Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 microm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.

/pdf/nonlinear-magic-multiphoton-microscopy-in-the-biosciences-3fszv5aq8z.pdf

Nonlinear magic: multiphoton microscopy in the biosciences

We report focusing of coherent light through opaque scattering materials by control of the incident wavefront. The multiply scattered light forms a focus with a brightness that is up to a factor of 1000 higher than the brightness of the normal diffuse transmission.

https://ps.brillouinzone.com/sites/default/files/Focusing_light_OptLett.pdf

Focusing coherent light through opaque strongly scattering media

http://www.dmphotonics.com/Femtosecond_Imaging/143.pdf

Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience

Fluorescence polarization was first applied in biochemistry almost 6 decades ago, when Gregorio Weber described his studies on bovine serum albumin and ovalbumin conjugated with 1-dimethylaminonaphthalene-5-sulfonyl chloride (dansyl chloride)1,2 (Figure 1). (For an overview of Gregorio Weber's wide-ranging contributions to fluorescence see3). Polarization methods became increasingly popular during the decades following Weber's work. During the past few decades, however, the increase in the number and diversity of fluorescence polarization studies has been astonishing and the method is now extremely wide-spread in the clinical and biomedical fields. The virtual explosion of polarization studies, which began during the mid-1980's, was due to several factors, including the availability of commercial instruments equipped with polarizers, the commercial availability of a great many fluorescence probes (largely due to the company Molecular Probes – now part of Invitrogen) and, in the clinical chemistry area, the introduction of the TDx instrument (and associated reagents) by Abbott Laboratories. The reasons for the popularity of fluorescence polarization in clinical and high-throughput assays are manifold. Firstly, polarization assays are homogeneous, i.e., there is no necessity for separation of free and bound ligand (these type of assays are often referred to as “mix and measure” assays). Secondly, and one of the original motivations for the development of fluorescence-based assays, there is no need for radioisotopes. Thirdly, polarization assays are reproducible and facilely automated. In this review we shall briefly trace the history of the technique and discuss in detail both theoretical and practical aspects. We shall also present copious examples from the literature, which illustrate the scope of the method in areas such as ligand binding, immunoassays, high-throughput screening, and live cell imaging and hint at future directions and applications.



Figure 1

Gregorio Weber in Hawaii – 1989. Photograph courtesy of Prof. David Jameson.




2.0 Historical Overview
As mentioned above, fluorescence polarization is widely applied in diverse fields, especially in the life sciences. A rough survey using Pubmed reveals that publications on fluorescence polarization/anisotropy numbered around a dozen in the 1950's, less than 100 in the 1960s, several hundreds in the 1970s, more than two thousand in the 1980's, more than three thousand in the1990s and close to four thousand in the 2000s to date. Given this rising trend, one may be curious how it all got started. We shall thus present a brief history of fluorescence polarization.

2.1 Discovery of Polarization
In 1808, Etienne-Louis Malus observed sunlight reflected from the windows of the Luxemburg Palace in Paris through an Iceland spar (Calcite) crystal that he rotated (Erasmus Bartholin had discovered the double refraction of light by Iceland spar in 1669). Malus discovered that the intensity of the reflected light varied as he rotated the crystal and coined the term “polarized” to describe this property of light. He published his findings in 1809 4. Malus also derived an expression for calculating the transmission of light as a function of the angle (θ) between two polarizers. This equation (Malus' Law) is now written as: I(θ) = I0 (cos2θ). Some years later, David Brewster studied the relationship between refractive index and angle of incidence on the polarization of the reflected light 5. He discovered that for normal glass and visible light, an incidence angle of ∼56 degrees resulted in total reflection of one plane of polarization – this angle is now known as Brewster's Angle. This discovery allowed Brewster to construct a polarizer composed of a “pile of plates” (interestingly, when one of the authors – DMJ – first joined Gregorio Weber's laboratory as a graduate student, Weber showed him the “pile of plates” polarizer that he had used in some of his initial studies). In 1828, William Nicol joined two crystals of Iceland spar, cut at an angle of 68°, using Canada balsam, which allowed a spatial separation between the orthogonally polarized components. Other important calcite polarizers developed around this time include: Glan-Foucault; Glan-Thompson; Glan-Taylor; Wollaston; and Rochon. Most modern spectrofluorimeters today use Glan-Taylor type calcite polarizers, which have an air-gap between the two calcite crystals allowing for transmission deep into the ultraviolet, and which makes them less susceptible to photodamage at higher irradiance levels. But the Henry Ford of polarizers was Edwin Herbert Land. In 1929 Edwin Land patented the sheet polarizer (the J-sheet), consisting of crystals of iodoquinine sulfate embedded in nitrocellulose film followed by alignment of the crystals by stretching, which led to dichroism. In 1937 he founded the Polaroid Company and in 1938 he invented the H-sheet which comprised polyvinyl alcohol sheets with embedded iodine. Land's invention made him quite rich and also allowed the use of inexpensive polarizers in diverse applications such as sunglasses and photography filters.

/pdf/fluorescence-polarization-anisotropy-in-diagnostics-and-3u5kdf0yry.pdf

Fluorescence Polarization/Anisotropy in Diagnostics and Imaging

A solid tumor is an organ composed of cancer and host cells embedded in an extracellular matrix and nourished by blood vessels. A prerequisite to understanding tumor pathophysiology is the ability to distinguish and monitor each component in dynamic studies. Standard fluorophores hamper simultaneous intravital imaging of these components. Here, we used multiphoton microscopy techniques and transgenic mice that expressed green fluorescent protein, and combined them with the use of quantum dot preparations. We show that these fluorescent semiconductor nanocrystals can be customized to concurrently image and differentiate tumor vessels from both the perivascular cells and the matrix. Moreover, we used them to measure the ability of particles of different sizes to access the tumor. Finally, we successfully monitored the recruitment of quantum dot–labeled bone marrow–derived precursor cells to the tumor vasculature. These examples show the versatility of quantum dots for studying tumor pathophysiology and creating avenues for treatment.

/pdf/quantum-dots-spectrally-distinguish-multiple-species-within-4nhfvzkkor.pdf

Quantum dots spectrally distinguish multiple species within the tumor milieu in vivo

Summary Two-photon absorption and emission spectra for fluorophores relevant in cell imaging were measured using a 45 fs Ti:sapphire laser, a continuously tuneable optical parametric amplifier for the excitation range 580 ‐1150 nm and an optical multichannel analyser. The measurements included DNA stains, fluorescent dyes coupled to antibodies as well as organelle trackers, e.g. Alexa and Bodipy dyes, Cy2, Cy3, DAPI, Hoechst 33342, propidium iodide, FITC and rhodamine. In accordance with the two-photon excitation theory, the majority of the investigated fluorochromes did not reveal significant discrepancies between the two-photon and the one-photon emission spectra. However, a blue-shift of the absorption maxima ranging from a few nanometres up to considerably differing courses of the spectrum was found for most fluorochromes. The potential of non-linear laser scanning fluorescence microscopy is demonstrated here by visualizing multiple intracellular structures in living cells. Combined with 3D reconstruction techniques, this approach gives a deeper insight into the spatial relationships of subcellular organelles.

http://nathan.instras.com/MyDocsDB/doc-700.pdf

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

We present the application of a novel micro mirror array, which is based on a micro electro mechanical system (MEMS), as one- and two-dimensional phase-modulating spatial light modulator (SLM) for femtosecond pulse shaping in the spectral region from the deep-ultraviolet (DUV) to the near-infrared (NIR) (200–900 nm). Using such a high-resolution MEMS-SLM, we demonstrate one-dimensional pulse shaping at 400 nm, including THz-pulse train generation, chirp compensation, and phase wraps.

/pdf/micromirror-slm-for-femtosecond-pulse-shaping-in-the-4g5dfo4atf.pdf

Micromirror SLM for femtosecond pulse shaping in the ultraviolet

A novel liquid crystal display (LCD) with 640 stripes was successfully implemented and investigated for femtosecond pulse shaping. As compared to previously used devices, the large active area allows for operation in high-power laser systems. The increased number of pixels greatly enlarges the manifold of accessible pulse modulations, making the device an ideal tool for coherent control experiments and optical information processing.

A new high-resolution femtosecond pulse shaper

We demonstrate the adaptation of an iterative Fourier transform algorithm for the calculation of theoretical spectral phase functions required for pulse shaping applications. The algorithm is used to determine the phase functions necessary for the generation of different temporal intensity profiles. The performance of the algorithm is compared to two exemplary standard approaches. i.e. a Genetic Algorithm and a combination of a Simplex Downhill and a Simulated Annealing algorithm. It is shown that the iterative Fourier transform algorithm converges much faster than both alternative methods.

Iterative Fourier transform algorithm for phase-only pulse shaping.

We have investigated theoretically and experimentally the generation of shaped pulses at 400 nm by frequency-doubling of phase-modulated, ultrashort laser pulses. We present an analytical description of frequency-doubled pulses with a sinusoidal spectral phase modulation. It is shown that such a phase modulation can be transferred completely into a spectral amplitude modulation with a resolution determined only by the phase-modulating device. A criterion for achieving the maximum modulation contrast is given.

G. Stobrawa

Papers

Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging

Micromirror SLM for femtosecond pulse shaping in the ultraviolet

A new high-resolution femtosecond pulse shaper

Iterative Fourier transform algorithm for phase-only pulse shaping.

Frequency doubling of phase-modulated, ultrashort laser pulses