Seiichi Uchiyama

Intracellular temperature mapping with a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy

Cellular functions are fundamentally regulated by intracellular temperature, which influences biochemical reactions inside a cell. Despite the important contributions to biological and medical applications that it would offer, intracellular temperature mapping has not been achieved. Here we demonstrate the first intracellular temperature mapping based on a fluorescent polymeric thermometer and fluorescence lifetime imaging microscopy. The spatial and temperature resolutions of our thermometry were at the diffraction limited level (200 nm) and 0.18–0.58 °C. The intracellular temperature distribution we observed indicated that the nucleus and centrosome of a COS7 cell, both showed a significantly higher temperature than the cytoplasm and that the temperature gap between the nucleus and the cytoplasm differed depending on the cell cycle. The heat production from mitochondria was also observed as a proximal local temperature increase. These results showed that our new intracellular thermometry could determine an intrinsic relationship between the temperature and organelle function.

Hydrophilic Fluorescent Nanogel Thermometer for Intracellular Thermometry

The first methodology to measure intracellular temperature is described. A highly hydrophilic fluorescent nanogel thermometer developed for this purpose stays in the cytoplasm and emits stronger fluorescence at a higher temperature. Thus, intracellular temperature variations associated with biological processes can be monitored by this novel thermometer with a temperature resolution of better than 0.5 degrees C.

Development of fluorescent microgel thermometers based on thermo-responsive polymers and their modulation of sensitivity range

Fluorescent molecular thermometers based on thermo-responsive linear polymer molecules such as poly(N-isopropylacrylamide) (PNIPAM) labelled with a polarity-responsive fluorescent molecule benzofurazan (BD) are the most sensitive known. Thermo-responsive PNIPAM and some related polymer microgel particles labelled with BD by emulsion polymerization have been prepared and their fluorescence properties in water as fluorescent thermometers studied. All the cross-linked polymer microgel particles dispersed in water fluoresce strongly as soon as each threshold temperature is exceeded. The nine kinds of microgel dispersion developed in this work thoroughly cover the sensitivity range from 18 to 47 °C. They are not only more sensitive than the previous fluorescent molecular thermometers based on other principles but also highly reproducible in their behaviour.

Temperature-dependent fluorescence lifetime of a fluorescent polymeric thermometer, poly(N-isopropylacrylamide), labeled by polarity and hydrogen bonding sensitive 4-sulfamoyl-7-aminobenzofurazan.

Fluorescent molecular thermometers showing temperature-dependent fluorescence lifetimes enable thermal mapping of small spaces such as a microchannel and a living cell. We report the temperature-dependent fluorescence lifetimes of poly(NIPAM-co-DBD-AA), which is a random copolymer of N-isopropylacrylamide (NIPAM) and an environment-sensitive fluorescent monomer (DBD-AA) containing a 4-sulfamoyl-7-aminobenzofurazan structure. The average fluorescence lifetime of poly(NIPAM-co-DBD-AA) in aqueous solution increased from 4.22 to 14.1 ns with increasing temperature from 30 to 35 degrees C. This drastic change in fluorescence lifetime (27% increase per 1 degrees C) is the sharpest ever reported. Concentration independency, one of the advantages of fluorescence lifetime measurements, was seen in average fluorescence lifetime (13.7 +/- 0.18 ns) of poly(NIPAM-co-DBD-AA) at 33 degrees C over a wide concentration range (0.005-1 w/v%). With increasing temperature, polyNIPAM units in poly(NIPAM-co-DBD-AA) change their structure from an extended form to a globular form, providing apolar and aprotic environments to the fluorescent DBD-AA units. Consequently, the environment-sensitive DBD-AA units translate the local environmental changes into the extension of the fluorescence lifetime. This role of the DBD-AA units was revealed by a study of solvent effects on fluorescence lifetime of a model environment-sensitive fluorophore.

Multiplexing Sensory Molecules Map Protons Near Micellar Membranes

Fluorescent sensors have great potential to operate as molecular-level devices in nanospaces. Generally, a fluorescent sensor monitors a single parameter of its local environment, such as ion concentration. More functionalized systems which operate according to similar principles are molecular logic gates. These gates respond to multiple parameters simultaneously according to defined Boolean transformations. There are also a few examples of molecular sensors which respond to multiple parameters, each by a different analytical technique. Herein we demonstrate a new multiplexing fluorescent sensor which simultaneously monitors multiple parameters (local proton concentration and polarity in this instance) by multiple emission properties (intensity and wavelength, respectively). As the polarity of spherical micelles in water is expected to change largely monotonically along a radial coordinate, polarity data translate into positions. We can thus obtain local proton densities at various positions by scattering a series of multiplexing sensors widely over the aqueous micellar field. Therefore a nanoscaled mapping of proton concentration emerges for this simple membrane system. Proton concentration gradients are responsible for the subject of bioenergetics. Multiplexing sensors also correspond to nanoscale versions of robotic vehicles which go to humanly inaccessible spaces, map local properties and send information back to us. Scheme 1 shows the structures of the fluorescent multiplexing sensors 1–18 used in this study. These sensors consist of a polarity-sensitive fluorophore (blue), a proton receptor (orange), position tuners (red), and a spacer (green). The sensors function as follows: 1) The local proton concentration is examined by a DpKa value (pKa in micellar solution–pKa in water) of a conjugate acid of the receptor amine. This DpKa value is affected by electrostatic potential and dielectric constant at the sensor location but is independent of intrinsic acidity/basicity of the sensor. If local effective proton concentration is higher than that of bulk water, a positive DpKa value is obtained. [9] As our sensors possess a fluorescence “off–on” switching system by controlling photoinduced electron transfer processes with a fluorophore–spacer–receptor format, the DpKa values can be determined from fluorescence intensity, with pH profiles arising from titrations. 2) The local polarity is estimated from the emission wavelength of the polarity-sensitive fluorophore, 4-sulfamoyl-7aminobenzofurazan, as its emission wavelength is strongly red-shifted with increasing environmental polarity and is smoothly related to the dielectric constant e of the solvent. Thus, the relationship between the emission wavelength and the e value is obtained beforehand for each sensor from the fluorescence spectra in water, methanol, and so on (see the Supporting Information). 3) The position of a sensor near micellar membranes is altered by changing its substituents R–R. The sensor bearing more hydrophilic substituents is expected to stay at a more hydrophilic region in the nanospace. Finally, by collecting the environmental data for 1– 18, proton concentration maps near micellar membranes can be established in the form of DpKa–e diagrams. In the present study, Triton X-100 (neutral, radius: < 4.8 nm), octyl b-dglucopyranoside (OG; neutral, ~ 2.3 nm), sodium dodecylsulfate (SDS; anionic, < 3.6 nm), and cetyltrimethylammonium chloride (CTAC; cationic, < 3.5 nm) are used as micelle media in which the nanoscaled proton gradients are evaluated. The fluorescence properties of 9 in water and 18 in Triton X-100 aqueous solution during titrations are shown in Figure 1 as representatives of sensory functions. Regarding proton concentration, the DpKa value for 18 in the Triton XScheme 1. Fluorescent multiplexing sensors 1–18. The orders of 1!9 and 10!18 are determined by the logP (n-octanol/water partition coefficient) value of a corresponding amine RRNH (see the Supporting Information).

Cationic fluorescent polymeric thermometers with the ability to enter yeast and mammalian cells for practical intracellular temperature measurements.

An accurate method for measuring intracellular temperature is potentially valuable because the temperature inside a cell can correlate with diverse biological reactions and functions. In a previous study, we reported the use of a fluorescent polymeric thermometer to reveal intracellular temperature distributions, but this polymer required microinjection for intracellular use, such that it was not user-friendly; furthermore, it could not be used in small cells or cells with a cell wall, such as yeast. In the present study, we developed several novel cationic fluorescent copolymers, including NN-AP2.5 and NN/NI-AP2.5, which exhibited spontaneous and rapid entry (≤20 min) into yeast cells and subsequent stable retention in the cytoplasm. The fluorescence lifetime of NN-AP2.5 in yeast cells was temperature-dependent (6.2 ns at 15 °C and 8.6 ns at 35 °C), and the evaluated temperature resolution was 0.09-0.78 °C within this temperature range. In addition, NN-AP2.5 and NN/NI-AP2.5 readily entered and functioned within mammalian cells. Taken together, these data show that our novel cationic fluorescent polymeric thermometers enable accurate and practical intracellular thermometry in a wide range of cells without the need for a microinjection procedure.

Molecular Logic Gates and Luminescent Sensors Based on Photoinduced Electron Transfer

The competition between Photoinduced electron transfer (PET) and other de-excitation pathways such as fluorescence and phosphorescence can be controlled within designed molecular structures. Depending on the particular design, the resulting optical output is thus a function of various inputs such as ion concentration and excitation light dose. Once digitized into binary code, these input-output patterns can be interpreted according to Boolean logic. The single-input logic types of YES and NOT cover simple sensors and the double- (or higher-) input logic types represent other gates such as AND and OR. The logic-based arithmetic processors such as half-adders and half-subtractors are also featured. Naturally, a principal application of the more complex gates is in multi-sensing contexts.

Quantitative mapping of aqueous microfluidic temperature with sub-degree resolution using fluorescence lifetime imaging microscopy

The use of a water-soluble, thermo-responsive polymer as a highly sensitive fluorescence-lifetime probe of microfluidic temperature is demonstrated. The fluorescence lifetime of poly(N-isopropylacrylamide) labelled with a benzofurazan fluorophore is shown to have a steep dependence on temperature around the polymer phase transition and the photophysical origin of this response is established. The use of this unusual fluorescent probe in conjunction with fluorescence lifetime imaging microscopy (FLIM) enables the spatial variation of temperature in a microfluidic device to be mapped, on the micron scale, with a resolution of less than 0.1 degrees C. This represents an increase in temperature resolution of an order of magnitude over that achieved previously by FLIM of temperature-sensitive dyes.

A study of the relationship between the chemical structures and the fluorescence quantum yields of coumarins, quinoxalinones and benzoxazinones for the development of sensitive fluorescent derivatization reagents

To develop new fluorescent derivatization reagents, we investigated the relationship between the chemical structures and the fluorescence quantum yields (phi(f)) of coumarins, quinoxalinones and benzoxadinones. Forty-six compounds were synthesized and their fluorescence spectra were measured in n-hexane, ethyl acetate, methanol and water. The energy levels of these compounds were calculated by combination of the semi-empirical AM1 and INDO/S (CI = all) methods. The deltaE(Tn(n,pi*), S1(pi,pi*)) (the energy gap between the Tn(n,pi*) and S1(pi,pi*) states) values were well correlated with the phi(f) values, which enables us to predict the phi(f) values from their chemical structures. Based on this relationship, 3-phenyl-7-N-piperazinoquinoxalin-2(1H)-one (PQ-Pz) and 7-(3-(S)-aminopyrrolidin-1-yl)-3-phenylquinoxalin-2-(1H)-one (PQ-APy) were developed as fluorescent derivatization reagents for carboxylic acids. The derivatives of the carboxylic acids with PQ-Pz and PQ-APy showed large phi(f) values even in polar solvents, suggesting that these reagents are suitable for the microanalysis of biologically important carboxylic acids by reversed phase HPLC.

Environment‐Sensitive Fluorophores with Benzothiadiazole and Benzoselenadiazole Structures as Candidate Components of a Fluorescent Polymeric Thermometer

An environment-sensitive fluorophore can change its maximum emission wavelength (λ(em)), fluorescence quantum yield (Φ(f)), and fluorescence lifetime in response to the surrounding environment. We have developed two new intramolecular charge-transfer-type environment-sensitive fluorophores, DBThD-IA and DBSeD-IA, in which the oxygen atom of a well-established 2,1,3-benzoxadiazole environment-sensitive fluorophore, DBD-IA, has been replaced by a sulfur and selenium atom, respectively. DBThD-IA is highly fluorescent in n-hexane (Φ(f) =0.81, λ(em) =537 nm) with excitation at 449 nm, but is almost nonfluorescent in water (Φ(f) =0.037, λ(em) =616 nm), similarly to DBD-IA (Φ(f) =0.91, λ(em) =520 nm in n-hexane; Φ(f) =0.027, λ(em) =616 nm in water). A similar variation in fluorescence properties was also observed for DBSeD-IA (Φ(f) =0.24, λ(em) =591 nm in n-hexane; Φ(f) =0.0046, λ(em) =672 nm in water). An intensive study of the solvent effects on the fluorescence properties of these fluorophores revealed that both the polarity of the environment and hydrogen bonding with solvent molecules accelerate the nonradiative relaxation of the excited fluorophores. Time-resolved optoacoustic and phosphorescence measurements clarified that both intersystem crossing and internal conversion are involved in the nonradiative relaxation processes of DBThD-IA and DBSeD-IA. In addition, DBThD-IA exhibits a 10-fold higher photostability in aqueous solution than the original fluorophore DBD-IA, which allowed us to create a new robust molecular nanogel thermometer for intracellular thermometry.