Pixis 400br

A 7 mm diameter optical probe (EMVision, Loxahatchee, Florida) is used to deliver 80 mW of power from a 785 nm diode laser (Innovative Photonics Solutions, Monmouth Junction, New Jersey). Raman signal is acquired by detector fibres in the probe and delivered to a near-infrared-optimised spectrograph (LS785, Princeton Instruments, Princeton, New Jersey), and recorded by a deep depletion, thermo-electrically cooled CCD (Pixis 400BR, Princeton Instruments). The 3D scanner component of Marginbot consists of two motors and servomotors (Tower Pro, Shenzen City, China), which is controlled by a laptop. A customised program code written in LabVIEW (National Instruments, Austin, Texas) and MATLAB (Mathworks, Natick, Massachusetts) can (i) reconstruct a 3D diagram of the specimen margin based from captured images of it from all angles by a camera (Genius, Doral, Florida), (ii) control the movement of motors and servomotors, and (iii) receive feedback from these components via a signal relay port (Belkins, Playa Vista, California).

The transmittance spectra of Ag/Au alloyed nanoislands were measured with an inverted microscope (Carl Zeiss, Axiovert 200M) equipped with a commercial spectrometer (Princeton Instruments, MicroSpec 2300i) and a charge-coupled device camera (Princeton Instruments, PIXIS: 400BR). The transmittance spectra were collected with 50x objective lens.

The excised tumors were snap-frozen in liquid nitrogen and stored at -80 ^oC. The frozen tumors were thawed in PBS and fixed in 10% neutral buffered formalin prior to the Raman measurements. The formalin fixed tumors were washed in PBS and sandwiched between a quartz coverslip and an aluminum plate for Raman mapping measurements. A previously described fiber probe-based portable clinical Raman spectroscopy system was used to perform Raman mapping in the current study 50 (link). Briefly, the system is comprised of an 830 nm diode laser (Process Instruments, maximum power: 500 mW) for excitation, a spectrograph (Holospec f/1.8i, Kaiser Optical Systems), and a thermoelectrically cooled CCD camera (PIXIS 400BR, Princeton Instruments). The laser power of ca. 20 mW was delivered to the tissue via a fiber-optic probe mounted on a motorized 2-D translational stage (T-LS13M, Zaber Technologies Inc., travel range: 13 mm) to acquire spectra from distinct points on the flattened tumors approximately 1 mm apart. Each spectrum from spatially distinct points was acquired for 5 seconds (10 accumulations of 0.5 seconds each to prevent CCD saturation).

To explore possible molecular changes in spores under high vacuum, a special vacuum chamber was developed (Supplementary Fig. 1), which allows direct observation of individual spores under high vacuum using image-guided single-cell confocal Raman micro-spectroscopy.^{38 (link),39 (link)} Briefly, the spores (~1 μl of 10⁸ spores/ml in water) were air dried on a quartz coverslip (0.1 mm thick) that was sealed on a window of a vacuum chamber pumped with a Turbo pumping station (Pfeiffer Vacuum, Berlin, Germany). The bright-field or phase-contrast image of multiple individual spores adhered in random positions on the quartz coverslip was recorded with an imaging camera, and then analyzed by a MATLAB program to locate their centroid positions in a field of view. These coordinates were used to drive a pair of galvo-mirrors and steer a single laser beam to illuminate the individual spores located. Confocal micro-Raman spectroscopy of the illuminated spores was recorded by a multichannel charge-coupled device (CCD) spectrometer (Princeton Instruments, PIXIS 400BR).

The use of the same spectrometer and probe as in the integrated Raman/DESI‐MS imaging instrument ensured model transferability. We used the NIR Raman probe (RPB785, InPhotonics Inc.) to perform the bulk tissue measurements. The Raman probe was connected to a 785 nm laser (B&W Tek BRM‐785‐0.55‐1000.22‐FC, 600 mW) (≈200 mW on the sample) with a 105 µm excitation fiber and a NIR spectrometer (Princeton Instruments Acton LS785 using a Princeton Instruments PIXIS 400BR ≈750–1100 nm). Comprehensive software has been developed in the Matlab 2022a environment to provide real‐time data collection and analysis.

A slit-scanning confocal Raman microscope (RAMAN-11; Nanophoton, Osaka, Japan) was used for acquiring Raman spectra and Raman spectral images, as described in our previous studies23 (link)30 (link)49 (link)50 (link). A frequency doubled Nd:YAG laser (532 nm) was employed for excitation. The excitation laser beam was focused into a line (735 μm in length and approximately diffraction limit in width) on a sample through a cylindrical lens and a long working distance objective lens (UPlanFL N, ×10, NA = 0.3; Olympus, Tokyo, Japan). Raman scattering was collected with the same objective lens, focused onto the input slit of the spectrometer with a 600 grooves/mm grating, and detected with a two-dimensional image sensor (Pixis 400BR, −70 °C, 1340 × 400 pixels; Princeton Instruments, Trenton, NJ, USA). Owing to the line shaped focus of the excitation beam, a one-dimensional spectral image from the line-illuminated site was collected with the same objective lens (one pixel in space corresponds to 1.8 μm in length and approximately 0.89 μm in width). The excitation laser power was 235 μW/μm² on the sample plane. The exposure time for each line and the slit width of the spectrometer were respectively set at 10 s and 70 μm (corresponding spectral resolution was 7 cm⁻¹) for all experiments.

Raman spectra were obtained with a laser Raman confocal microscope [RAMAN-11 (Nanophoton, Osaka, Japan)] that has been described previously13 (link)14 (link). A frequency-doubled Nd:YAG laser operating at 532 nm was employed for excitation. The subepicardial heart tissue was illuminated with the point laser beam through an objective lens [UPLSAPO 10×, NA = 0.30 (Olympus, Tokyo, Japan)], and Raman spectra were obtained with a thermoelectrically cooled CCD camera [Pixis 400BR, 400 × 1340 pixels (Princeton Instruments, Trenton, NJ, USA)]. The entrance slit width of the spectrometer installed in the Raman microscope was set to 100 μm. The irradiated laser intensity at the sample plane and exposure time for each point were up to 20 mW/μm² and 10 s, respectively. Raman spectra were collected at 15 and 45 min after commencement of the coronary perfusion and at 10, 20, 30, 45, 60, 90, and 120 min after the stopped-flow under conditions so as not to change the focus for measurements over the course of experiments. In total, 20 hearts were examined: control (n = 3), SI group (n = 8), and IPC group (n = 9). At each time point, spectra were collected from more than 5 points (laser measurement diameter = ca. 0.8 μm). The impact of hemoglobin on the Raman spectra was considered to be negligible because the blood was adequately washed out from the heart14 (link).