In vivo medical imaging has made great progress due to advances in the engineering of imaging devices and developments in the chemistry of imaging probes Several modalities have been utilized for medical imaging, including X-ray radiography and computed tomography (x-ray CT), radionuclide imaging using single photons and positrons, magnetic resonance imaging (MRI), ultrasonography (US), and optical imaging In order to extract more information from imaging, “contrast agents” have been employed For example, organic iodine compounds have been used in X-ray radiography and computed tomography, superparamagnetic or paramagnetic metals have been used in MRI, and microbubbles have been used in ultrasonography Most of these, however, are non-targeted reagents

Molecular imaging is widely considered the future for medical imaging Molecular imaging has been defined as the in vivo characterization and measurement of biologic process at the cellular and molecular level1, or more broadly as a technique to directly or indirectly monitor and record the spatio-temporal distribution of molecular or cellular processes for biochemical, biologic, diagnostic, or therapeutic application2 Molecular imaging is the logical next step in the evolution of medical imaging after anatomic imaging (eg x-rays) and functional imaging (eg MRI) In order to attain truly targeted imaging of specific molecules which exist in relatively low concentrations in living tissues, the imaging techniques must be highly sensitive Although MRI, US, and x-ray CT are often listed as molecular imaging modalities, in truth, radionuclide and optical imaging are the most practical modalities, for molecular imaging, because of their sensitivity and the specificity for target detection

Radionuclide imaging, including gamma scintigraphy and positron emission tomography (PET), are highly sensitive, quantitative, and offer the potential for whole body scanning However, radionuclide imaging methods have the disadvantages of poor spatial and temporal resolution3 Additionally, they require radioactive compounds which have an intrinsically limited half life, and which expose the patient and practitioner to ionizing radiation and are therefore subject to a variety of stringent safety regulations which limit their repeated use4

Optical imaging, on the other hand, has comparable sensitivity to radionuclide imaging, and can be “targeted” if the emitting fluorophore is conjugated to a targeting ligand3 Optical imaging, by virtue of being “switchable”, can result in very high target to background ratios “Switchable” or activatable optical probes are unique in the field of molecular imaging since these agents can be turned on in specific environments but otherwise remain undetectable This improves the achievable target to background ratios, enabling the detection of small tumors against a dark background5,6 This advantage must be balanced against the lack of quantitation with optical imaging due to unpredictable light scattering and absorption, especially when the object of interest is deep within the tissue Visualization through the skin is limited to superficial tissues such as the breast7-9 or lymph nodes10,11 The fluorescence signal from the bright GFP-expressing tumors can be seen in the deep organ only in the nude mice 12,13 However, optical molecular imaging can also be employed during endoscopy14 or surgery 15,16

/pdf/new-strategies-for-fluorescent-probe-design-in-medical-1mcbc28raf.pdf

New Strategies for Fluorescent Probe Design in Medical Diagnostic Imaging

This critical review covers the advances made using the 4-bora-3a,4a-diaza-s-indacene (BODIPY) scaffold as a fluorophore in the design, synthesis and application of fluorescent indicators for pH, metal ions, anions, biomolecules, reactive oxygen species, reactive nitrogen species, redox potential, chemical reactions and various physical phenomena. The sections of the review describing the criteria for rational design of fluorescent indicators and the mathematical expressions for analyzing spectrophotometric and fluorometric titrations are applicable to all fluorescent probes (206 references).

Fluorescent indicators based on BODIPY

When a molecule absorbs a photon of appropriate energy, a chain of photophysical events ensues, such as internal conversion or vibrational relaxation (loss of energy in the absence of light emission), fluorescence, intersystem crossing (from singlet state to a triplet state) and phosphorescence, as shown in the Jablonski diagram for organic molecules (Fig. 1). Each of the processes occurs with a certain probability, characterized by decay rate constants (k). It can be shown that the average length of time τ for the set of molecules to decay from one state to another is reciprocally proportional to the rate of decay: τ = 1/k. This average length of time is called the mean lifetime, or simply lifetime. It can also be shown that the lifetime of a photophysical process is the time required by a population of N electronically excited molecules to be reduced by a factor of e. Correspondingly, the fluorescence lifetime is the time required by a population of excited fluorophores to decrease exponentially to N/e via the loss of energy through fluorescence and other non-radiative processes. The lifetime of photophycal processes vary significantly from tens of femotoseconds for internal conversion1,2 to nanoseconds for fluorescence and microseconds or seconds for phosphorescence.1






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Figure 1


Jablonski diagram and a timescale of photophysical processes for organic molecules.

/pdf/fluorescence-lifetime-measurements-and-biological-imaging-xfalzh4w7e.pdf

Fluorescence Lifetime Measurements and Biological Imaging

Design strategies for water-soluble small molecular chromogenic and fluorogenic probes.

5. Cyanine-Based pHi Indicators 2717 5.1. Design of pH-Sensitive Cyanine Dyes 2717 5.2. Near-Neutral Cyanine-Based pH Indicators 2718 5.3. Acidic Cyanine-Based pH Indicators 2719 6. Miscellaneous Small Molecule pHi Indicators 2719 6.1. Various Indicators for Near-Neutral pH Values 2719 6.1.1. Europium Complex 2719 6.1.2. Fluorene Derivative 2719 6.1.3. 1,4-Dihydroxyphthalonitrile (1,4-DHPN) 2720 6.1.4. 8-Hydroxypyrene-1,3,6-trisulfonic acid (HPTS) 2720

Fluorescent indicators for intracellular pH.

A long-term goal of cancer diagnosis is to develop tumor-imaging techniques that have sufficient specificity and sensitivity. To achieve this goal, minimizing the background signal originating from nontarget tissues is crucial. Here we achieve highly specific in vivo cancer visualization by using a newly designed targeted 'activatable' fluorescent imaging probe. This agent is activated after cellular internalization by sensing the pH change in the lysosome. Novel acidic pH-activatable probes based on the boron-dipyrromethene fluorophore were synthesized and then conjugated to a cancer-targeting monoclonal antibody. As proof of concept, ex vivo and in vivo imaging of human epidermal growth factor receptor type 2-positive lung cancer cells in mice was performed. The probe was highly specific for tumors with minimal background signal. Furthermore, because the acidic pH in lysosomes is maintained by the energy-consuming proton pump, only viable cancer cells were successfully visualized. The design concept can be widely adapted to cancer-specific, cell surface-targeting molecules that result in cellular internalization.

Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes.

A light source device for endoscopes includes a light source for emitting white light containing the components of light belonging to a visible region and wavelength bands other than the visible region; at least, a rotary color separating filter on a field sequential system; and an infrared cutoff filter for blocking light in an infrared region, of a wavelength band of the white light. When the rotary color separating filter is disposed in an optical path from the light source to a light guide, an interference filter in which an interference film is applied to a transparent base plate is placed in the optical path. This arrangement obviates the defect of burning the entrance end of the light guide or of breaking the infrared cutoff filter.

Light source device for endoscopes

An endoscope system is disclosed for detecting fluorescent light emitted in the near-infrared region by a plurality of fluorescent labeling materials introduced into a living tissue. An illumination system generates illumination light in the wavelength range 600 nm-2000 nm which serves as excitation light for the plurality of fluorescent labeling materials, and a detection system that can separately detect different ones of the plurality of fluorescent light emissions that are emitted at different wavelengths from among the plurality of fluorescent labeling materials is provided. The endoscope system may include a conventional-type endoscope having an insertion section, or a capsule endoscope that wirelessly transmits image data. By superimposing the image data obtained using reflected light in the visible region and fluorescent light emitted by the fluorescent labeling materials, improved diagnostic capabilities are provided.

Endoscope system for fluorescent observation

An imaging apparatus for endoscopes includes an endoscope relaying an object image through an observing optical system, and an imaging section removably mounted to the rearmost end of the endoscope, including a stop unit provided with a variable stop capable of changing an aperture size thereof, an imaging lens system, a focus lens system, and an image sensor. The relative positions of the variable stop, the imaging lens system, and the image sensor are constant, and the focus lens system can be moved along the optical axis of the imaging section. In this way, the imaging apparatus for endoscopes can be provided which is simple in structure, small in size, and low in cost.

Imaging apparatus for endoscopes

A light source device is disclosed having a lamp which emits light along an optical path, a condensing lens, and optical filters that may be selectively positioned in the optical path in a manner that at least three of four enumerated illumination modes can be selected. Also disclosed is a diagnosing method of a living body lesion in combination with three enumerated observation methods. Also disclosed is an endoscope that includes an imaging unit at a tip portion of an insertable part wherein the imaging unit includes a spatial frequency conversion means. The imaging unit performs at least one of the following three functions: (1) imaging fluorescence from in vivo tissue; (2) imaging arrays of capillary vessels in the mucosal layer of in vivo tissue; and (3) imaging arrays of blood vessels and lymphatics in the lower layer of the mucosa of in vivo tissue.

Akira Hasegawa

Papers

Selective molecular imaging of viable cancer cells with pH-activatable fluorescence probes.

Light source device for endoscopes

Endoscope system for fluorescent observation

Imaging apparatus for endoscopes

Endoscope apparatus and diagnosing method using it