Spectral and Lifetime Imaging

Spectral imaging is the collection and display of the spectral components of a fluorescence image. LOCI is currently developing a combined spectral/lifetime detector that is optimized for low-light level multiphoton imaging. The detector works in photon counting mode and essentially sorts detected photons into spectral and temporal bins. This detector is being developed primarily for the Optical Workstation but will also be used with the high-speed multiphoton imaging system currently under development.

Benefits of spectral imaging

Most commercial confocal and multiphoton microscopes currently have the ability to collect two or three pre-specified colors simultaneously. However, there is often a need for more complete spectral information to allow the detection of more fluorophores and to facilitate the setting of spectral windows to optimize detection of a specific fluorophore. The main goal of the detection system is to collect the desired signal in the presence of noise (detection noise, system noise, fluorescent background, etc.). Background fluorescence from endogenous fluorophores or from another interfering exogenous fluorophores can severely reduce or interpretation of the image signal. With multiple labeled samples, the signal from one fluorophore is often much stronger than another and can spill over to an adjacent channel. This problem is exacerbated by fluorophores with extended red tails such as DAPI. In these instances it is often better to move the spectral detection windows as far apart as possible to aid discrimination between the two fluorophores being studied rather than choosing spectral windows to give the maximum signal in each channel.

The use of multiple fluorescent labels has long been commonplace in the study of fixed specimens, and is now becoming established for in vivo studies. Not so long ago only three fluorophores were in widespread use (fluorescein, rhodamine and DAPI).  Now there is a plethora of fluorophores available, each with its own unique spectral characteristics. This has generated a considerable problem for fluorescence microscopists in that many different filter sets are required for double- or triple-labeled samples. Filter sets use expensive interference filters and dichroic mirrors and are often difficult to interchange. Ideally, filters should be continuously adjustable so that for any particular combination of fluorophores used, an optimal set of band-pass assignments can be selected for each detection channel.  This would minimize signal bleed-through and maximize the signal-to-noise ratio. Perhaps the greatest power of collecting the entire spectrum is this allows fluorophores to be identified and separated computationally (by comparison to reference fluorophore spectra) in the presence of high levels of background.

Most biological tissue is autofluorescent. Molecules such as NAD(P)H, elastin, and chlorophyll act as endogenous fluorophores. Often, these endogenous fluorophores can be identified by their characteristic spectra. A spectral imaging system is of considerable use in identifying endogenous fluorophores and specifying spectral windows that would either maximally accept or reject these signals depending on the application. Additional information may obtained by comparing spectra obtained at different excitation wavelengths.

The use of engineered fluorescent probes as physiological indicators has become a well-established technique. Some probes indicate the presence of a bound ligand by changes in fluorescence intensity (e.g. Calcium Green 1) while others use spectral shifts (e.g. Indo 1). The latter are favored because ratio imaging at two different wavelengths may be used to provide quantitative measurements that are independent of the concentration of the indicator molecule. Spectral detection allows an optimum set of spectral windows to be used for ratio imaging.

Fluorescence resonant energy transfer (FRET) is a powerful technique for measuring intermolecular distances in vivo (dos Remedios & Moens, 1995. J. Struct. Biol. 115, 175). This technique also requires custom filter sets that are matched to the donor and acceptor molecule's emission spectra. Ratiometric measurements are used to measure the extent of resonance transfer. FRET is proving to be a valuable technique for the in vivo visualization of the docking of a receptor with its ligand, and it is the basis of operation of a new GFP based calcium indicator, Cameleon (Miyawaki et al., 1997 Nature 388, 882-887).

Fluorescence in situ hybridyzation (FISH) is another very significant area where multiple fluorophores and ratiometric techniques are used (Dauwerse et al., 1992 Hum. Mol. Genet. 1, 593). Often the main requirement in this application is to spectrally resolve as many separate fluorescent probes as possible (Schröck et al., 1996 Science 273:494).

The following list summarizes the main advantages of a spectral imaging system over a conventional, filter-based three-channel detector:

  • Dynamic identification of auto-fluorescence and optimization of windows for rejection or imaging as required
  • Dynamic optimization of spectral windows for multiple labels
  • Dynamic background subtraction of reference spectra before the image is even displayed
  • Identification of spectral shifts of fluorophores in different environments e.g. bodipy ceramide Golgi marker or acridine orange binding to RNA or DNA
  • Full signal optimization for any given ratiometric indicator
  • Permits fluorophore separation after data collection if full spectral image is taken
  • Permits evaluation of standard histological procedures for MPLSM analysis for identification of tissue-specific spectral shifts of staining


Applications of lifetime and spectral imaging

At LOCI we are interested in lifetime measurements as a means of providing another dimension of information from fluorescent probes used in vivo. We find that in most applications where probes are viewed in 4-dimensions in vivo, we would benefit from more or better information. Specifically we anticipate that the combined MP spectral and lifetime imaging system will provide the following benefits to our collaborators:

  • More accuracy in ratiometric probe measurements. This will be achieved by choosing optimal spectral and lifetime parameters that give the maximum shift with the probe target signal (e.g. Ca++ concentration).
  • Lifetime imaging will enable some popular, non-ratiometric probes, such as calcium green to be used in a way that is concentration independent, thereby facilitating calibration of readings.
  • In conjunction with spectral imaging, lifetime imaging will improve the rejection of background fluorescence from endogenous fluorophores by the specification of optimum spectral and temporal windows. This is becoming an increasing important requirement for detecting low levels of GFP probe amid a background of autofluorescence.
  • Lifetime measurements add extra information that can be used in conjunction with spectral measurements for fluorophore identification. This will be useful when there is significant spectral overlap between probes. This technique should allow the use of a greater number of probes simultaneously, such as combinations of GFP variants.
  • Increasingly cell biological studies are using FRET for studies of protein/protein interactions or physiological parameters in vivo. There is usually a striking change in the lifetime of the donor and acceptor fluorophores undergoing a FRET interaction. Lifetime imaging may well prove superior to spectral ratio imaging for measuring FRET interactions. A combination of lifetime and spectral imaging will probably be better still.


Spectral and lifetime fluorescence imaging can be done using the SLIM MPLSM.