The array AFM system is adapted from a MultiMode AFM (Bruker) with Nanoscope III controller by customizing both illumination and deflection beam paths. A supercontinuum laser (Extreme; NKT Photonics. Inc.) is used to illuminate cantilever arrays through a dispersive grating and an objective lens. The AFM head is customized to have a top opening for illumination and side opening for deflection beam detection. Two QPDs (Skyhunt) are used to monitor the deflected beams from two cantilevers, respectively. The two deflected beams are separated by a dichroic mirror (Semrock). Two programmable amplifiers (Alligator Technologies)/locking-in amplifiers (SR830 DSP; Stanford Research Systems) amplify the signal from QPDs and send it to AFM through a Signal Access Module. The system is operated with passive cantilever array, and all of the AFM images are taken in constant height mode. In our current complementation, the supercontinuum laser was operated with around 300-ps pulse duration at 78-MHz repetition frequency. The peak power was around 40 times the average power. Considering the comparatively high peak energy and large laser spot size, special attention has to be paid when imaging photosensitive specimens, and the laser power, pulse duration, and wavelength range should be adjusted accordingly (50 , 51 (link)).
Dichroic mirror
Manufactured by IDEX Corporation
Sourced in United States
A dichroic mirror is an optical component that selectively reflects certain wavelengths of light while allowing others to pass through. It is made by depositing thin, alternating layers of dielectric materials onto a transparent substrate. This creates a wavelength-dependent reflective and transmissive behavior, which can be tailored to specific applications in optical systems.
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Lab products found in correlation
Live cell imaging solution, by Thermo Fisher Scientific (2 mentions)
35 mm glass bottom dishes, by MatTek (2 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (2 mentions)
Ix71 inverted microscope, by Olympus (2 mentions)
Multimode afm, by Bruker (1 mentions)
Sr830 dsp, by Stanford Research Systems (1 mentions)
Raman spectrometer, by Oxford Instruments (1 mentions)
Gaasp photomultiplier tube, by Hamamatsu Photonics (1 mentions)
Deepsee, by Spectra-Physics (1 mentions)
Picoharp 300, by PicoQuant (1 mentions)
Band pass filter, by IDEX Corporation (1 mentions)
Spec 10, by Teledyne (1 mentions)
Superk extreme, by NKT Photonics (1 mentions)
Pcie 7842r, by National Instruments (1 mentions)
Beam splitter, by Thorlabs (1 mentions)
Plan fluorite objective, by Olympus (1 mentions)
Photomultiplier tube, by Hamamatsu Photonics (1 mentions)
Phantom v9, by Ametek (1 mentions)
Sp8 2 photon microscope, by Leica (1 mentions)
Mai tai hp deepsee laser, by Spectra-Physics (1 mentions)
15 protocols using dichroic mirror
1
Customized Array Atomic Force Microscopy
2
Raman Spectra of Mouse PASMC
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Raman spectra of mouse WT and AOX PASMC were measured using a home-built microscope equipped with a ×40 magnification microscope objective (Olympus, Tokyo, Japan), a Raman spectrometer (Andor Technology, Belfast, UK), and a laser with an excitation wavelength of 532 nm (Altechna, Vilnius, Lithuania). The Raman signal was filtered out from the laser light using a filter cube containing a dichroic mirror (Semrock, Rochester, NY, USA) and a 532-nm edge filter (Semrock). An in-house–designed gas-tight microfluidic system was used to register Raman spectra on the cultured PASMC at different oxidation states, i.e., normoxic and hypoxic conditions (48 (link)). The sample was illuminated with a laser output power of 3.1 mW with an integration time of 60 s. Acquired Raman data were processed in two steps. First, Cosmic rays were removed using the 2D second difference (49 (link)). Second, an optimal reconstruction was done using a Savitzky-Golay filter (50 ). To confirm the Raman signal from the single live cells, background spectra of the microfluidic system and the buffer were taken.
3
Mitochondrial Dynamics Imaging in MEF Cells
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Confluent MEF cells were harvested, seeded onto 35 mm glass-bottom dishes (MatTek Life Sciences) coated with poly-D-lysine (0.1 mg/mL) and allowed to grow overnight at 37 °C under 5% CO2. For visualization of mitochondria, cells were stained with 50 nM MitoTrackerTM Deep Red FM (Thermo Fisher Scientific) at 37 °C for 15 min. Following three rounds of washes with 1X PBS, cells were placed in Live Cell Imaging Solution (Invitrogen). Imaging relating to Fig. EV3 was performed using Zeiss Axio Observer Z1 Advanced Marianas™ Microscope system, an Alpha Plan-Apochromat ×100/1.46 NA Oil TIRF Objective M27 and Prime 95B scientific CMOS camera (Photometrics). MitoTrackerTM-stained mitochondria were imaged using “Cy5” filter set (Cy5-4040C, Excitation: 628/40 nm [608-648 nm], Emission: 692/40 nm [672–712 nm], Dichroic Mirror: 660 nm) (Semrock). Temperature, humidity, and CO2 concentrations were controlled with an Okolab Microscope Stage Incubator System. Image acquisition and processing were done using SlideBookTM6 (Intelligent Imaging Innovations, Inc., Denver, CO) and Fiji (Schindelin et al, 2012 (link)). Time-lapse videos of stained mitochondria were taken at one frame per 30 s for a duration of 5 min.
4
Visualizing Mitochondrial Dynamics in Confluent MEF Cells
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Confluent MEF cells were harvested, seeded onto 35 mm glass-bottom dishes (MatTek Life Sciences) coated with poly-D-lysine (0.1 mg/mL) and allowed to grow overnight at 37°C under 5% CO2. For visualization of mitochondria, cells were stained with 50nM MitoTracker™ Deep Red FM (Thermo Fisher Scientific) at 37°C for 15 mins. Following three rounds of washes with 1X PBS, cells were placed in Live Cell Imaging Solution (Invitrogen). All fluorescence imaging was performed using Zeiss Axio Observer Z1 Advanced Marianas™ Microscope system, an Alpha Plan-Apochromat 100×/1.46 NA Oil TIRF Objective M27 and Prime 95B scientific CMOS camera (Photometrics). MitoTracker™-stained mitochondria were imaged using “Cy5” filter set (Cy5–4040C, Excitation: 628/40 nm [608–648nm], Emission: 692/40 nm [672–712nm], Dichroic Mirror: 660nm) (Semrock). Temperature, humidity, and CO2 concentrations were controlled with an Okolab Microscope Stage Incubator System. Image acquisition and processing were done using SlideBook™6 (Intelligent Imaging Innovations, Inc, Denver, CO) and Fiji (Schindelin et al., 2012 (link)). Time-lapse videos of stained mitochondria were taken at one frame per 30 seconds for a duration of 5 mins.
5
Two-photon Imaging of Striosomal and Matrix Neurons
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Two-photon Ca++ imaging procedures were as described previously53 (link). Briefly, GCaMP6s and tdTomato fluorescence was imaged through a LUMPlan FL, ×40, 0.8NA objective using galvo-galvo scanning with a Prairie Ultima IV 2-photon microscopy system (Bruker). Excitation light at 910 nm was provided by a tunable Ti:Sapphire laser equipped with dispersion compensation (Mai Tai Deep See, Spectra-Physics). Green and red fluorescence emission signals were split with a dichroic mirror (Semrock) and directed to GaAsP photomultiplier tubes (Hamamatsu). Images were acquired at a frame rate of 5 Hz. Laser power at the sample ranged from 11 to 42 mW, depending on the imaging depth and level of GCaMP6s expression. We selected fields of view (FOVs) that allowed simultaneous imaging of striosomal and matrix neurons. The FOVs had both clearly labeled GCaMP6s-expressing cells in striosomes, as defined by dense tdTomato signal in the neuropil, as well as in areas free of tdTomato labeling. Cells were classified as striosomal or matrix depending on whether they were found inside the tdTomato-expressing neuropil zones. In total, we imaged 75 FOVs in 13 mice.
6
Imaging Cortical Dynamics in ArcLight Mice
7
Photoluminescence Spectroscopy of Single Crystals
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Photoluminescence (PL) spectra and lifetimes in single crystals were measured at the Center for Nanoscale Materials (CNM) at Argonne National Laboratory, using a home-made fluorescence microscope fitted with a liquid nitrogen cooled, continuous flow cryostat (Janis ST-500UC) in CNM. The instrument was based on an Olympus IX-71 inverted microscope. A pulsed 375 nm laser (PicoQuant, DC375M), operated at 4 MHz, was used to excite the sample through a ThorLabs LMU-15X-NUVobjective that was used to both focus the incoming laser light and collect the emitted PL. The collected PL was separated from the exciting laser using a dichroic mirror and a bandpass filter (both Semrock). A low pass filter (LPF) of 468 nm was used for both the PL spectra and TCSPC measurements. The PL was then routed either to a spectrograph (Princeton Instruments, SpectraPro-300) fitted with a CCD camera (Princeton Instruments, PIXIS) or, for lifetime measurements, to a fiber-coupled single photon avalanche diode (SPAD) (Micro Photon Devices, PDM). The output from the SPAD and a trigger pulse from the laser power supply were fed to the two input channels of a time-correlated single photon counting (TCSPC) system (PicoQuant, PicoHarp 300). The relevant lifetimes are provided in Table S4. The emission spectra and normalized emission decay plots are provided in Figs. 2(a) –2(d) .
8
Miniaturized Fluorescence Microscope Design
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The miniaturized microscope was designed for both bright-field and fluorescence imaging, and fabricated by assembling a CMOS camera (FLIR, Inc., Victoria, British Columbia, Canada), a dichroic mirror (Semrock, Inc., Rochester, NY, USA), an excitation filter (Semrock) with a 474 nm center wavelength, an emission filter (Semrock) with a 525 nm center wavelength, a long-pass filter (Edmund Optics, Inc., NJ, USA) with a 500 nm cut-on wavelength, a liquid lens (Optotune, Inc., Zurich, Switzerland), a white LED (JENO Corp., Seoul, Korea), and a UV LED (LED Engin, Inc., San Jose, CA, USA) (Figure 1 and Figure S1 ). The housing for the optical components was printed with the 3D printer. The long-pass filter was placed between the white LED and microfluidic chamber to prevent the UV light from unintentionally illuminating a phosphor coated on the emitter of the white LED. Thus, this optical setup enables clear fluorescence imaging without a mechanical shutter. The liquid lens was used for rapid autofocusing during bright-field and fluorescence cell imaging, allowing for the rapid acquisition of multiple in-focus images. In addition, the incorporation of an electronic on–off switch enables easy transition between the bright-field and fluorescence imaging mode. The field of view (FOV) of the miniaturized microscope was 0.61 mm × 0.46 mm.
9
Raman Microscopy with Polarized Light
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The microscope and cryostat used for high-speed camera recording were also used for the Raman microscopy. The output of a continuous-wave diode laser (λ = 785 nm) was coupled to the microscope, directed toward the sample by a dichroic mirror (Semrock), and focused onto the sample by a 50 × IR objective lens (Olympus)44 . Scattered light from the sample was collected by the same objective lens and imaged on the entrance slits of an imaging monochromator (Acton Research, SpectraPro 2150) through a long-pass filter (Semrock) that blocked the excitation laser. The spectra were recorded by a liquid-nitrogen-cooled back-illuminated CCD camera (Princeton Instruments, Spec10, 1340 pixels × 400 pixels) attached to the monochromator. For the polarized Raman microscopy measurements, polarizers were placed before and after the sample.
10
Near-Infrared Fluorescence Spectroscopy of OCC-DNAs
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