Tissue preparation- healthy mouse brain
Fluorescein and fluorescein 5(6)-isothiocyanate (FITC) were used to validate MALDI MS imaging of mouse brain vasculature using fluorescence microscopy and MALDI MSI. The mice were injected with 400 mg/kg fluorescein sodium salt (Sigma-Aldrich, St. Louis, MO) or FITC (Sigma-Aldrich, St. Louis, MO) in PBS from the tail vein and sacrificed after three minutes. The mouse brain was flash frozen in liquid nitrogen and stored in −80°C freezer. For tissue sectioning, the frozen mouse brain was mounted with minimal amount of optimal cutting medium (OCT) compound, such that OCT did not come into contact with the cryotome blade, and sectioned at 10 μm thickness using a Microm HM550 cryostat (Mikron Instruments Inc, Vista, CA). The specimen temperature was set at −19°C and chamber temperature at −20°C. Tissue sections were thaw-mounted on indium tin oxide (ITO) coated glass slides (BrukerDaltonics, Germany) for mass spectrometry imaging, and sister sections were mounted independently on optical slides (Fisher, Pittsburgh PA) for histochemistry. The slides were first analyzed using fluorescence microscopy, followed by MALDI time-of-flight (TOF) mass spectrometry imaging of the same section.
Tissue preparation- mouse orthotopic models
Human U87- Animal models were prepared by intracranial injections of human U87 glioma cells or murine PVL311 neural stem cells (derived from embryonic brains of p53-null mice and transformed to express human B-RAF V600E point mutation), both lines engineered to overexpress luciferase. Tumor growth was monitored by in vivo bioluminescence imaging, after intra-peritoneal (I.P.) injection of luciferin at 225 mg/kg (mice sedated via inhalation of ~3% isoflurane). The animals were imaged using a CCD camera after 10 minutes. After documentation of tumor growth, the animals were treated by oral gavage of therapeutic levels of drug, and sacrificed at half-point to the in vivo half-life of the active compound. The animals were promptly dissected and liver, kidney, and brain were flash frozen in liquid nitrogen. Sections with 10 μm thickness were collected for mass spectrometry and histochemistry analysis. All animal experiments were approved by the Dana Farber Animal Care and Use Committee and distress to animals was minimized. Tissue sections were transferred using a brush onto indium tin oxide (ITO) coated glass slides for mass spectrometry imaging or optical slides for immunohistochemistry and hematoxylin and eosin (H&E) staining.
The ImagePrep (BrukerDaltonics, Germany), which can spray matrix solution homogeneously, was used for matrix deposition on ITO coated slides. The matrix solution was 30 mg/mL 2,5-dihydroxybenzoic acid dissolved in 50% HPLC grade methanol, 50% HPLC grade water and 0.2% trifluoroacetic acid. The solution was sonicated for 5 min and centrifuged at 10,000 rpm for 10 min before transferred to the ImagePrep. The ITO coated slides from −80°C freezer were dehydrated in a dessicator for 15 min upon thawing. The matrix was sprayed onto the slides by piezoelectric nebulization in ImagePrep with approximately 85 thin layers of matrix deposition. Chemicals were purchased from Sigma (Sigma-Aldrich, St. Louis, MO).
MALDI TOF mass spectrometry
MALDI TOF imaging was performed using the UltrafleXtreme MALDI TOF/TOF (Bruker Daltonics, Germany) in positive reflectron ion mode with a 1 KHz smartbeam laser. The instrument was calibrated with peptide calibration standard (Bruker Daltonics, Germany) for m/z 700–1800. Tissue sections were imaged with spatial resolution from 25–100 μm. Each spectrum was acquired from 200 or 300 laser shots. The MALDI images were displayed using the software FlexImaging 3.0.
MALDI FTICR mass spectrometry
The tissue sections analyzed by MALDI q-FTICR mass spectrometry (Apex-Ultra, Bruker, MA) were imaged with 45 μm to 220 μm laser raster. Instruments with magnetic fields from 7.0–12.0 T were used, with the infinity ion cyclotron resonance cell geometry36
, dual MALDI/electrospray source (Apollo II), and vacuum elements with readings of below 4 × 10−10
mbar in the analyzer region. The instrument was calibrated in ESI mode using 0.01 mg/mL sodium formate (Sigma-Aldrich, St. Louis, MO) solution in 50% acetonitrile 0.1% formic acid. However, the imaging experiment was performed under MALDI mode. Mass spectrometry experiments involved the following steps. Initially MALDI source and transfer parameters were optimized for maximum signal magnitude. Next, excitation amplitude was tuned for maximum accuracy under internal calibration conditions37
. Sidekick trapping38
was employed. Chirp excitation and image charge detection was performed. The free induction decay (FID) was multiplied by a sine bell apodization function and was fast Fourier transformed. The mass range for the filters was m/z
± 0.001 and the intensity range were 100,000 to 1,000,000 which was set to avoid picking noise signal and was tested in negative controls. The theoretical masses and molecule fine structures were calculated and predicted by a lab-developed software39
Fluorescence imaging was performed on the same tissue as for MALDI MSI prior to MALDI matrix spraying. Fluorescein and fluorescein isothiocyanate (FITC), two BBB impermeable fluorophores have excitation maximum of 490 nm and 492 nm, and emission maximum at 518 nm and 514 nm respectively. Fluorescence images were acquired using a fluorescent microscope Observer.Z1 with the X-Cite 120Q series light source and AxioCam MRm mounted camera (Zeiss, Germany) with 5× objective, optovar of 1.6, and 1 s exposure time.
The tissue morphological information was revealed on sister sections of the ones for MS imaging using standard hematoxylin and eosin staining (H&E Staining). All the reagents used for staining were from Sigma (Sigma-Aldrich, St. Louis, MO). After the sections were dried, toluene was applied and the slides and covered with glass coverslips. The optical images of tissues were scanned by Axio Imager M1 microscope (Zeiss, Chester, VA) at 40× magnification. The detailed morphology information of healthy sections and tumors was evaluated on the Mirax Digital Slide Desktop Server system.
Data analysis and 3D reconstruction
For the images obtained from MALDI imaging, heme and various drugs have identical maximum absolute intensity threshold respectively. The minimum intensity threshold is based on examining individual imaging pixels until showing reasonable S/N ratio in spectrum for each image. 3D Doctor is used for 3D reconstruction of MS images and optical images. For RAF265 study, the 3D models of heme, drug and histological images are built individually. However, for BKM120, heme and drug images from the same brain tissue are considered as consecutive sections and reconstructed within one model. In reconstructed models, heme and drug distributions are highlighted using the function of “interactive segment” in 3D Doctor (Able Software Corp., Lexington, MA) without displaying intensity discrepancy. The reconstruction of optical images is based on drawing outlines of regions of interest manually.