Ultra 897

One-photon trans-craniotomy imaging was performed on awake head fixed mice, voluntarily running on an air-floating Styrofoam sphere, or immobilised in a MAG-1 mouse holder (Narishige, Japan). A self-build microscope (Cerna, Thorlabs) equipped with a 4x objective (RMS4X-PF, Olympus) was used. Fluorescence was recorded with a cooled EMCCD camera (Andor iXon Ultra897) at indicated frame rates. jRGECO and mClY were excited using a 580 nm and 470 nm LED (CoolLED pE-4000), respectively, and filtered by a dual band filter set (ET488/561x, ET488/561rdc, Chroma). Emitted light was first filtered with a 694 nm short pass filter to block the light from the behaviour LED (FF02-694/SP-25, Semrock) and further filtered with a 500 nm (ET500LP, Chroma) and 575 nm (AT575LP) long pass filter for mClY and jRGECO emission, respectively. Images were collected with μManager^{71 (link)} (Version 2.0) and stored as 16-bit uncompressed tiff files.

Imaging was performed on a custom-made inverted single-molecule fluorescence microscope built around a commercial microscope frame (Olympus IX73). The illumination laser wavelength was at 488 nm (Coherent Sapphire) for excitation of the YFP derivate Venus in combination with a 525/45 emission filter (Semrock) and a dichroic beam splitter (Chroma ZT405/488/561/640rpc). The laser beam was circularly polarized to excite fluorescent proteins homogeneously regardless of their orientation. The microscope was equipped with an EM-CCD camera (Andor iXon Ultra 897) with effective pixel size on the sample of 118 nm. A 100x NA = 1.49 oil immersion TIRF objective (Olympus UAPON100XOTIRF) was used.

The LightPath optical imaging system (Lightpoint Medical Ltd., Chesham, UK) is developed for intraoperative margin assessment using the Cerenkov radiation induced by β-emitting radionuclides. This system is equipped with a camera lens (F/0.95, 512 × 512 pixels) coupled through optics to a − 80 °C cooled electron multiplying charge coupled device (EMCCD; Andor iXon Ultra 897). The EMCCD is shielded with a tungsten plate and folded optics are used to reduce the number of gamma photons striking the EMCCD sensor. A standard optical camera (F/1.4 lens, 1600 × 1200 pixels) was used to acquire white-light reference images. The system has a light-tight imaging chamber to shield from ambient light.
In this study, images were initially acquired using the previously published protocol for ¹⁸F with an exposure time of 300 s and 8-pixel binning (E300B8) [16 (link)]. To find a suitable protocol for clinical application using ⁶⁸Ga, acquisition settings were varied with exposure times of 60, 120, and 300 s without or with the use of 2-, 4-, or 8-pixel binning. Acquisition protocols applied for both radionuclides. Unless otherwise specified, images were acquired without an optical filter. Data analysis was performed using MATLAB R2017b (The MathWorks, Natick, 2017).

Imaging was performed on our lab-built inverted fluorescence microscope. Cells were imaged through a Nikon CFI Plan Apo VC 100x 1.4 NA objective. A retractable external phase plate (Ti-C CLWD Ph3 Annulus Module) was inserted into the light path during phase-contrast imaging but removed for fluorescence imaging to avoid decreased signal due to the neutral density annulus on the phase plate.
For fluorescence imaging, we excite GFP and YPet proteins using a Coherent Sapphire 50 mW 488 nm or 150 mW 514 nm CW laser (see Table 2). The beam diameter is expanded, providing uniform illumination over the field of view. An Acousto-Optic Tunable Filter (AOTF, AA Opto-Electronic AOTFnC-400.650) controls the laser excitation intensity. Images were collected on an iXon Ultra 897 512x512 pixel EMCCD camera. The microscope system is controlled by Micro-Manager.

Epifluorescence imaging was performed on a fully automated iMIC-based microscope from FEI/Till Photonics, using an Olympus 100 × 1.4 NA objective and DPSS lasers at 488 nm (Cobolt Calypso, 75 mW) and 561 nm (Cobolt Jive, 150 mW) as light sources. Lasers were selected through an AOTF and directed through a broadband fiber to the microscope. A galvanometer-driven two-axis scan head was used to adjust laser incidence angles. Images were collected using an Imago-QE Sensicam camera. Acquisition was controlled by LiveAcquisition software (Till Photonics). Fluorescence Recovery After Photobleaching (FRAP) of actin-GFP was performed using a third galvanometer-controlled mirror (Polytrope) to switch between wide-field and FRAP modalities. Ablation experiments were carried out on an iMIC setup equipped with a pulsed 355 nm picosecond UV laser (Sepia, PicoQuant) as previously described (Raabe et al., 2009 (link)). Confocal microscopy was performed on an iMIC42 setup equipped with a spinning disk unit (Andromeda) using Olympus 20x air (NA 0.75) and 60x oil immersion (NA 1.49) objectives. Images were taken using typical filter settings for excitation and emission of fluorescence probes/proteins and recorded on EMCCD cameras (Andor iXon Ultra 897).