siFLIM: single-image frequency-domain FLIM provides fast and photon-efficient lifetime data.

We developed single-image fluorescence lifetime imaging microscopy (siFLIM), a method for acquiring quantitative lifetime images from a single exposure. siFLIM takes advantage of a new generation of dedicated cameras that simultaneously record two 180°-phase-shifted images, and it allows for video-rate lifetime imaging with minimal phototoxicity and bleaching. siFLIM is also inherently immune to artifacts stemming from rapid cellular movements and signal transients.

Modulated electron-multiplied fluorescence lifetime imaging microscope: all-solid-state camera for fluorescence lifetime imaging.

We have built an all-solid-state camera that is directly modulated at the pixel level for frequency-domain fluorescence lifetime imaging microscopy (FLIM) measurements. This novel camera eliminates the need for an image intensifier through the use of an application-specific charge coupled device design in a frequency-domain FLIM system. The first stage of evaluation for the camera has been carried out. Camera characteristics such as noise distribution, dark current influence, camera gain, sampling density, sensitivity, linearity of photometric response, and optical transfer function have been studied through experiments. We are able to do lifetime measurement using our modulated, electron-multiplied fluorescence lifetime imaging microscope (MEM-FLIM) camera for various objects, e.g., fluorescein solution, fixed green fluorescent protein (GFP) cells, and GFP-actin stained live cells. A detailed comparison of a conventional microchannel plate (MCP)-based FLIM system and the MEM-FLIM system is presented. The MEM-FLIM camera shows higher resolution and a better image quality. The MEM-FLIM camera provides a new opportunity for performing frequency-domain FLIM.

Photon budget analysis for fluorescence lifetime imaging microscopy.

We have constructed a mathematical model to analyze the photon efficiency of frequency-domain fluorescence lifetime imaging microscopy (FLIM). The power of the light source needed for illumination in a FLIM system and the signal-to-noise ratio of the detector have led us to a photon "budget." These measures are relevant to many fluorescence microscope users and the results are not restricted to FLIM but applicable to widefield fluorescence microscopy in general. Limitations in photon numbers, however, are more of an issue with FLIM compared to other less quantitative types of imaging. By modeling a typical experimental configuration, examples are given for fluorophores whose absorption peaks span the visible spectrum from Fura-2 to Cy5. We have performed experiments to validate the assumptions and parameters used in our mathematical model. The influence of fluorophore concentration on the intensity of the fluorescence emission light and the Poisson distribution assumption of the detected fluorescence emission light have been validated. The experimental results agree well with the mathematical model. This photon budget is important in order to characterize the constraints involved in current fluorescent microscope systems that are used for lifetime as well as intensity measurements and to design and fabricate new systems.

Microfluidic chip of fast DNA hybridization using denaturing and motion of nucleic acids.

This study presents the effect of fluidic temperatures and velocities on improving DNA hybridization. The efficiency of hybridization could be improved by introducing elevated temperature in the hot region and velocity in the cold region. Compared with the conventional methods, this hybridization microchip was able to increase the hybridization signal 4.6-fold within 30 min. The 1.4-kb single-stranded target DNA was tested. The increasing tendency of the fluorescence intensity was apparent when the temperature was higher than 82 degrees C, and the fluorescence intensity reached an asymptotic value at T>90 degrees C. A mathematical model was proposed to relate the fluorescence intensity of DNA hybridization with the hot-region temperature and the cold-region velocity. Based on these results, the new hybridization chip with the processes of temperature and velocity differences will improve efficiency of DNA detection. The microchip combined with hot-region temperature and cold-region bulk flow velocity effects could provide additional efficiency in DNA hybridization.