The image intensity in most fluorescent microscopes is proportional to the concentration of the fluorophore being used. This provides localization information, but the resolution is limited by diffraction of about a micron, depending on the wavelength.
“What we’re trying to do is get more information from that sample,” explains Paul French, Ph.D., head of photonics group, Imperial College, London. “We are applying multidimensional fluorescence imaging and analysis to tissue autofluorescence.” His group is accomplishing this by developing and enhancing instruments, such as combining multibeam, multiphoton microscopes with fluorescent lifetime imaging.
A portable wide-field fluorescent lifetime imaging (FLIM) scanner based on time-gated imaging is being developed to provide faster in vivo diagnostic screening. This has potential to monitor disease and treatment response by using the autofluorescence of tissues. By evaluating the fluorescent lifetime of the autofluorescence, it has potential to show changes in cancerous vs. normal tissues.
FLIM can be quite slow because it must collect a lot of photons to make an accurate measurement and the widely used technique of time-correlated single-photon counting imposes a maximum photon detection rate. Wide field imaging can be faster because it collects photons for all the pixels in parallel.
“We are combining wide field time-gated imaging with Nipkow spinning disc microscopy to do high-speed FLIM to accelerate research based on FLIM and FLIM-FRET,” states Dr. French.
His group is working toward goals to develop clinical diagnostic instruments exploiting autofluorescence, to apply techniques like FLIM and FRET to automated imaging in a 96-well format, and to move to super resolution using a technique called stimulated emission depletion and combined with FLIM for protein-protein interaction analysis on a small scale to look at microclusters of signaling.
Optical imaging is being applied in the cancer drug development process to provide important information. Various in vivo imaging techniques are used in pre-clinical models to monitor disease progress and therapeutic efficacy, and to demonstrate target-oriented drug effects.
Werner Scheuer, Ph.D., research leader, preclinical imaging, Roche Diagnostics, is utilizing near infrared optical imaging because it has several advantages over that of other methods such as: no radioactive isotopes required, acquisition time between one and five seconds, and good penetration of tissue—roughly one centimeter. In addition, it is much less expensive, and training technical personnel can be accomplished in a reasonable time period.
His group is using optical imaging for pharmacodynamic (Pd) studies and pharmacokinetic (Pk) studies. Quenched probes, which are activated by tumor-associated proteases and allow monitoring of cell viability, are used for Pd studies. Therapeutic antibodies labeled with organic fluorophores like Cy5 are used for Pk studies. Monitoring signal intensity of the tumor area provides an indication of how fast the labeled antibody enters the tumor tissue and biodistribution of labeled antibodies to organs can be followed up. Verification of tumor-associated surface molecule expressed in different human xenografts in vivo is possible by using fluorescence labeled monoclonal antibodies. This approach allows the selection of an appropriate xenograft for further evaluation of an antitumor efficacy of target-orientated drugs.
One of the main limitations of optical imaging is exact quantification. “If you want to know exact quantification of tumor cell destruction, that’s hard to perform. But, since we’ve been using it for the past three years, it can accelerate the drug development process.” Dr. Scheuer says he is applying optical imaging in attempts to monitor apoptosis in vivo. “We are looking for other techniques or other proteins to identify apoptotic cells over time in tumor carrying mice.”
Studies are under way to evaluate labeled Annexin V for monitoring induction of apoptosis in vivo, allowing earlier estimates of drug efficacy. Also, quantum dots are suitable to address specific questions due to their photophysical properties like stronger signals and no bleaching.
“This field is growing fast, and the impact of optical imaging is becoming greater in the drug development process and in the clinic,” Dr. Scheuer says.