This presentation describes the development of the optical macroscanner and its application for metabolic imaging of large areas of tumors in mice. The scanner allows to interrogate areas as large as 15x15mm with the lateral resolution on the order of 15 micrometers. Acquisition times range from a few seconds for low pixel numbers to
several minutes for high-resolution images. We present data for NAD(P)H imaging of tumor with genetically encoded mKate2. In addition, using macroscanner we demonstrated the possibility of visualizing caspase-3 activity using the FRET-biosensor TR23K, which is based on a pair of proteins - a red fluorescent protein as a donor and a chromoprotein as an acceptor. The in vivo assay was noninvasive and could be applied in strongly and weakly fluorescent subcutaneous xenografts in mice using the FLIM-FRET method.
Caspase-3 is known for its role in apoptosis and programmed cell death regulation. We detected caspase-3 activation in vivo in tumor xenografts via shift of mean fluorescence lifetimes of a caspase-3 sensor. We used the genetically encoded sensor TR23K based on the red fluorescent protein TagRFP and chromoprotein KFP linked by 23 amino acid residues (TagRFP-23-KFP) containing a specific caspase cleavage DEVD motif to monitor the activity of caspase-3 in tumor xenografts by means of fluorescence lifetime imaging-Forster resonance energy transfer. Apoptosis was induced by injection of paclitaxel for A549 lung adenocarcinoma and etoposide and cisplatin for HEp-2 pharynx adenocarcinoma. We observed a shift in lifetime distribution from 1.6 to 1.9 ns to 2.1 to 2.4 ns, which indicated the activation of caspase-3. Even within the same tumor, the lifetime varied presumably due to the tumor heterogeneity and the different depth of tumor invasion. Thus, processing time-resolved fluorescence images allows detection of both the cleaved and noncleaved states of the TR23K sensor in real-time mode during the course of several weeks noninvasively. This approach can be used in drug screening, facilitating the development of new anticancer agents as well as improvement of chemotherapy efficiency and its adaptation for personal treatment.
CdSe-core, ZnS-capped semiconductor nanoparticles - quantum dots (QDs) - have been at the forefront of biomedical
nanotechnology research thanks to their unique optical Η photophysical properties. In the present study the impact of the
particle coating and size on their in vivo fate after intravenous (IV) injection into mice was studied by fluorescence
methods. For this study, we compared organ-selective biodistribution and elimination routes of synthesized QDs coated
with 3-mercaptopropionic acid (QD MPA) and commercially available Qtracker 705 nontargeted quantum dots with
poly(ethylene glycol) coating (QD PEG). We observed primary accumulation of these QDs in lung. Experiments
demonstrated that QD MPA and QD PEG have both remained fluorescent in lung after at least 24 hours postinjection.
Moreover, QDs was seen to deposit mainly in liver, spleen, kidney and lymph nodes. We also concluded that QDs MPA
and QDs 705 are both sequestered and not excreted with feces or urine.
FRET-sensor with nonfluorescent protein as an acceptor was synthesized to observe caspase-3 activity in lifetime mode.
We inserted caspase-3 cleavable linker between red highly fluorescent protein TagRFP and chromoprotein KFP.
Dynamic light scattering was used to determine size of the fusion protein. Incubation with caspase-3 lead to increase
both fluorescence intensity and lifetime of the construction. Cleavage of the linker between proteins was confirmed by
electrophoresis and immunoblotting. FLIM-microscopy showed the differences between fluorescence decays of A549
cell line expressed TagRFP and TagRFP-23-KFP.
The investigations of photodynamic activity of the dibiotinylated aluminium sulphophthalocyanine in vitro and in vivo were performed. The results obtained showed that in vitro dibiotinylated aluminium sulphophthalocyanine provides an effective damage of small cell lung carcinoma OAT-75. In vivo dibiotinylated aluminium sulphophthalocyanine induces a total damage of Erlich carcinoma with expressed vascular damage even in a concentration 0.5 mg/kg of body weight.