Exploring and understanding biological and pathological changes are of great significance for early diagnosis and therapy of diseases. Optical sensing and imaging approaches have experienced major progress in this field. Particularly, an emergence of various functional optical nanoprobes has provided enhanced sensitivity, specificity, targeting ability, as well as multiplexing and multimodal capabilities due to improvements in their intrinsic physicochemical and optical properties. However, one of the biggest challenges of conventional optical nanoprobes is their absolute intensity-dependent signal readout, which causes inaccurate sensing and imaging results due to the presence of various analyte-independent factors that can cause fluctuations in their absolute signal intensity. Ratiometric measurements provide built-in self-calibration for signal correction, enabling more sensitive and reliable detection. Optimizing nanoprobe designs with ratiometric strategies can surmount many of the limitations encountered by traditional optical nanoprobes. This review first elaborates upon existing optical nanoprobes that exploit ratiometric measurements for improved sensing and imaging, including fluorescence, surface enhanced Raman scattering (SERS), and photoacoustic nanoprobes. Next, a thorough discussion is provided on design strategies for these nanoprobes, and their potential biomedical applications for targeting specific biomolecule populations (e.g. cancer biomarkers and small molecules with physiological relevance), for imaging the tumor microenvironment (e.g. pH, reactive oxygen species, hypoxia, enzyme and metal ions), as well as for intraoperative image guidance of tumor-resection procedures.

Ratiometric optical nanoprobes enable accurate molecular detection and imaging

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.

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Imaging cellular ultrastructures using expansion microscopy (U-ExM).

The advent of compact, high brightness supercontinuum radiation sources employing solid core photonic crystal fibres is beginning to make an impact across the field of applied spectroscopy research In this article we focus on the use of supercontinuum sources to construct novel instrumentation for chemical sensing A brief overview is given on the mechanisms of supercontinuum generation in solid core photonic crystal fibres, and then we review recent, and present new, results from our own research We present examples on gas phase sensing applications, permitting wide bandwidth molecular spectra to be gathered at ultrahigh speed Furthermore we demonstrate the design and construction of a wide bandwidth microscope for wavelength flexible hyperspectral confocal imaging We conclude with an outlook and a summary of where and how we think the field may develop over coming years

Supercontinuum radiation for applications in chemical sensing and microscopy

Determining the structure and composition of macromolecular assemblies is a major challenge in biology. Here we describe ultrastructure expansion microscopy (U-ExM), an extension of expansion microscopy that allows the visualization of preserved ultrastructures by optical microscopy. This method allows for near-native expansion of diverse structures in vitro and in cells; when combined with super-resolution microscopy, it unveiled details of ultrastructural organization, such as centriolar chirality, that could otherwise be observed only by electron microscopy.U-ExM enables near-native expansion microscopy of samples in vitro and in cells. The combination of U-ExM with confocal microscopy and HyVolution revealed details of centriole chirality that were previously accessible only by electron microscopy.

Imaging cellular ultrastructures using expansion microscopy (U-ExM)

A method and apparatus wherein small, two- or three-dimensional structures are formed by multiple-photon-absorbed photopolymerization and/or cross-linking of a precursor composition, that is, photopolymerization using multi-photon excitation. Use of multi-photon excitation allows fabrication of structures and structural features having at least one dimension of less than about 1 micron, preferably less than about 500 nm, more preferably less than about 250 nm, and most preferably of less than about 100 nm, in bulk phase as well as in solution, and from a wide variety of organic and inorganic precursor subunits, including synthetic polymers and biological polymers such as proteins, lipids, oligonucleotides, and the like.

Free-form nanofabrication using multi-photon excitation

True confocal microscopy requires point-shaped illumination and detection. To generate an image, a diffraction limited spot is moved over the sample. Single spot scanning has suffered in the past from low image rates; a solution is the employment of very fast scanning devices (resonant scanners) for x-movement. In the process of introducing resonant scanning devices, it was found that both signal yield is improved and bleaching is decreased-in contrary to the assumed performance. This article will show by a simple and well understood model a straightforward explanation for the potential increase of signal yield and decrease in photobleaching. The time that is ruling the dose-rate effects is the effective time; a fluorochrome is illuminated. This time depends on the diameter of the spot that is moved over the sample and the speed at which the spot moves. In essence, the scan process causes a pulsed illumination of the fluorochromes. Various schemes of pulsed illumination are simulated with a fluorescence model. The model includes a dark state, where fluorochromes will exit the fluorescence process and slowly decay back into the ground state. Upon splitting a single dose into two pulses separated by a dark time-reflecting an increased scan speed-the amount of fluorescence emission is increased and bleaching is reduced. These results show a potential increase of fluorescence and a lower photobleaching upon higher scan speed. As illumination during the bleach-phase in a FRAP-experiment is similar to a light pulse, the findings also suggest to critically consider the very beginning of fluorescence recovery in terms of triplet relaxation process that potentially could falsify the measurements.

MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching.

Modern microscopy in life sciences is ruled by development and exploration of new dyes and stains (probes for histochemical staining, quantum dots, fluorescent proteins etc.) on one side, and technological improvements and innovations for fluorescence microscopy-especially high resolution and optical sectioning microscopy-on the other side. Concerning the technical innovations, several ingenious inventions have been made available for confocal microscopy. First, the acousto optical tunable filter, which allows switching and dimming of laser lines. Second the spectral detector, employing mirror sliders in front of the detectors which allow continuous tuning of the spectral emission band detected by the sensor. Third, the most challenging task: a substitute to the classical beam splitter-the device which is restricting fluorescence microscopy most. This was solved by introduction of the acousto optical beam splitter. The very last device which is still lacking flexibility is the laser source, operating only at non-equidistant frequencies and requiring a set of quite different laser sources as gas lasers, solid state lasers or diode lasers. A new approach by supercontinuum light sources is presented and discussed, which significantly enhances flexibility and coverage of the excitation spectra of typical, rare and natural fluorochromes.

Closing the spectral gap: The transition from fixed-parameter fluorescence to tunable devices in confocal microscopy

“White,” when describing a physical parameter, means constant distribution over the observed range. The white confocal refers to a concept that allows tuning any wavelength or light pattern from a white laser source for illumination of the sample, simultaneously transmitting a white spectrum to the detector and finally collecting at any selection of tunable bands within the visible spectrum. In this chapter, such a technology is described. It is composed of lasers based on photonic crystal fibers and acousto-optical devices for selection (AOTF) of small bandlets and guiding them onto the sample. Simultaneously, a second acousto-optical device (AOBS) performs white transmission of the emission into a tunable multiband detection system, based on a spectrophotometer concept. This concept is the ultimate solution for efficient recording and optimal separation of multiparameter fluorescence in confocal microscopy.

The White Confocal: Continuous Spectral Tuning in Excitation and Emission

Super-resolution refers to any device or method that can resolve better than the classical Abbe limit. Apart from infinite super-resolution techniques such as STED (stimulated emission depletion) and SMLM (single-molecule localization methods) that can theoretically resolve to any detail, there are also methods for limited super-resolution. Here we present HyVolution by Leica, which merges optical super-resolution and computational super-resolution. The optical part is provided by confocal microscopy, and the computational part by deconvolution. Lateral resolution of 140 nm is demonstrated. HyVolution offers multiple fluorescence recording in truly simultaneous mode.

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HyVolution – the Smart Path to Confocal Super-Resolution

The new Leica TCS SP5 spectral confocal and multiphoton microscope system is based on the widely used SP® spectral detection module. This module allows selection of any spectral emission band in the visible range. Five spectral bands can be selected simultaneously. As an extension, two of these detectors may operate as a sensor for fluorescence lifetime imaging microscopy (FLIM). This SP-FLIM combination opens up a completely new dimension in wavelength-dependent lifetime imaging.

Rolf Borlinghaus

Papers

MRT letter: high speed scanning has the potential to increase fluorescence yield and to reduce photobleaching.

Closing the spectral gap: The transition from fixed-parameter fluorescence to tunable devices in confocal microscopy

The White Confocal: Continuous Spectral Tuning in Excitation and Emission

HyVolution – the Smart Path to Confocal Super-Resolution

Spectral fluorescence lifetime imaging microscopy: new dimensions with Leica TCS SP5