Raman spectroscopy for physiological investigations of tissues and cells.

Raman micro-spectroscopy provides a convenient non-destructive and location-specific means of probing cellular physiology and tissue physiology at sub-micron length scales. By probing the vibrational signature of molecules and molecular groups, the distribution and metabolic products of small molecules that cannot be labeled with fluorescent dyes can be analyzed. This method works well for molecular concentrations in the micro-molar range and has been demonstrated as a valuable tool for monitoring drug-cell interactions.

A smart and versatile theranostic nanomedicine platform based on nanoporphyrin.

Multifunctional nanoparticles with combined diagnostic and therapeutic functions show great promise towards personalized nanomedicine. However, attaining consistently high performance of these functions in vivo in one single nanoconstruct remains extremely challenging. Here we demonstrate the use of one single polymer to develop a smart 'all-in-one' nanoporphyrin platform that conveniently integrates a broad range of clinically relevant functions.

Arginine starvation-associated atypical cellular death involves mitochondrial dysfunction, nuclear DNA leakage, and chromatin autophagy.

Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, excessive autophagy could be cytotoxic, contributing to cell death, but its mechanism remains elusive. Arginine starvation has emerged as a potential therapy for several types of cancers, owing to their tumor-selective deficiency of the arginine metabolism.

A highly sensitive nanoscale pH-sensor using Au nanoparticles linked by a multifunctional Raman-active reporter molecule.

Chemical sensing on the nanoscale has been breaking new ground since the discovery of surface enhanced Raman scattering (SERS). For nanoparticles, controlled particle aggregation is necessary to achieve the largest SERS enhancements. Therefore, aggregating agents such as salts or linker molecules are used in conjunction with chemically sensitive reporters in order to develop robust environmentally sensitive SERS probes.

Rapid 3D fluorescence imaging of individual optically trapped living immune cells.

We demonstrate an approach to rapidly characterize living suspension cells in 4 dimensions while they are immobilized and manipulated within optical traps. A single, high numerical aperture objective lens is used to separate the imaging plane from the trapping plane. This facilitates full control over the position and orientation of multiple trapped cells using a spatial light modulator, including directed motion and object rotation, while also allowing rapid 4D imaging.

Controlling the diameter, monodispersity, and solubility of ApoA1 nanolipoprotein particles using telodendrimer chemistry.

Nanolipoprotein particles (NLPs) are nanometer-scale discoidal particles that feature a phospholipid bilayer confined within an apolipoprotein "scaffold," which are useful for solubilizing hydrophobic molecules such as drugs and membrane proteins. NLPs are synthesized either by mixing the purified apolipoprotein with phospholipids and other cofactors or by cell-free protein synthesis followed by self-assembly of the nanoparticles in the reaction mixture. Either method can be problematic regarding the production of homogeneous and monodispersed populations of NLPs, which also currently requires multiple synthesis and purification steps.

Label-free analysis of cellular biochemistry by Raman spectroscopy and microscopy.

We review the biomedical applications of Raman spectroscopy at the single cell and tissue level. Raman scattering is the inelastic scattering of light by molecular bonds resulting in a wealth of spectral bands, which enable the identification of biological materials and the nondestructive analysis of dynamic changes in their biochemistry. We briefly review the basics behind highly sensitive Raman spectroscopy and highlight recent applications to biomedical research.

Coherent anti-Stokes emission from gold nanorods and its potential for imaging applications.

We used coherent anti-Stokes scattering (CAS) to characterize individual gold nanorods (GNRs) and GNR aggregates. By creating samples with different densities of GNRs on silicon wafer substrates, we were able to determine surface coverage by scanning electron microscopy (SEM) and then correlate the coverage to the CAS intensities of the samples. The observed CAS signal intensity was quadratically dependent on the number of particles.

Novel single-cell functional analysis of red blood cells using laser tweezers Raman spectroscopy: application for sickle cell disease.

Laser tweezers Raman spectroscopy was used to characterize the oxygenation response of single normal adult, sickle, and cord blood red blood cells (RBCs) to an applied mechanical force. Individual cells were subjected to different forces by varying the laser power of a single-beam optical trap, and the intensities of several oxygenation-specific Raman spectral peaks were monitored to determine the oxygenation state of the cells. For all three cell types, an increase in laser power (or mechanical force) induced a greater deoxygenation of the cell.

Characterization of de novo synthesized GPCRs supported in nanolipoprotein discs.

The protein family known as G-protein coupled receptors (GPCRs) comprises an important class of membrane-associated proteins, which remains a difficult family of proteins to characterize because their function requires a native-like lipid membrane environment. This paper focuses on applying a single step method leading to the formation of nanolipoprotein particles (NLPs) capable of solubilizing functional GPCRs for biophysical characterization. NLPs were used to demonstrate increased solubility for multiple GPCRs such as the Neurokinin 1 Receptor (NK1R), the Adrenergic Receptor â2 (ADRB2) and the Dopamine Receptor D1 (DRD1).

Synthesis and characterization of a disulfide reporter molecule for enhancing pH measurements based on surface-enhanced Raman scattering.

In this paper, we describe the synthesis and characterization of 2,5-dimercaptobenzoic acid as a novel pH-sensitive disulfide reporter molecule for surface-enhanced Raman scattering (SERS) capable of inducing the controlled aggregation of gold (Au) colloids in solution without the addition of salts. While weak acids have been shown to yield some pH sensitivity as reporter molecules for SERS measurements, the reproducibility and signal strength of nanoparticle probes based on such molecules can vary greatly. This limited reproducibility depends greatly on the salt-induced aggregation of the colloidal nanoprobes, which is required in order to obtain SERS signals strong enough to probe individual clusters.

Establishment of rules for interpreting ultraviolet autofluorescence microscopy images for noninvasive detection of Barrett's esophagus and dysplasia.

The diagnostic potential of autofluorescence (AF) microscopy under ultraviolet (UV) excitation is explored using ex vivo human specimens. The aim is to establish optical patterns (the rules for interpretation) that correspond to normal and abnormal histologies of the esophagus, spanning from early benign modifications (Barrett's esophagus) to subsequent dysplastic change and progression toward carcinoma. This was achieved by developing an image library categorized by disease progression.

Label-free identification and characterization of human pluripotent stem cell-derived cardiomyocytes using second harmonic generation (SHG) microscopy.

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) are a potentially unlimited source of cardiomyocytes (CMs) for cardiac transplantation therapies. The establishment of pure PSC-CM populations is important for this application, but is hampered by a lack of CM-specific surface markers suitable for their identification and sorting. Contemporary purification techniques are either non-specific or require genetic modification.

Raman spectroscopy of individual monocytes reveals that single-beam optical trapping of mononuclear cells occurs by their nucleus.

We show that laser-tweezers Raman spectroscopy of eukaryotic cells with a significantly larger diameter than the tight focus of a single beam laser trap leads to optical trapping of the cell by its optically densest part, i.e. typically the cell's nucleus.

Lipid-cell interactions in human monocytes investigated by doubly-resonant coherent anti-Stokes Raman scattering microscopy.

We demonstrate that doubly-resonant coherent anti-Stokes Raman scattering can provide enhanced and highly specific contrast for molecules containing unique Raman-active small molecular groups. This combination provides contrast for molecules that can otherwise be difficult to discriminate by Raman spectroscopy. Here, human monocytes were incubated with either deuterated oleic acid or 17-octadecynoic acid (a fatty acid with an end terminal acetylene group).

Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy.

Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex.

Parallel analysis of individual biological cells using multifocal laser tweezers Raman spectroscopy.

We report on the development and characterization of a multifocal laser tweezers Raman spectroscopy (M-LTRS) technique for parallel Raman spectral acquisition of individual biological cells. Using a 785-nm diode laser and a time-sharing laser trapping scheme, multiple laser foci are generated to optically trap single polystyrene beads and suspension cells in a linear pattern. Raman signals from the trapped objects are simultaneously projected through the slit of a spectrometer and spatially resolved on a charge-coupled device (CCD) detector with minimal signal crosstalk between neighboring cells.

Label-free imaging and analysis of the effects of lipolysis products on primary hepatocytes.

The increased accumulation of intracellular lipid droplets within hepatocytes is a pathologic hallmark of liver injury of various etiologies, especially non-alcoholic steatohepatitis (NASH). The dynamics, subcellular origin, and chemical composition of lipid droplets under various pathophysiologic conditions, however, remain poorly understood. We used coherent Raman microscopy and spontaneous Raman spectroscopy to monitor and analyze the formation of lipid droplets in living primary rat hepatocytes exposed to triglyceride-rich lipoprotein (TGRL) lipolysis products.

Manipulating CD4+ T cells by optical tweezers for the initiation of cell-cell transfer of HIV-1.

Cell-cell interactions through direct contact are very important for cellular communication and coordination - especially for immune cells. The human immunodeficiency virus type I (HIV-1) induces immune cell interactions between CD4(+) cells to shuttle between T cells via a virological synapse. A goal to understand the process of cell-cell transmission through virological synapses is to determine the cellular states that allow a chance encounter between cells to become a stable cell-cell adhesion.

Signal generation and Raman-resonant imaging by non-degenerate four-wave mixing under tight focusing conditions.

The authors demonstrate Raman-resonant imaging based on the simultaneous generation of several nonlinear frequency mixing processes resulting from a 3-color coherent anti-Stokes Raman scattering (CARS) experiment. The interaction of three coincident short-pulsed laser beams simultaneously generates both 2-color (degenerate) CARS and 3-color (non-degenerate) CARS signals, which are collected and characterized spectroscopically - allowing for resonant, doubly-resonant, and non-resonant contrast mechanisms. Images obtained from both 2-color and 3-color CARS signals are compared and found to provide complementary information.

Physico-chemical characterization of polylipid nanoparticles for gene delivery to the liver.

Polylipid nanoparticles (PLNP) have been shown to be very effective in delivering antioxidative genes in the treatment of liver injury in mice. To build on our previous studies and to further characterize PLNP formulated from polycationic lipid (PCL) and cholesterol, we report here the synthesis of multigram quantities of PCL and employ analytical tools, such as Raman spectroscopy of single PLNP and live-cell imaging of lipofection, for the physicochemical characterization of PCL, PLNP, and the transfection process. Mass spectrometry demonstrates the characteristics of polymeric lipids.

Raman microscopy based on doubly-resonant four-wave mixing (DR-FWM).

Doubly-resonant four-wave mixing (DR-FWM) is a nondegenerate four-wave mixing process in which four photons interact to coherently probe two distinct Raman resonances. We demonstrate DR-FWM microscopy as a label-free and nondestructive molecular imaging modality with high chemical specificity on the submicron scale by imaging alkyne-substituted oleic acid in both aqueous and lipid-rich environments. DR-FWM microscopy is contrasted to coherent anti-Stokes Raman scattering (CARS) microscopy and it is shown that the coherent addition of two simultaneously probed Raman resonances leads to a significant increase in signal without increasing the non-resonant background.

Label-free biochemical characterization of stem cells using vibrational spectroscopy.

Raman and infrared (IR) spectroscopy are two complementary vibrational spectroscopic techniques that have experienced a tremendous growth in their use in biological and biomedical research. This is, in large part, due to their unique capability of providing label-free intrinsic chemical information of living biological samples at tissue, cellular, or sub-cellular resolutions. This article reviews recent developments in applying these techniques for the characterization of stem cells.

Fluorescence lifetime imaging microscopy: in vivo application to diagnosis of oral carcinoma.

A compact clinically compatible fluorescence lifetime imaging microscopy (FLIM) system was designed and built for intraoperative disease diagnosis and validated in vivo in a hamster oral carcinogenesis model. This apparatus allows for the remote image collection via a flexible imaging probe consisting of a gradient index objective lens and a fiber bundle. Tissue autofluorescence (337 nm excitation) was imaged using an intensified CCD with a gate width down to 0.

Raman spectroscopy of DNA packaging in individual human sperm cells distinguishes normal from abnormal cells.

Healthy human males produce sperm cells of which about 25-40% have abnormal head shapes. Increases in the percentage of sperm exhibiting aberrant sperm head morphologies have been correlated with male infertility, and biochemical studies of pooled sperm have suggested that sperm with abnormal shape may contain DNA that has not been properly repackaged by protamine during spermatid development. We have used micro-Raman spectroscopy to obtain Raman spectra from individual human sperm cells and examined how differences in the Raman spectra of sperm chromatin correlate with cell shape.