Fluorescence Lifetime Imaging Microscopy: Homodyne Technique Using High-speed Gated Image Intensifier

In the previous sections we demonstrated imaging of intracellular Ca2+ using our approach to FLIM. What other analytes can be imaged using FLIM? We have now characterized the lifetime of a good number of ion indicators. Based on these studies we know that Cl- can be imaged using FLIM with probes such as SPQ or MQAE, pH can be imaged using resorufin and probes of the SNAFL and SNARF (Molecular Probes) series, and Mg2+ can be imaged using Magnesium Green, Mag-quin-2, or Mag-quin-1 (Molecular Probes). At present, the probe for K+, as PBFI, are just adequate as a lifetime probe, but it seems likely that newer probes for Na+ (Sodium Green) and K+ will be practical for effective imaging. Of course, imaging of oxygen is possible using a wide variety of fluorophores. It should be noted that a wide variety of substances and/or phenomena are known to alter decay times, acting as quenchers. These include the phenomena of resonance energy transfer, collisional quenching, temperature effects, and viscosity effects. Also, the FLIM method is not limited to microscopic objects but can be possibly used in remote imaging of any object. Hence, FLIM will allow the imaging of the chemical and physical properties of objects based on the effects of the local environment on the decay kinetics of fluorophores. The instrumentation for FLIM is presently complex and requires a moderately complex laser source, a gain-modulated image intensifier, and a slow-scan CCD camera. However, one can readily imagine the instrumentation becoming rather compact, and even all solid-state, owing to advances in laser and CCD technologies and, more importantly, advances in probe chemistry. To be specific, the dye laser shown in Fig. 1 may be replaced by a simpler UV laser, such as the 354 nm HeCd laser which has become available (Fig. 11). Intensity modulation of a continuous wave sources can be accomplished with acoustooptic modulators. The scientific slow-scan CCD cameras are presently rather expensive, but they are used in the present instrumentation because of their linearity and high dynamic range. However, the increasing use of CCD detectors suggest that even the scientific-grade CCD cameras will soon become less costly. Additionally, the frame rates of these detectors continue to increase in response to the need for faster imaging. Furthermore, the performance of the video CCD cameras is increasing, as seen by the introduction of 10-bit video analog-to-digital (A/D) converters.(ABSTRACT TRUNCATED AT 400 WORDS)