Media 1: An all-fiber-optic endoscopy platform for simultaneous OCT and fluorescence imaging

Originally published in Biomedical Optics Express on 01 November 2012 (boe-3-11-2851

GPQuest: A Spectral Library Matching Algorithm for Site-Specific Assignment of Tandem Mass Spectra to Intact N‑glycopeptides

Glycoprotein changes occur in not only protein abundance but also the occupancy of each glycosylation site by different glycoforms during biological or pathological processes. Recent advances in mass spectrometry instrumentation and techniques have facilitated analysis of intact glycopeptides in complex biological samples by allowing the users to generate spectra of intact glycopeptides with glycans attached to each specific glycosylation site. However, assigning these spectra, leading to identification of the glycopeptides, is challenging. Here, we report an algorithm, named GPQuest, for site-specific identification of intact glycopeptides using higher-energy collisional dissociation (HCD) fragmentation of complex samples. In this algorithm, a spectral library of glycosite-containing peptides in the sample was built by analyzing the isolated glycosite-containing peptides using HCD LC-MS/MS. Spectra of intact glycopeptides were selected by using glycan oxonium ions as signature ions for glycopeptide spectra. These oxonium-ion-containing spectra were then compared with the spectral library generated from glycosite-containing peptides, resulting in assignment of each intact glycopeptide MS/MS spectrum to a specific glycosite-containing peptide. The glycan occupying each glycosite was determined by matching the mass difference between the precursor ion of intact glycopeptide and the glycosite-containing peptide to a glycan database. Using GPQuest, we analyzed LC-MS/MS spectra of protein extracts from prostate tumor LNCaP cells. Without enrichment of glycopeptides from global tryptic peptides and at a false discovery rate of 1%, 1008 glycan-containing MS/MS spectra were assigned to 769 unique intact N-linked glycopeptides, representing 344 N-linked glycosites with 57 different N-glycans. Spectral library matching using GPQuest assigns the HCD LC-MS/MS generated spectra of intact glycopeptides in an automated and high-throughput manner. Additionally, spectral library matching gives the user the possibility of identifying novel or modified glycans on specific glycosites that might be missing from the predetermined glycan databases

Imaging of N‑Linked Glycans from Formalin-Fixed Paraffin-Embedded Tissue Sections Using MALDI Mass Spectrometry

Aberrant glycosylation is associated with most of the diseases. Direct imaging and profiling of N-glycans on tissue sections can reveal tissue-specific and/or disease-associated N-glycans, which not only could serve as molecular signatures for diagnosis but also shed light on the functional roles of these biomolecules. Mass spectrometry imaging (MSI) is a powerful tool that has been used to correlate peptides, proteins, lipids, and metabolites with their underlying histopathology in tissue sections. Here, we report an MSI technique for direct analysis of N-glycans from formalin-fixed paraffin-embedded (FFPE) tissues. This technique consists of sectioning FFPE tissues, deparaffinization, and rehydration of the sections, denaturing tissue proteins, releasing N-linked glycans from proteins by printing peptide-N-glycosidase F over the sections, spray-coating the tissue with matrix, and analyzing N-glycans by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Brain sections from a C57BL/6 mouse were imaged using this technique at a resolution of 100 μm. Forty-two N-glycans were analyzed from the mouse brain section. The mass spectrometry images were used to study the relative abundance of oligomannose, nonfucosylated, and fucosylated complex N-glycans in different brain areas including isocortex, hippocampal formation, and brainstem and specific glycans associated with different areas of the brain were identified. Furthermore, glioblastoma tumor xenografts in a NOD/SCID mouse were imaged. Several glycans with differential expression in tumor versus normal brain tissues were identified. The MSI technique allows for imaging of N-glycans directly from FFPE sections. This method can potentially identify tissue-specific and/or disease-associated glycans coexpressed with other molecular signatures or within certain histological structures

Label-Free Metabolic Imaging In Vivo by Two-Photon Fluorescence Lifetime Endomicroscopy

NADH intensity and fluorescence lifetime characteristics have proved valuable intrinsic biomarkers for profiling the cellular metabolic status of living biological tissues. To fully leverage the potential of NADH fluorescence lifetime imaging microscopy (FLIM) in (pre)clinical studies and translational applications, a compact and flexible endomicroscopic embodiment is essential. Herein we present our newly developed two-photon fluorescence (2PF) lifetime imaging endomicroscope (2p-FLeM) that features an about 2 mm diameter, subcellular resolution, and excellent emission photon utilization efficiency and can extract NADH lifetime parameters of living tissues and organs reliably using a safe excitation power (∼30 mW) and moderate pixel dwelling time (≤10 μs). In vivo experiments showed that the 2p-FLeM system was capable of tracking NADH lifetime dynamics of cultured cancer cells and subcutaneous mouse tumor models subject to induced apoptosis, and of a functioning mouse kidney undergoing acute ischemia–reperfusion perturbation. The complementary structural and metabolic information afforded by the 2p-FLeM system promises functional histological imaging of label-free internal organs in vivo and in situ for practical clinical diagnosis and therapeutics applications

3151408.pdf

Supplemental documen

cycloid_suppvideo.avi

Video of multi-volume sequence acquired with cycloid scanner

In vitro studies of ICG loaded monocytes.

<p>(<i>A</i>, <i>B</i>) Representative images, RAW 264.7 mouse monocytes incubated in media alone and stained with DAPI. (<i>A</i>) Brightfield image. (<i>B</i>) DAPI blue nuclear staining and no NIR fluorescence (pseudocolor green). (<i>C</i>, <i>D</i>) Representative images, Monocytes after incubation in media with ICG solution and stained with DAPI. (<i>C</i>) Brightfield image. (<i>D</i>) DAPI fluorescence (blue) and NIR ICG fluorescence (pseudocolor green). (<i>E</i>) <i>Ex </i><i>vivo</i> cellular fluorescence over time. Monocytes incubated in media with ICG display an average NIR fluorescence of 5.22±0.34 arbitrary units (a.u.) above background (control non-ICG loaded cells) following the loading procedure, decreasing toward background over the following 12 hours (N=10). (<i>F</i>, <i>G</i>) Yield and viability of monocytes after ICG loading procedure. (<i>F</i>) Cell yield, after maintaining the cells <i>ex </i><i>vivo</i> in culture media and washing and centrifuging at each time point, decreases over the first 24 hours after loading with ICG. (<i>G</i>) Viability of cells remains greater than 80% for more than 12 hours (N=10). (<i>H</i>) ICG loaded monocyte chemotactic capacity. Chemotactic index is the number of cells migrating through the chemotaxis filter toward media with the chemoattractant MCP-1 relative to the number of cells migrating through the chemotaxis filter toward media alone. The average number of migrated ICG-loaded monocytes per five high powered fields was 86.0±27.8 with MCP-1 in the bottom well, and 48.9±16.5 with chemotaxis media alone in the bottom well (N=3). All error bars represent SEM. *<i>P</i><0.05.</p

Normalized fluorescence for in vivo imaging of ICG labeled cells and ICG solution.

<p>(<i>A</i>) Normalized fluorescence of inoculation area. Normalized fluorescence is significantly higher in the infection model (N=8) than in the inflammation model (N=8) as early as 2 hours after cellular injection (p<0.05). After injection of ICG solution, normalized fluorescence in the infection model (N=8) is significantly higher than in the inflammation model (N=8) at the 8, 12, and 24 hour time points (p<0.05). Normalized fluorescence after injection of cells was significantly higher than after injection of ICG solution from the 6 hour time point onward (p<0.05). Fluorescence intensity was normalized to the intensity at 0 hours after injection. (<i>B</i>) Normalized fluorescence of contralateral control area. Normalized fluorescence generally decreases throughout the study in all groups (N=8 each). All error bars represent SEM.</p

Near infrared images taken after systemic injection of ICG-loaded monocytes or ICG solution.

<p>Top row: Infection model, cellular injection. Second row: Infection model, solution injection. Third row: Inflammation model, cellular injection. Fourth row: Inflammation model, solution injection. Red arrow indicates inoculation site with Complete Freund’s adjuvant in the inflammation model, or Group A Streptococcus in the infection model. Asterisk indicates ICG being excreted through the bowel.</p