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Plan apochromat objective

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The Plan-Apochromat objective is a high-performance optical lens designed for microscopy applications. It is characterized by its excellent chromatic correction, providing superior image quality with minimal color aberrations. The lens features a flat field of view, ensuring that the image remains sharp and in focus across the entire field of view.

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Lab products found in correlation

Sp8 falcon microscope, by Leica (2 mentions) Anti nc82, by Developmental Studies Hybridoma Bank (1 mentions) Anti dlg1, by Developmental Studies Hybridoma Bank (1 mentions) Sp8 laser scanning confocal microscope, by Leica (1 mentions) Draq5, by Abcam (1 mentions) Rabbit anti γh2ax, by Cell Signaling Technology (1 mentions) Cy sc007, by Cytoskeleton (1 mentions) Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions) Sp8 inverted confocal microscope, by Leica (1 mentions)

7 protocols using plan apochromat objective

1

Analyzing Neuromuscular Junction Morphology

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To analyze the NMJ synapse length on larval muscle 8 and surface area of muscle 8, third instar larvae were dissected in HL3 buffer and fixed in Bouin’s for 3 min. After permeabilization and blocking (10% goat serum), immunostaining was performed with anti-Brp (anti-nc82; Developmental Studies Hybridoma Bank (DSHB), clone name: nc82, 1/100) or anti-Discs large 1 (anti-dlg1; DSHB, clone name: 4F3, 1/200) in larvae that selectively express membrane-tethered GFP in motor neurons (OK371-GAL4, UAS-mCD8::GFP; UAS-mCD8::GFP). Images were taken of muscle 8 in abdominal segment 5 using a Leica SP8 laser scanning confocal microscope with 20× Plan-Apochromat objective (0.8 NA). Maximum intensity projections of z-stacks comprising the entire NMJ were used to measure the synapse length and the surface area of muscle 8.
Ignacio B.J., Dijkstra J., Mora N., Slot E.F., van Weijsten M.J., Storkebaum E., Vermeulen M, & Bonger K.M. (2023). THRONCAT: metabolic labeling of newly synthesized proteins using a bioorthogonal threonine analog. Nature Communications, 14, 3367.
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2

Immunofluorescent Detection of γH2AX

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Sections were deparaffinised in xylene and rehydrated through a graded series of 100 – 50% alcohol solutions, followed by immersion in MilliQ water. Antigen retrieval was performed using 0.01 M citrate buffer, pH 6.0. Sections were blocked in a 4% (w/v) solution of BSA in 1x TBS-T (0.01 M Tris, 150 mM NaCl, and 0.1% (v/v) Tween-20) and then incubated with 1:250 dilution of rabbit anti-γH2AX (Cell Signalling) in blocking buffer overnight at 4°C. After rinsing in 1x TBS-T, primary antibodies were detected with a 10 μg/ml solution of Alexa Fluor 488-conjugated goat anti-rabbit IgG in blocking buffer for 2 h. This solution also contained a 1:1000 dilution of the DNA counterstain DRAQ5 (Abcam). Finally, sections were mounted with Mowiol mounting medium (0.1 M Tris, pH 8.5, 10% (w/v) Mowiol 4-88, 25% (v/v) glycerol and 10 μg/ml 1,4-diazabicyclo(2,2,2)octane (DABCO)). Images were captured by confocal microscopy using the Leica S5 microscope equipped with a 40x PlanApochromat objective. Images were rendered in ImageJ.
McMahon M., Frangova T.G., Henderson C.J, & Wolf C.R. (2016). Olaparib, alone or in combination with ionizing radiation, exacerbates DNA damage in normal tissues, as revealed by a new p21 reporter mouse. Molecular cancer research : MCR, 14(12), 1195-1203.
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3

Super-resolution Imaging of HCT116 Cells

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HCT116 cells were seeded onto high-performance cover glass (Carl Zeiss) where they were grown and silenced as detailed above). Cells were fixed 48 h post-transfection with filtered 4% paraformaldehyde, and DNA was counterstained using SiR-Hoechst (STED compatible [103 (link)]; Cytoskeleton, Inc; CY-SC007). Images were acquired with a Falcon SP8 microscope (Leica) equipped with a 100× oil immersion Plan-Apochromat objective (1.40 numerical aperture) and a 775 nm STED laser. The excitation laser was set to 635 nm and the signal was detected by an HyD detector set to a 650–700 nm interval with 0.3–9.9 ns time gating. The pinhole was set to 1 a.u. and images were acquired using 16 times line averaging, 2 frame accumulations and a pixel size in the xy plane of 28.41 × 28.41 nm.
Jeusset L.M., Guppy B.J., Lichtensztejn Z., McDonald D, & McManus K.J. (2021). Reduced USP22 Expression Impairs Mitotic Removal of H2B Monoubiquitination, Alters Chromatin Compaction and Induces Chromosome Instability That May Promote Oncogenesis. Cancers, 13(5), 1043.
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4

Quantitative Analysis of Hippocampal Neuron Subsets

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Fluorescent images were acquired using a Leica TCS SP5 Confocal Laser Scanning Microscope with a Plan-Apochromat objective (40x/1.2 Oil immersion). Multitrack acquisition was performed with scanning speed of 400 Hz, frame size of 1024 × 1024 pixels, X phase correction of −30, and same pinhole setting for all three channels. The images were obtained from consecutive sections containing the hippocampus (8–12 sections per mouse) in both hemispheres. All confocal images were blindly processed using Image J US NIH (http://imagej.nih.gov/ij). The total amount of c-Fos positive cells was obtained, and subsequently discriminated according to their co-immunolocalization with vGAT or vGLUT, and used as reference for localization of ANKG: length of AIS, and distance from soma (MAP2) was measured. The total amount of slices processed were: 71–89 for c-Fos counting, in CA1 region 85–108 were used for measurements of ANKG vGAT cells, and 39–55 in vGLUT cells, in DG region 74–98 were processed for measurements of ANKG in vGAT cells, and 14–59 in vGLUT cells. All neuronal measurements were grouped by animal for further statistical analysis.
Ruiz S.A., Tikochinsky E., Rubovitch V., Pick C.G, & Attali B. (2023). Contextual fear response is modulated by M-type K+ channels and is associated with subtle structural changes of the axon initial segment in hippocampal GABAergic neurons. AIMS Neuroscience, 10(1), 33-51.
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5

Time-lapse Imaging of Ciliary Dynamics

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3D z-stacks covering entire cilia were collected at 5 min intervals using an 40x oil immersion Plan-Apochromat objective (Leica, NA = 1.3) on a Leica SP8 Falcon microscope at 2x the Nyquist limit and 1 Airy disc. Donor sGFP fluorescence was excited by a pulsed (40 MHz) white light laser tuned at 488 nm, and emitted photons between 490 nm and 550 nm were collected with line repetition = 8.
Sheu S.H., Upadhyayula S., Dupuy V., Pang S., Deng F., Wan J., Walpita D., Pasolli H.A., Houser J., Sanchez-Martinez S., Brauchi S.E., Banala S., Freeman M., Xu C.S., Kirchhausen T., Hess H.F., Lavis L., Li Y., Chaumont-Dubel S, & Clapham D.E. (2022). A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility. Cell, 185(18), 3390-3407.e18.
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6

Confocal Microscopy Imaging and FRET Analysis

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The cell-seeded microscope slides were washed twice with the MEM medium supplemented with 4% FCS for 2 hours prior to acquiring images using a Leica SP8 Inverted confocal microscope with 63 × 1.4 NA Plan-Apochromat objective. Excitation wavelengths were 561 nm for Cy3 and 633 nm for Cy5 and Alexa Fluor 647. Emission was collected in the bands 566–618 nm for Cy3 and 638–791 nm for Cy5 and Alexa Fluor 647. Cy3 images in Fig. S9 in the ESI† were deconvolved using Huygens software (SVI) with standard settings. The FRET efficiency was calculated in an ROI containing the whole cell as FRETeff = (Ipost − Ipre)/Ipost where Ipre and Ipost are the total, background subtracted fluorescence of the ROI in the Cy3 channel before and after Cy5 bleaching. Co-localisation was quantified as Pearson's coefficients calculated in ROIs containing the cytoplasm or the nucleus using Huygens software.
Le T.T., Bruckbauer A., Tahirbegi B., Magness A.J., Ying L., Ellington A.D, & Cass A.E. (2020). A highly stable RNA aptamer probe for the retinoblastoma protein in live cells. Chemical Science, 11(17), 4467-4474.
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7

Time-lapse Imaging of Ciliary Dynamics

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3D z-stacks covering entire cilia were collected at 5 min intervals using an 40x oil immersion Plan-Apochromat objective (Leica, NA = 1.3) on a Leica SP8 Falcon microscope at 2x the Nyquist limit and 1 Airy disc. Donor sGFP fluorescence was excited by a pulsed (40 MHz) white light laser tuned at 488 nm, and emitted photons between 490 nm and 550 nm were collected with line repetition = 8.
Sheu S.H., Upadhyayula S., Dupuy V., Pang S., Deng F., Wan J., Walpita D., Pasolli H.A., Houser J., Sanchez-Martinez S., Brauchi S.E., Banala S., Freeman M., Xu C.S., Kirchhausen T., Hess H.F., Lavis L., Li Y., Chaumont-Dubel S, & Clapham D.E. (2022). A serotonergic axon-cilium synapse drives nuclear signaling to alter chromatin accessibility. Cell, 185(18), 3390-3407.e18.
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