Optosplit 2

Manufactured by Cairn Research

Sourced in United Kingdom

The Optosplit II is a high-performance image splitter that allows the simultaneous acquisition of multiple images from a single detector. It is designed to work with a variety of microscopy techniques, providing users with the ability to split and record multiple views or channels within a single image.

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Lab products found in correlation

Axio observer z1, by Zeiss (3 mentions) Metafluor, by Molecular Devices (2 mentions) Eclipse ti inverted microscope, by Nikon (2 mentions) Na1.40 plan apo oil immersion lens, by Nikon (2 mentions) Prior nanoscan z, by Oxford Instruments (2 mentions) Metafluor software, by Molecular Devices (2 mentions) Spectra x light engine, by Lumencor (2 mentions) Di01 t405 488 568 647, by IDEX Corporation (2 mentions) Csu10, by Yokogawa (2 mentions) Te2000 e, by Nikon (2 mentions) Xenon lamp, by Nikon (1 mentions) Ixon3 897 emccd camera, by Oxford Instruments (1 mentions) Eclipse ti u fluorescence microscope, by Nikon (1 mentions) Monochromator, by Cairn Research (1 mentions) Orca er digital camera, by Hamamatsu Photonics (1 mentions) Te2000 u inverted microscope, by Nikon (1 mentions) Imagem 1k camera, by Hamamatsu Photonics (1 mentions) Sr apo tirf, by Nikon (1 mentions) Ti e inverted microscope, by Nikon (1 mentions) Metamorph imaging software, by Molecular Devices (1 mentions)

21 protocols using optosplit 2

Simultaneous Imaging of Neuron-Astrocyte Dynamics

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An inverted Zeiss Axiovert 200 microscope (63x 1.4 NA oil objective), attached to an Evolve (EMCCD) camera (Photometrics), fitted with an image splitter (Optosplit II, Cairn Research), allowed simultaneous acquisition of images at two separate emission wavelengths. Videos were recorded at 8.5 Hz using Micro-manager software (Edelstein et al., 2010) . Excitation was achieved through a D470/40X filter (Chroma) and emission was split using a 565DCXR dichroic beam-splitter (Chroma), subsequently collecting with HQ522/40M and HQ607/75M (Cairn Research) filters for GFP and RGECO, respectively. A Grass S9 stimulator and a stimulation bath (Warner Instruments) allowed field stimulation (10 Hz for 10 s) of neuronastrocyte co-cultures prepared as described previously. Movies were aligned using the Cairn Image Splitter plugin in ImageJ. Graphs showing F/F0 were plotted using Graph pad prism.
Regions of interest were manually drawn and fluorescence was normalized to the first 10 frames before stimulation.

Gupta S., Bazargani N., Drew J., Modi S., Marie H., Attwell D., & Kittler J.T. (2020). The non-adrenergic imidazoline-1 receptor protein Nischarin is a key regulator of astrocyte glutamate uptake.

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Single-molecule FRET Experiments on DNA

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The smFRET experiments were carried out on a customized inverted microscope (IX51, Olympus), with a high NA TIRF objective (100X, 1.49 NA, oil immersion, Olympus). Flow chambers were passivated with PEG and 30 mg/mL BSA before immobilizing 50 pM duplex DNA through biotin/neutravidin interactions, as previously described (Zhao et al., 2019 (link)). Then chambers were washed with reaction buffer to remove any free DNA not specifically immobilized to the surface. The reaction buffer contained 20 mM Tris-HCl [pH 7.5], 5 mM MgCl₂, 25 mM KCl, 0.25 mM DTT, 0.25 mM EDTA, 0.1 mg/ml BSA and an oxygen scavenging system composed of 0.8% (w/v) glucose, 0.5 mg/mL glucose oxidase, 0.4 μg/mL catalase, and 5 mM Trolox. The Cy3/Cy5 labeled duplex DNA were excited by a 532 solid-state laser (OEM Laser Systems), and the emission from the two dyes was split using a dichroic (FF660, Semrock) and narrow-band bandpass filters (582/75 and 680/42, Semrock) inside an Optosplit II (Cairn Research). Thousand-frame movies were recorded with an exposure time of 30 ms via an EMCCD camera (iXon+, Andor) at room temperature. Custom written MATLAB scripts were applied to view and analyze the fluorescence time trajectories and then to extract the FRET efficiency. Each normalized smFRET distribution was generated from a minimum of 200 trajectories obtained from at least two independent experiments.

Sharma S., Anand R., Zhang X., Francia S., Michelini F., Galbiati A., Williams H., Ronato D.A., Masson J.Y., Rothenberg E., Cejka P, & d’Adda di Fagagna F. (2021). MRE11-RAD50-NBS1 Complex Is Sufficient to Promote Transcription by RNA Polymerase II at Double-Strand Breaks by Melting DNA Ends. Cell Reports, 34(1), 108565.

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Real-time FRET Imaging of cAMP in Cardiac Myocytes

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The same Ringer solution as described above for Ca²⁺ transients and cell‐shortening measurements was used in these experiments. Images were captured every 5 seconds using the 40× oil immersion objective of a Nikon TE 300 inverted microscope connected to a software‐controlled (Metafluor, Molecular Devices, Sunnyvale, CA) cooled charge coupled (CCD) camera (Sensicam PE; PCO). Cyan fluorescent protein (CFP) was excited during 150 to 300 ms by a Xenon lamp (100 W; Nikon) using a 440/20BP filter and a 455LP dichroic mirror. Dual‐emission imaging of CFP and yellow fluorescent protein (YFP) was performed using an Optosplit II emission splitter (Cairn Research) equipped with a 495LP dichroic mirror and BP filters 470/30 and 535/30, respectively. A region of interest including the entire cell was used for measurement of average CFP and YFP intensity. CFP and YFP intensities were background corrected and the YFP emission was corrected for CFP bleed through. The ratio of CFP over corrected YFP was used as an index of cAMP concentration.

Molina C.E., Johnson D.M., Mehel H., Spätjens R.L., Mika D., Algalarrondo V., Slimane Z.H., Lechêne P., Abi‐Gerges N., van der Linde H.J., Leroy J., Volders P.G., Fischmeister R, & Vandecasteele G. (2014). Interventricular Differences in β‐Adrenergic Responses in the Canine Heart: Role of Phosphodiesterases. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 3(3), e000858.

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Fluorescence Anisotropy Imaging for Protein Dynamics

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A linear sheet polarizer was placed after the excitation filter in the filter cube, aligned to polarize light in the vertical direction. An Optosplit II (Cairn Research, Kent, England) with a polarizing beam splitter was attached to the left port of the IX83 frame. The field of view was reduced using apertures and the parallel and perpendicular components of the emission light were projected on two halves of the camera chip, allowing simultaneous recording.
The parallel and perpendicular channels were segmented out and aligned with a fully automated image processing pipeline. Background subtraction of the images was performed. Anisotropy, r, was calculated using the formula

where I_par is the intensity of the parallel channel and I_perp the intensity of the perpendicular channel, and G is the correction factor for any bias in observation between the parallel and perpendicular channels. G is calculated such that the anisotropy of fluorescein in aqueous buffer is zero.
Δanisotropy is defined as Δr = r_t – r₀ for a time series, where r₀ is the mean anisotropy value for a cell at the 0th time point and r_t is the mean anisotropy value at any given time point. The retention index is defined as the ratio of the intensity at the site of damage to the intensity outside the site of damage, as defined in the PARP1 and PCNA experiments.

Kesavan P.S., Bohra D., Roy S, & Mazumder A. (2020). Monitoring global changes in chromatin compaction states upon localized DNA damage with tools of fluorescence anisotropy. Molecular Biology of the Cell, 31(13), 1403-1410.

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Dual-Color Time-Lapse Microscopy of Cell Propulsion

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The fluorescence imaging of cells and particles was performed at 488 nm using a Nikon Eclipse Ti-U fluorescence microscope (Nikon Instrument, Japan) using a 40x objective and a dual emission image beam splitter with 525/50 nm and 647/57 nm mounted emission filters (Optosplit II; Cairn Research, UK) connected to an iXon3 897 EMCCD camera (Andor Technology Ltd, UK) at 10 frames per second. This setup enabled dual-color time-lapse microscopy of green fluorescent cells and red fluorescent particles. From these data, the propulsion orientation (push or pull) of the cell and the respective swimming speed was determined. The same data set was used to analyze the particle attachment along the cell body, with a particle attached within the first fifth of the cell body being considered as polar attachment.

Schauer O., Mostaghaci B., Colin R., Hürtgen D., Kraus D., Sitti M, & Sourjik V. (2018). Motility and chemotaxis of bacteria-driven microswimmers fabricated using antigen 43-mediated biotin display. Scientific Reports, 8, 9801.

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Quantifying Kinetochore Kinase Activity

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Live fluorescence ratio imaging was collected using a live cell microscopy imaging system built on a Nikon Ti-Eclipse inverted microscope equipped with a 100 × / NA1.40 Plan Apo oil immersion lens (Nikon), a charge-coupled device camera (Andor), a spinning disk confocal (Yokogawa), an XY-piezo Z stage (Prior NanoScan Z), and a laser merge module (ILE, Andor) controlled by iQ3 software (Andor). For live-cell imaging of FRET sensors, TFP was excited at 445 nm, while TFP and YFP (FRET channel) emissions were simultaneously acquired with a beam splitter (Optosplit II, Cairn Research Ltd). Images of cells were collected as confocal image stacks (five planes, 0.5-µm spacing). For data analysis images with YFP and CFP signal were cropped in ImageJ first, and then the FRET emission ratio (TFP/YFP) was calculated by using custom software written in MATLAB as previous described (Liu et al., 2009 (link)). Individual kinetochore was defined automatically from images presented as maximal intensity projections of confocal stacks, and FRET emission ratio was calculated on each kinetochore. According to the design of the FRET-based sensors, a higher FRET emission ratio indicates a lower kinase activity.

Xu L., Ali M., Duan W., Yuan X., Garba F., Mullen M., Sun B., Poser I., Duan H., Lu J., Tian R., Ge Y., Chu L., Pan W., Wang D., Hyman A., Green H., Li L., Dou Z., Liu D., Liu X, & Yao X. (2021). Feedback control of PLK1 by Apolo1 ensures accurate chromosome segregation. Cell reports, 36(2), 109343.

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Quantifying Kinetochore Kinase Activity

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Single-cell cAMP Measurement in Intestinal L-cells

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Single‐cell measurements of cAMP levels were made using intestinal cultures from transgenic mice expressing the Förster resonance energy transfer‐based sensor Epac2‐camps (Nikolaev et al. 2004) under the control of the proglucagon promoter. Briefly, primary colonic L‐cells, continuously perfused with saline solution with or without test reagents at a rate of ∼1 ml min⁻¹, were visualized with a 40× oil immersion objective on an inverted microscope (Olympus IX71). Excitation at 435 nm was achieved using a xenon arc lamp coupled to a monochromator (Cairn Research) controlled by MetaFluor software (Molecular Devices). Cyan fluorescent protein (CFP) emission at 470 nm and yellow fluorescent protein (YFP) emission at 535 nm were monitored using an Optosplit II beam splitter (Cairn Research) and an Orca‐ER digital camera (Hamamatsu Photonics KK) and expressed as the CFP/YFP fluorescence ratio. Data were smoothened with a sliding average across 30 s. Peak CFP/YFP ratios were determined at baseline (30 s period prior to test condition) and after test reagent application, and the response to test agent was expressed relative to baseline.

Pais R., Rievaj J., Meek C., De Costa G., Jayamaha S., Alexander R.T., Reimann F, & Gribble F. (2016). Role of enteroendocrine L‐cells in arginine vasopressin‐mediated inhibition of colonic anion secretion. The Journal of Physiology, 594(17), 4865-4878.

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Calcium Signaling Dynamics in spd1 Roots

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spd1 was crossed with YC3.6 stable lines, and the F₂ plants were scored. The CNGC15b RNAi vector, which utilized the pK7GW1WG2D (II) backbone containing a DsRED selection marker, was expressed in spd1^+/− YC3.6 through hairy root transformation. Heterozygous or homozygous spd1 carrying YC3.6 root cells were imaged on a TE2000-U inverted microscope (Nikon) equipped with a Spectra-X Light Engine (Lumencor). Cameleon was excited at a wavelength of 458 nm and captured with an electron-multiplying charge-coupled device camera (model ImagEM-1K camera, Hamamatsu). Emitted fluorescence was separated using an image splitter with a dichroic mirror (model Optosplit II, Cairn Research) and then passed through a cameleon filter set. Images were collected every 5 s with 1-s exposure and analyzed using MetaFluor software (Molecular Devices). The time-lapse images were saved as TIF graphic files and the pseudocolor ratio images were processed using ImageJ software (41 ).

Liu H., Lin J.S., Luo Z., Sun J., Huang X., Yang Y., Xu J., Wang Y.F., Zhang P., Oldroyd G.E, & Xie F. (2022). Constitutive activation of a nuclear-localized calcium channel complex in Medicago truncatula. Proceedings of the National Academy of Sciences of the United States of America, 119(34), e2205920119.

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Multicolor 3D Imaging of Live Cells

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Experiments were performed on a modified Andor Revolution system spinning disk confocal microscope (Andor Technology, Belfast, UK) (Figure A1). The system is built using a Nikon TE2000E (Nikon, Tokyo, Japan) microscopy body, a spinning-disk unit (CSU10; Yokogawa Electric Corporation, Musashino, Japan), an OptoSplit II (Cairn Research Ltd., Faversham, UK) for separating the eGFP and mCherry emissions and an EMCCD camera (DU-897 Ixon, Andor Technology, Belfast, UK) for detection. The excitation was controlled using an acousto-optic tunable filter (Gooch and Housego, Ilminster, UK) and the excitation and fluorescence emission were separated using a quadruple-band dichroic beam splitter (Di01-T405/488/568/647; Semrock, Rochester, NY, USA). The eGFP and mCherry signals were separated using a dichroic beamsplitter (BS562) and the respective emission filters (HC525/50, and ET605/70), all purchased from AHF Analysentechnik AG (Tübingen, Germany). Z-stacks were recorded over 20 min with an exposure time of 130 ms/frame/plane and 15–25 z positions spaced by 300 nm were acquired per z-stack. This resulted in a complete three-dimensional (3D) image every ~3 to 5 s.

Dupont A., Glück I.M., Ponti D., Stirnnagel K., Hütter S., Perrotton F., Stanke N., Richter S., Lindemann D, & Lamb D.C. (2020). Identification of an Intermediate Step in Foamy Virus Fusion. Viruses, 12(12), 1472.

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