4. Survey of Novel Multiphoton Active Materials 1257 4.1. Multiphoton Absorbing Systems 1257 4.2. Organic Molecules 1257 4.3. Organic Liquids and Liquid Crystals 1259 4.4. Conjugated Polymers 1259 4.4.1. Polydiacetylenes 1261 4.4.2. Polyphenylenevinylenes (PPVs) 1261 4.4.3. Polythiophenes 1263 4.4.4. Other Conjugated Polymers 1265 4.4.5. Dendrimers 1265 4.4.6. Hyperbranched Polymers 1267 4.5. Fullerenes 1267 4.6. Coordination and Organometallic Compounds 1271 4.6.1. Metal Dithiolenes 1271 4.6.2. Pyridine-Based Multidentate Ligands 1272 4.6.3. Other Transition-Metal Complexes 1273 4.6.4. Lanthanide Complexes 1275 4.6.5. Ferrocene Derivatives 1275 4.6.6. Alkynylruthenium Complexes 1279 4.6.7. Platinum Acetylides 1279 4.7. Porphyrins and Metallophophyrins 1279 4.8. Nanoparticles 1281 4.9. Biomolecules and Derivatives 1282 5. Nonlinear Optical Characterizations of Multiphoton Active Materials 1282

Multiphoton Absorbing Materials : Molecular Designs, Characterizations, and Applications

Multiphoton Excitation Provides Optical Sections from Deeper within Scattering Specimens than Confocal Imaging

Mitochondria play a pivotal role in energy metabolism, programmed cell death and oxidative stress. Mutated mitochondrial DNA in diseased cells compromises the structure of key enzyme complexes and, therefore, mitochondrial function, which leads to a myriad of health-related conditions such as cancer, neurodegenerative diseases, diabetes and aging. Early detection of mitochondrial and metabolic anomalies is an essential step towards effective diagnoses and therapeutic intervention. Reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) play important roles in a wide range of cellular oxidation-reduction reactions. Importantly, NADH and FAD are naturally fluorescent, which allows noninvasive imaging of metabolic activities of living cells and tissues. Furthermore, NADH and FAD autofluorescence, which can be excited using distinct wavelengths for complementary imaging methods and is sensitive to protein binding and local environment. This article highlights recent developments concerning intracellular NADH and FAD as potential biomarkers for metabolic and mitochondrial activities.

/pdf/intracellular-coenzymes-as-natural-biomarkers-for-metabolic-3iwj1v2nj2.pdf

Intracellular coenzymes as natural biomarkers for metabolic activities and mitochondrial anomalies.

Multiphoton‐absorbing organic materials for microfabrication, emerging optical applications and non‐destructive three‐dimensional imaging

Reduced nicotinamide adenine dinucleotide, NADH, is a major electron donor in the oxidative phosphorylation and glycolytic pathways in cells. As a result, there has been recent resurgence in employing intrinsic NADH fluorescence as a natural probe for a range of cellular processes that include apoptosis, cancer pathology, and enzyme kinetics. Here, we report on two-photon fluorescence lifetime and polarization imaging of intrinsic NADH in breast cancer (Hs578T) and normal (Hs578Bst) cells for quantitative analysis of the concentration and conformation (i.e., free-to-enzyme-bound ratios) of this coenzyme. Two-photon fluorescence lifetime imaging of intracellular NADH indicates sensitivity to both cell pathology and inhibition of the respiratory chain activities using potassium cyanide (KCN). Using a newly developed non-invasive assay, we estimate the average NADH concentration in cancer cells (168 ± 49 μM) to be ∼1.8-fold higher than in breast normal cells (99 ± 37 μM). Such analyses indicate changes in energy metabolism and redox reactions in normal breast cells upon inhibition of the respiratory chain activity using KCN. In addition, time-resolved associated anisotropy of cellular autofluorescence indicates population fractions of free (0.18 ± 0.08) and enzyme-bound (0.82 ± 0.08) conformations of intracellular NADH in normal breast cells. These fractions are statistically different from those in breast cancer cells (free: 0.25 ± 0.08; bound: 0.75 ± 0.08). Comparative studies on the binding kinetics of NADH with mitochondrial malate dehydrogenase and lactate dehydrogenase in solution mimic our findings in living cells. These quantitative studies demonstrate the potential of intracellular NADH dynamics (rather than intensity) imaging for probing mitochondrial anomalies associated with neurodegenerative diseases, cancer, diabetes, and aging. Our approach is also applicable to other metabolic and signaling pathways in living cells, without the need for cell destruction as in conventional biochemical assays.

/pdf/two-photon-autofluorescence-dynamics-imaging-reveals-43k1irzrpn.pdf

Two-photon autofluorescence dynamics imaging reveals sensitivity of intracellular NADH concentration and conformation to cell physiology at the single-cell level.

Two‐photon laser scanning fluorescence microscopy‐from a fluorophore and specimen perspective

We report the experimental results of our using irregularly shaped diamond microparticles as handles for laser tweezers. Because of their irregular optical shape, control of the rotation of diamond microparticles can easily be achieved in a gradient force optical trap by use of a fixed linearly polarized beam with a fundamental Gaussian mode. By changing the laser focal plane upon a diamond particle near the liquid surface or interfaces, one can fully manipulate both the direction and speed of the rotation. The ability to manipulate a diamond-particle-tagged biological specimen by optical tweezers is discussed. The application of these particles as handles for optical tweezers is demonstrated by optical manipulation of biological cells. Independent movement of linear translation and rotation, with controllable rotation directions and speeds, is successfully achieved.

/pdf/cell-manipulation-by-use-of-diamond-microparticles-as-hst6wnjnaz.pdf

Cell manipulation by use of diamond microparticles as handles of optical tweezers

The non-linear nature of multi-photon fluorescence excitation restricts the fluorescing volume to the vicinity of the focal point. As a result, the technology has the capacity for micro- spectroscopy of biological specimen at high spatial resolution. Chloroplasts in mesophyll protoplast of Arabidopsis thaliana and maize stem sections were used to demonstrate the feasibility of multi-photon fluorescence micro-spectroscopy at subcellular compartments. Time-lapse spectral recording provides a means for studying the response of cell organelles to high intensity illumination.

Multi-photon micro-spectroscopy of biological specimens

Summary from only given. We present a study using a multi-modal nonlinear microscopy, which reveals nonlinear optical activities in those naturally occurring biophotonic crystalline structures. This technique, based on a femtosecond Cr:forsterite laser at 1230 nm, combines different imaging modalities including second-, third-harmonic generations, and multi-photon fluorescence.

P. C. Cheng

Papers

Two‐photon laser scanning fluorescence microscopy‐from a fluorophore and specimen perspective

Cell manipulation by use of diamond microparticles as handles of optical tweezers

Multi-photon micro-spectroscopy of biological specimens

Biophotonic crystal effects in multi-modal nonlinear microscopy