Photons in - numbers out: perspectives in quantitative fluorescence microscopy for in situ protein counting.

The full understanding of cellular functions requires information about protein numbers for various biomolecular assemblies and their dynamics, which can be partly accessed by super-resolution fluorescence microscopy. Yet, many protein assemblies and cellular structures remain below the accessible resolution on the order of tens of nanometers thereby evading direct observation of processes, like self-association or oligomerization, that are crucial for many cellular functions. Over the recent years, several approaches have been developed addressing concentrations and copy numbers of biomolecules in cellular samples for specific applications. This has been achieved by new labeling strategies and improved sample preparation as well as advancements in super-resolution and single-molecule fluorescence microscopy. So far, none of the methods has reached a level of general and versatile usability due to individual advantages and limitations. In this article, important requirements of an ideal quantitative microscopy approach of general usability are outlined and discussed in the context of existing methods including sample preparation and labeling quality which are essential for the robustness and reliability of the methods and future applications in cell biology.

Time-resolved molecule counting by photon statistics across the visible spectrum.

In the past few years quantification of fluorescently labeled (bio-) molecules has become of increasing importance and several approaches have been developed to address this task. Counting by photon statistics measures the distribution of multiple photon detection events that carry information about the number and brightness of independently emitting fluorophores. The method enables absolute and non-destructive quantification, with the quality of estimates critically depending on the ability to accurately measure said photon statistics. Here, we present a combination of simulations and experiments that relate fundamental properties of fluorophores, i.e. their molecular brightness and photostability, to important experimental conditions, i.e. excitation power and acquisition time. Thereby, experimental settings and analysis parameters can be quantitatively evaluated, making counting by photon statistics a robust method for absolute counting of the number of emitters in a diffraction limited observation volume. We show that the time-resolution of counting varies with the fluorophore brightness and can be as fast as 10-100 ms. At the same time, the range of suitable fluorophores can be easily assessed. We evaluated the brightness and photostability of 16 organic dyes across the visible spectrum, providing information crucial for a range of single-molecule spectroscopy applications. This opens up exciting possibilities to analyze absolute stoichiometries in dynamic multi-component complexes.

Monitoring hydroquinone-quinone redox cycling by single molecule fluorescence spectroscopy.

Current research in the field of single-molecule chemistry is increasingly focused on the development of reliable experimental approaches for investigating chemical processes on a molecular level using single-molecule fluorescence spectroscopy (SMFS). Herein, we report on single-molecule observations of the copper(II)/air mediated oxidation of fluorescently labeled hydroquinone-based probe molecules followed by their reduction with cysteine. The redox cycle is signaled by quenching/recovery of fluorescence emission after addition of the oxidant/reductant, respectively. The experiments were realized by immobilizing the probe on a glass cover slide to allow single-molecule observation by means of total-internal-reflection fluorescence microscopy. Besides detection of successful oxidation and reduction events on single probe molecules, individual molecular intensity trajectories revealed dynamic processes and formation of intermediate states upon reaction. For the experimental design presented, we envision further reaction studies of catalytic redox-processes of single hydroquinone-moieties by means of SMFS.

Capillary array scanner for time-resolved detection and identification of fluorescently labelled DNA fragments.

The first capillary array scanner for time-resolved fluorescence detection in parallel capillary electrophoresis based on semiconductor technology is described. The system consists essentially of a confocal fluorescence microscope and a x,y-microscope scanning stage. Fluorescence of the labelled probe molecules was excited using a short-pulse diode laser emitting at 640 nm with a repetition rate of 50 MHz. Using a single filter system the fluorescence decays of different labels were detected by an avalanche photodiode in combination with a PC plug-in card for time-correlated single-photon counting (TCSPC). The time-resolved fluorescence signals were analyzed and identified by a maximum likelihood estimator (MLE). The x,y-microscope scanning stage allows for discontinuous, bidirectional scanning of up to 16 capillaries in an array, resulting in longer fluorescence collection times per capillary compared to scanners working in a continuous mode. Synchronization of the alignment and measurement process were developed to allow for data acquisition without overhead. Detection limits in the subzeptomol range for different dye molecules separated in parallel capillaries have been achieved. In addition, we report on parallel time-resolved detection and separation of more than 400 bases of single base extension DNA fragments in capillary array electrophoresis. Using only semiconductor technology the presented technique represents a low-cost alternative for high throughput DNA sequencing in parallel capillaries.

Multiplex dye DNA sequencing in capillary gel electrophoresis by diode laser-based time-resolved fluorescence detection.

A new one-lane, four-dye DNA sequencing method was developed which is based on time-resolved detection and identification of fluorescently labeled primers. For fluorescent labels, we used two newly synthesized rhodamine derivatives (MR200-1, JA169), a new oxazine derivative (JA242), and a commercially available cyanine dye (CY5). The dye fluorescence was excited by a pulsed diode laser emitting at 630 nm. The fluorescence decay was detected by an avalanche photodiode using a single-filter system. The dyes used here, so-called multiplex dyes, can be distinguished and identified via their fluorescence decay patterns. The DNA fragments were labeled at the primer using linkers of various lengths and positions. For separation of the enzymatically generated DNA fragments, capillary gel electrophoresis (CGE) with a 5% linear polyacrylamide gel was employed. On covalent attachment to oligonucleotides, the dyes exhibit fluorescence decay times of 3.7 (MR200-1), 2.9 (JA169), 2.4 (JA242), and 1.6 ns (CY5) measured during CGE. The CGE mobility of the labeled DNA fragments could be controlled and nearly equalized by the coupling position and the linker length. First, time-resolved, one-lane, four-dye DNA sequencing runs in CGE are presented. The sequence information of 660 bp was determined with a probability of correct classification of > 90%. This result was obtained directly from the raw data without any of the mobility corrections that are necessary with other methods.