dcxr, HQ525lp, Chroma Technology). The LSCI employed a diode laser (CrystaLaser,

λ = 638 nm

, 25 mW) to coherently illuminate the imaging site with an incident angle of about 30 deg. Images were acquired by the CCD camera (DP72, Olympus) after light passed through the objective lens (LD Plan-Neofluar,

20 \times

,

NA = 0.4

, Carl Zeiss). For each imaging site, five wide-field fluorescence images were taken followed by 200 frames of raw speckle images with sizes of

1024 \times 1360 pixels

, captured at a frequency of 15 Hz. The exposure time was set to 75 ms for fluorescence images and 20 ms for speckle images.

Yan J., Kang Y., Xu S., Ong L.L., Zhuo S., Bunte R.M., Chen N., Asada H.H., So P.T., Wanless I.R, & Yu H. (2014). In vivo label-free quantification of liver microcirculation using dual-modality microscopy. Journal of Biomedical Optics, 19(11), 116006.

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10

T Cell Cytotoxicity Imaging Assay

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For imaging of T cell cytotoxic efficiency, T cell and peptide-loaded APCs were cultured as described for Granule polarization imaging, except 2×10⁵ JR209 T cells and 1×10⁵ peptide-loaded T2-A2Kb APCs were imaged for 8 hours, acquired at 3 minutes intervals with a 40× air objective (LD Plan-Neofluar, 0.6 NA, Zeiss). For analysis, initial T cell/APC conjugation and APC death were manually identified - T cell/APC conjugates lasting longer than 10 min were analyzed. Cytotoxic efficiency was calculated as: [(T cell/APC conjugates that result in APC lysis) / (total stable T cell/APC conjugates observed)]. Image acquisition and analysis was performed using MetaMorph® Microscopy Automation & Image Analysis Software (Molecular Devices). Cytotoxic efficiency was calculated as described in Materials and Methods.

Moogk D., Zhong S., Yu Z., Liadi I., Rittase W., Fang V., Dougherty J., Perez-Garcia A., Osman I., Zhu C., Varadarajan N., Restifo N.P., Frey A.B, & Krogsgaard M. (2016). Constitutive Lck activity drives sensitivity differences between CD8+ memory T cell subsets. Journal of immunology (Baltimore, Md. : 1950), 197(2), 644-654.

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Ld plan neofluar

Lab products found in correlation

12 protocols using ld plan neofluar

Targeted Chloroplast Ablation in Moss Cells

Imaging Granulomas Using Epifluorescence and Confocal Microscopy

Three-Dimensional Dark-Field Microscopy Imaging

Immunostaining of Fixed Brain Slices

3D Imaging of Mouse Cortical Layers

Rapid Cell Deformability Characterization

Imaging Granulomas Using Epifluorescence and Confocal Microscopy

Femtosecond Laser Tissue Ablation

In Vivo Speckle and Fluorescence Imaging

T Cell Cytotoxicity Imaging Assay

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