Optoscan monochromator, by Cairn Research - Product details

1

Holographic Photostimulation and Widefield Imaging

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An analogous holographic photostimulation path was coupled with widefield epifluorescence imaging on a second system, here denoted as setup 2 (see Supplementary Fig. S6).
This system was built around an Olympus BX51WI upright microscope, capable of widefield epifluorescence imaging using illumination with an arc lamp, (OptoSource Illuminator, Cairn Research, coupled with a monochromator, Optoscan Monochromator, Cairn Research), and an Orca Flash 4.0 Hamamatsu CCD camera for epifluorescence imaging. The native infrared differential-interference contrast (DIC) path of the Olympus microscope allowed DIC imaging on the CCD.
The holographic photoactivation laser source consisted of a conventional pulsed Ti:Sapphire laser, used at 920 nm (pulse width: 100 fs, repetition rate: 80 MHz, Mai-Tai, Deep-See, Spectra Physics).
The holographic path was analogous to the one described for setup 1: a beam expander enlarged the beam in front of the spatial light modulator (LCOS-SLM X10468-02), whose plane was projected at the back focal plane of a 40×-NA 0.8 objective (LUM PLAN FI/IR, Olympus) by an afocal telescope (f=750mm, Thorlabs #AC508-750-B and f=500mm Thorlabs #AC508-500-B). The holographic beam was coupled to the optical axis of the microscope by a dichroic mirror (FF670, SDi01, 25×36 mm, Semrock). Photostimulation light pulses were generated by a Pockels cell (350-80, Conoptics).

Shemesh O.A., Tanese D., Zampini V., Linghu C., Piatkevich K., Ronzitti E., Papagiakoumou E., Boyden E.S, & Emiliani V. (2017). Temporally precise single-cell resolution optogenetics. Nature neuroscience, 20(12), 1796-1806.

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2

Holographic Photostimulation and Widefield Imaging

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An analogous holographic photostimulation path was coupled with widefield epifluorescence imaging on a second system, here denoted as setup 2 (see Supplementary Fig. S6).
This system was built around an Olympus BX51WI upright microscope, capable of widefield epifluorescence imaging using illumination with an arc lamp, (OptoSource Illuminator, Cairn Research, coupled with a monochromator, Optoscan Monochromator, Cairn Research), and an Orca Flash 4.0 Hamamatsu CCD camera for epifluorescence imaging. The native infrared differential-interference contrast (DIC) path of the Olympus microscope allowed DIC imaging on the CCD.
The holographic photoactivation laser source consisted of a conventional pulsed Ti:Sapphire laser, used at 920 nm (pulse width: 100 fs, repetition rate: 80 MHz, Mai-Tai, Deep-See, Spectra Physics).
The holographic path was analogous to the one described for setup 1: a beam expander enlarged the beam in front of the spatial light modulator (LCOS-SLM X10468-02), whose plane was projected at the back focal plane of a 40×-NA 0.8 objective (LUM PLAN FI/IR, Olympus) by an afocal telescope (f=750mm, Thorlabs #AC508-750-B and f=500mm Thorlabs #AC508-500-B). The holographic beam was coupled to the optical axis of the microscope by a dichroic mirror (FF670, SDi01, 25×36 mm, Semrock). Photostimulation light pulses were generated by a Pockels cell (350-80, Conoptics).

Shemesh O.A., Tanese D., Zampini V., Linghu C., Piatkevich K., Ronzitti E., Papagiakoumou E., Boyden E.S, & Emiliani V. (2017). Temporally precise single-cell resolution optogenetics. Nature neuroscience, 20(12), 1796-1806.

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3

Measuring Cardiomyocyte Ca2+ Dynamics

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To measure SR Ca²⁺ concentration ([Ca²⁺]_SR), freshly isolated rabbit cardiomyocytes were loaded with 8 μmol/L Mag-Fluo-4-AM (Invitrogen, Carlsbad, CA, USA) with 0.2% Pluronic F-127 (Biotium, Hayward, CA, USA) for 2 h at room temperature. Subsequently, cells were washed twice in fresh Tyrode’s solution for 30 min to allow de-esterification to occur. Then, cardiomyocytes were placed in a narrow bath chamber with embedded field stimulation electrodes (RC-27NE2, Warner Instruments) and stimulated at 0.5 Hz frequency in Tyrode’s solution at room temperature (22 ± 1 °C). Mag-Fluo-4 was excited at 480 nm wavelength using an Optoscan monochromator (Cairn Research, Faversham, UK) and fluorescence emission was collected at 535 ± 15 nm.
To measure [Ca²⁺]_i, cardiomyocytes were loaded with 10 μmol/L Rhod2-AM (ThermoFisher, Waltham, MA, USA) for 10 min at room temperature and subsequently left to de-esterify in fresh Tyrode’s solution for a minimum of 30 min. Then, cardiomyocytes were placed in a RC-27NE2 recording chamber and stimulated at 0.5 Hz frequency in Tyrode’s solution at room temperature. Rhod2 was excited at 561 nm wavelength using an Optoscan monochromator, and fluorescence was collected at 530 ± 20 nm. Fluorescence signals were recorded after steady state was reached in the cell during pacing.

Hegyi B., Pölönen R.P., Hellgren K.T., Ko C.Y., Ginsburg K.S., Bossuyt J., Mercola M, & Bers D.M. (2021). Cardiomyocyte Na+ and Ca2+ mishandling drives vicious cycle involving CaMKII, ROS, and ryanodine receptors. Basic Research in Cardiology, 116(1), 58.

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4

Calcium Imaging with Channelrhodopsin Activation

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The growth medium was supplemented with 2 µM Fura-2-acetoxymethyl ester (Life Technologies GmbH, Darmstadt, Germany) 45 min, prior to measurements. The excitation light, sourced from an Optoscan Monochromator (Cairn Research, Kent, UK), was coupled to the optical path of an inverted microscope Olympus IX70 (Olympus, Tokyo, Japan) equipped with an Olympus ×40 water immersion objective (Olympus, Tokyo, Japan) and a FF493/574 dichroic mirror (AF-Analysetechnik, Tübingen, Germany) for fluorescence imaging with a pco.panda 4.2 sCMOS camera (Kelheim, Germany). For ChR activation, the Optoscan Monochromator was operated at a centre wavelength of 470 ± 5 nm for 10 s with an intensity of ~0.08 mW/mm². For Fura-2 excitation, consecutive 50 ms exposure flashes of 340 ± 10 nm (0.02 mW/mm²) and 380 ± 10 nm (0.14 mW/mm²) were applied with the Optoscan Monochromator. The sampling rate of the Fura-2 signal was 0.5 Hz. The bath solution used for Fura-2-AM imaging contained 1 mM [NaCl], 1 mM [KCl], 1 mM [CsCl], 2 mM [MgCl₂], 70 mM [CaCl₂] and 10 mM [HEPES], adjusted to pH 7.2 and 320 mOsm using glucose.

Fernandez Lahore R.G., Pampaloni N.P., Peter E., Heim M.M., Tillert L., Vierock J., Oppermann J., Walther J., Schmitz D., Owald D., Plested A.J., Rost B.R, & Hegemann P. (2022). Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling. Nature Communications, 13, 7844.

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5

Fura-2 AM Calcium Imaging and Tas1r1 Expression Analysis

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Prior to imaging, the slices were incubated for 75–95 min with 1.25 μM Fura-2 AM (Fura-2 dissolved in 20% Pluronic F-127 solution in DMSO, and diluted 1:800 into low-glucose aCSF; Life Technologies, UK). After incubation, the slices were transferred into a holding chamber with low-glucose aCSF to wash out excess Fura-2 AM and DMSO/pluronic. For imaging, they were transferred to a flow chamber on an Olympus BX51 microscope equipped with a 60× water immersion objective (NA 1.0). Ratiometric imaging of Fura-2 emissions at excitation wavelengths of 340 and 380 nm (provided by a Cairn Research Optoscan monochromator) was performed under the control of the MetaFluor software.
Brain samples for Tas1r1 expression imaging were obtained from the German Institute of Human Nutrition Potsdam-Rehbruecke. We used the tissue of Tas1r1-Cre/eR26-tauGFP mice that expressed GFP at the site of Tas1r1 [22] (link), [23] (link). The tissue was cut at 35 μm using Bright OTF 5000 cryostat and mounted on slides with VECTASHIELD (Vector Laboratories, US) containing DAPI. We used Leica SP5 confocal laser microscope for imaging and FIJI software for further analysis.

Lazutkaite G., Soldà A., Lossow K., Meyerhof W, & Dale N. (2017). Amino acid sensing in hypothalamic tanycytes via umami taste receptors. Molecular Metabolism, 6(11), 1480-1492.

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6

Redox Imaging of FAD and NADH in Cells

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Redox (FAD/FAD+NADH) imaging was performed using a customized fluorescence microscope with a 40×/0.8 W objective (Olympus) for time-lapse imaging. The endogenous autofluorescence images^{16 (link)} were excited with an Optoscan monochromator (Cairn Research Ltd., Kent, UK). The pseudocolor of the FAD channel is red (excited by 430-nm light with 20-nm bandwidth and collected with 525–575-nm bandpass filter), whereas the NADH+FAD channel is green (sequentially excited by 340-nm light with 20-nm bandwidth and collected with 420-nm long pass filter). In addition, the fluorescence images were acquired with an Evolve 512 EMCCD (Photometrics Ltd., Tucson, UK). Time-lapse imaging of BA was performed in 4 ml of Tyrode’s solution at 33 °C. Sixty frames were recorded in a time-lapse imaging at 30-s intervals. NE (0.1 μM) was injected as early as the 11th frame of time-lapse imaging for the redox studies in BA. The other conditions of imaging, data acquisition and analysis for the redox study were the same as the conditions in the thermogenic study.

Xie T.R., Liu C.F, & Kang J.S. (2017). Sympathetic transmitters control thermogenic efficacy of brown adipocytes by modulating mitochondrial complex V. Signal Transduction and Targeted Therapy, 2, 17060-.

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7

Cytoplasmic Calcium Imaging Protocol

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Cytoplasmic [Ca²⁺] imaging was performed using the customized fluorescence microscope with a 40×/0.8 W objective (Olympus) for time-lapse imaging and an Optoscan monochromator (Cairn Research Ltd.) as a light source. Cells were stained with 5 μM Fura2-AM in Tyrode’s solution for 30 min at 37 °C. Then, Fura2-AM was washed out using Tyrode’s solution. The pseudocolor of the 380-nm channel is red (excited by 380-nm light with 10-nm bandwidth and collected with 505–535-nm bandpass filter), and the 340-nm channel is green (sequentially excited by 340-nm light with 10-nm bandwidth and collected with 505–535-nm bandpass filter). Ratiometric values of the 340-nm channel to the 380-nm channel were calculated pixel by pixel to represent the relative [Ca²⁺] of the sample. The other conditions of imaging, data acquisition and analysis for the cytoplasmic [Ca²⁺] study were the same as the conditions in the redox study.

Xie T.R., Liu C.F, & Kang J.S. (2017). Sympathetic transmitters control thermogenic efficacy of brown adipocytes by modulating mitochondrial complex V. Signal Transduction and Targeted Therapy, 2, 17060-.

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8

Quantifying Astrocytic Nitric Oxide

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Optical recordings of NO production in cultured astrocytes were performed using an inverted epifluorescence Olympus microscope, equipped with a cooled CCD camera (Clara model; Andor). The cells were loaded with the NO sensitive fluorescent probe DAR-4M-AM (Sigma; 10 µM, 30 min incubation at room temperature) or transduced to express the genetically encoded NO sensor geNOp. Recordings were performed in a custom-made flow-through imaging chamber at ~32°C in aCSF saturated with 95% O₂ / 5% CO₂ (pH 7.4). The rate of chamber perfusion with aCSF was 4 ml min^-1. DAR-4M fluorescence was excited by using a Xenon arc lamp and an Optoscan Monochromator (Cairn Research) at 560/10 and the fluorescence emission was recorded at 590 nm. geNOp fluorescence was excited at 488/10 nm and the fluorescence emission was recorded at 535 nm.
Hypoxic conditions in vitro were induced by the displacement of oxygen in the medium by argon. In all experiments in cell cultures and organotypic slices the hypoxic challenge was applied for 5-15 min. A representative profile of PO₂ changes in the recording chamber during argon displacement is illustrated by Figure 2F. All test drugs were applied ~10 min before the hypoxic challenge. Imaging data were collected and analyzed using Andor iQ3 software (Andor).

Christie I.N., Theparambil S.M., Braga A., Doronin M., Hosford P.S., Brazhe A., Mascarenhas A., Nizari S., Hadjihambi A., Wells J.A., Hobbs A., Semyanov A., Abramov A.Y., Angelova P.R, & Gourine A.V. (2023). Astrocytes produce nitric oxide via nitrite reduction in mitochondria to regulate cerebral blood flow during brain hypoxia. Cell Reports, 42(12), 113514.

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9

Redox Imaging with FAD and NADH

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Redox (FAD/FAD + NADH) imaging was performed using a customized fluorescence microscope with a 40/0.8 W objective (Olympus) for time-lapse imaging. The endogenous autofluorescence images were excited with Optoscan monochromator (Cairn Research Ltd., UK). FAD channel is excited by 430 nm light with 20 nm bandwidth and collected with 525–575 nm band pass filter whilst NADH + FAD channel sequentially excited by 340 nm light with 20 nm bandwidth and collected with 420 nm long pass filter. The fluorescence images were acquired with Evolve 512 EMCCD (Photometrics Ltd., UK). Time-lapse images of the cells were performed in 4 mL Tyrode’s solution at 33 °C. MATLAB (MathWorks Inc. USA) and ImageJ (NIH, USA) were applied to analyze images.

Shen L., Xie T.R., Yang R.Z., Chen Y, & Kang J.S. (2018). Application of a dye-based mitochondrion-thermometry to determine the receptor downstream of prostaglandin E2 involved in the regulation of hepatocyte metabolism. Scientific Reports, 8, 13065.

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10

Wide-field epifluorescence microscopy for live imaging

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We used wide-field epifluorescence microscopy for all live imaging experiments (Fig. 1A). An Optoscan monochromator (Cairn Research, Faversham, UK) uniformly illuminated the preparations with 488 ± 15-nm light using either a Leica DM-LFS (Leica Microsystems) or an Olympus X50WI compound microscope (Olympus, Center Valley, PA). Emitted light passed through standard green fluorescent protein (GFP) emission filters before reaching an Andor DU897 EMCCD camera (Andor Technologies, Belfast, UK). Images were captured at 5 or 10 Hz with Andor IQ software and constant gain settings. Optical and electrical recordings were synchronized with pulses generated during each camera exposure. Images were stabilized against lateral shifts in ImageJ (NIH, Bethesda, MD). Fluorescence values were extracted from regions of interest (ROIs) in thoracic (T2–T3) and abdominal (A1–A8/9) ganglia with ImageJ or custom MATLAB scripts. T1 and the SOG were obscured by the brain and were not analyzed. Extracted optical signals were analyzed in ImageJ, MATLAB, and Spike2. Signals are expressed as the percent change in fluorescence from baseline, ΔF/F. Changes of 50% ΔF/F were typical in all segments.

Pulver S.R., Bayley T.G., Taylor A.L., Berni J., Bate M, & Hedwig B. (2015). Imaging fictive locomotor patterns in larval Drosophila. Journal of Neurophysiology, 114(5), 2564-2577.

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Optoscan monochromator

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10 protocols using optoscan monochromator

Holographic Photostimulation and Widefield Imaging

Holographic Photostimulation and Widefield Imaging

Measuring Cardiomyocyte Ca2+ Dynamics

Calcium Imaging with Channelrhodopsin Activation

Fura-2 AM Calcium Imaging and Tas1r1 Expression Analysis

Redox Imaging of FAD and NADH in Cells

Cytoplasmic Calcium Imaging Protocol

Quantifying Astrocytic Nitric Oxide

Redox Imaging with FAD and NADH

Wide-field epifluorescence microscopy for live imaging

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