The combination of pulsed-mode excitation multifocal multiphoton microscopy with a high-repetition, time-gated intensified CCD camera enables efficient three-dimensional (3D) fluorescence lifetime imaging. With a 200-ps gate opening at 76 MHz repetition rate, fluorescence decay can be traced in a sequence of images with varying delays between pulse and gate. Fluorophore lifetimes are measured with a precision of a few picoseconds. As an application we show that, upon two-photon excitation at 800 nm, certain pollen samples feature a multiexponential fluorescence relaxation. Our results indicate that efficient four-dimensional microscopy with hundreds of nanometer spatial and tens of picoseconds temporal resolution is within reach.

Fluorescence lifetime three-dimensional microscopy with picosecond precision using a multifocal multiphoton microscope

A scanning microscope utilizing two-photon excitation in combination with fluorescence lifetime contrast is presented. The microscope makes use of a tunable femtosecond titanium:sapphire laser enabling the two-photon excitation of a broad range of fluorescent molecules, including UV probes. Importantly, the penetration depth of the two-photon exciting (infra)red light is substantially greater than for the corresponding single-photon wavelength while photobleaching is significantly reduced.



The time structure of the Ti:Sa laser can be employed in a straightforward way for the realization of fluorescence lifetime imaging. The fluorescence lifetime is sensitive to the local environment of the fluorescent molecule. This behaviour can be used for example to quantify concentrations of ions, such as pH and Ca2+, or pO2 and pCO2. In the set-up presented here the fluorescence lifetime imaging is accomplished by time-gated single photon counting.



The performance and optical properties of the microscope are investigated by a number of test measurements on fluorescent test beads. Point-spread functions calculated from measurements on 230-nm beads using an iterative restoration procedure compare well with theoretical expectations.



Lifetime imaging experiments on a test target containing two different types of test bead in a fluorescent buffer all with different lifetimes (2.15 ns, 2.56 ns and 3.34 ns) show excellent quantitative agreement with reference values obtained from time correlated single photon counting measurements. Moreover, the standard deviation in the results can be wholly ascribed to the photon statistics.



Measurements of acridine orange stained biofilms are presented as an example of the potential of two-photon excitation combined with fluorescence lifetime contrast. Fluorescence lifetime and intensity images were recorded over the whole sample depth of 100 μm. Fluorescence intensity imaging is seriously hampered by the rapid decrease of the fluorescence signal as a function of the depth into the sample. Fluorescence lifetime imaging on the other hand is not affected by the decrease of the fluorescence intensity.

Time-gated fluorescence lifetime imaging and microvolume spectroscopy using two-photon excitation

The Acid-Base Status of the Blood

Saliva and dental caries

A Tunable Ratiometric pH Sensor Based on Carbon Nanodots for the Quantitative Measurement of the Intracellular pH of Whole Cells.

The oral cavity is the most complex and accessible microbial ecosystem in the human body. It is the entrance to the respiratory and gastrointestinal tracts and, as such is exposed to unique environmental constraints. The human mouth is home to a myriad of microorganisms, many of which are exclusively found in this unique habitat. These microbial inhabitants can establish themselves and thrive in this environment by attaching to the various surfaces of the oral cavity. Following attachment, they form three-dimensional, complex and highly-integrated microbial communities. Despite their complexity and natural fluctuations in environmental parameters, in health, these communities remain relatively stable over time. This stability is termed microbial homeostasis. Disruption of the microbial homeostasis occurs as a result of regular or prolonged challenges in the form of an altered environment. These disruptions favour a shift in the microbial populations, suppressing the metabolism of the beneficial inhabitants and allowing unfavourable microorganisms to thrive in the lack of competition. This change in the oral microbiota facilitates the progression from oral health to disease. Despite continuing research and development in preventative measures dental caries, characterised by localised dissolution of the dental hard tissues, remain one of the most prevalent disorders affecting man today. This decay occurs as a result of strong organic acids produced by the microbiota within dental plaque following exposure to fermentable carbohydrates. Furthermore, prolonged and regular exposure to acids suppresses the growth of 'beneficial' bacteria allowing acidogenic, aciduric microorganisms, such as Streptococcus mutans and lactobacilli to thrive in the lack of competition. The presence of these acidogenic microorganisms causes an increase in acid production and an increase in the duration of exposure to those acids. Although rarely life-threatening, they create an enormous economic burden to healthcare providers worldwide and cause significant physical and social impact on those affected, including diet, communication and self-esteem. Greater understanding is required to appreciate the dynamic relationship that exists between the environment, the microbiota and the host. To gain a greater understanding of how the microbial ecology is affected, it is necessary to be able to determine how pH changes during and following fermentation and the effect these perturbations have upon the microbial community. At present, the most commonly employed methods include the use of microelectrodes, whether through insertion into laboratory grown biofilm or incorporated within in vivo prosthetics devices. These methods are not without their drawbacks. In the act of measurement, microelectrodes are inserted into the biofilm resulting in, at least partial, disruption of the biofilm and this may have a detrimental effect on the results. In vivo prosthetics provides measurement at the biofilm interfaces and in physiological conditions, however almost certainly require partially dentate individuals and are difficult to use. Novel methods are required to investigate pH within biofilms which provide a multidimensional determination, including temporal, and do not cause a detrimental effect upon the biofilm. Here, I examine two optical methods which utilised different properties of fluorescence to investigate pH microgradients within biofilms designed to mimic cariogenic dental plaque. The two methods are dual-fluorophore, ratiometric, pH-sensitive nanosensors imaged through confocal laser scanning microscopy and SNARF®-4F 5-(and-6)-carboxylic acid imaged through time-correlated single-photon counting and fluorescence-lifetime imaging microscopy. The nanosensors were designed, produced and characterised prior to calibration. The nanosensors were applied to biofilms with limited success, likely due to poor penetration. The optical properties of SNARF®-4F 5-(and-6)-carboxylic acid were characterised, including the two-photon molecular excitation wavelength for use here. The fluorophore was calibrated and applied to bacterial sediment and biofilms and the localised environmental pH assessed following exposure to a fermentable carbohydrate to decrease the pH. Many of the drawbacks experienced with currently employed methods have been addressed by these methods, however further research and development is required.

Dallas Peter Roulston

Papers

An investigation of optical methods for the mapping of microgradients of hydrogen-ion concentration within dental biofilms