FLIM system

FLIM systems can be purchased as an add-on for a standard fluorescence microscope. Such a system will consist of a CCD camera coupled to a modulatable image intensifier, an LED light source, and driver electronics. This system will modulate the LED and image intensifier while shifting the phase between them as it takes a series of images (Fig. 2.1). 

Instrumentally, spectral FLIM generates a spectrally resolved set of lifetimes by either introducing filters to provide spectral resolution or a spectrograph between the sample and image intensifier. The first such system was created for looking at the long lifetimes of lanthanide dyes [37]. Later, a spectral FLIM system was described for measuring from a two-dimensional (2D) area of a microscope field... 

Introducing the spectrograph is relatively straightforward compared with the difficulty of assembling and programming a FLIM system and may be completed at reasonable cost (Fig. 2.2). 

Introduction of metalized neutral density filters in FLIM systems should be done cautiously as artifacts have been observed due to single or multiple reflections between pairs of ND filters. 

Once familiar with methods for calibrating the FLIM system, it is worthwhile to verify the range over which a given FLIM system performs well. This is particularly useful for persons new to the method to... 

Fig. 3.9. Principle of a wide field FLIM system with simultaneous detection of two time gates. The fluorescence image is split into two images and one of the images is optically delayed with respect to the other. Both images are detected simultaneously with the same time-gated detector.

Although new emerging technologies may provide more efficient applications in the future [29, 30], so far in all wide field TG-based FLIM systems, the gating process results in the loss of photons and a consequent reduction of the sensitivity (photon-economy). 

The upgrade of a frequency-domain fluorescence lifetime imaging microscope (FLIM) to a prismless objective-based total internal reflection-FLIM (TIR-FLIM) system is described. By off-axis coupling of the intensity-modulated laser from a fiber and using a high numerical aperture oil objective, TIR-FLIM can be readily achieved. The usefulness of the technique is demonstrated by a fluorescence resonance energy transfer study of Annexin A4 relocation and two-dimensional crystal formation near the plasma membrane of cultured mammalian cells. Possible future applications and comparison to other techniques are discussed. 

Since TIRF produces an evanescent wave of typically 80 nm depth and several tens of microns width, detection of TIRF-induced fluorescence requires a camera-based (imaging) detector. Hence, implementing TIRF on scanning FLIM systems or multiphoton FLIM systems is generally not possible. To combine it with FLIM, a nanosecond-gated or high-frequency-modulated imaging detector is required in addition to a pulsed or modulated laser source. In this chapter, the implementation with of TIRF into a frequency-domain wide-field FLIM system is described. 

Described below is how we upgraded our frequency-domain wide-held FLIM system in Amsterdam to incorporate TIRF. A TIRF upgrade to an existing wide-held FLIM setup is marginal both with respect to cost and time. Furthermore, after the upgrade, the system can still be used as a regular wide-held FLIM system. 

However, below I describe some simple troubleshooting experience of the TIRF-FLIM system in Amsterdam. 

As described in the previous section, the femtosecond fluorescence up-conversion microscope enabled us to visualize microscopic samples based on position-depen-dent ultrafast fluorescence dynamics. However, in the imaging measurements using the fluorescence up-conversion microscope, XY scanning was necessary as when using FLIM systems. To achieve non-scanning measurements of time-resolved fluorescence images, we developed another time-resolved fluorescence microscope. 

The time-resolved techniques that are usually used for FLIM are based on electronic-basis detection methods such as the time-correlated single photon counting or streak camera. Therefore, the time resolution of the FLIM system has been limited by several tens of picoseconds. However, fluorescence microscopy has the potential to provide much more information if we can observe the fluorescence dynamics in a microscopic region with higher time resolution. Given this background, we developed two types of ultrafast time-resolved fluorescence microscopes, i.e., the femtosecond fluorescence up-conversion microscope and the... 

Fig. 5.74 Fluorescence lifetime image recorded with the Leica SP2 D-FLIM system. One-photon excitation hy 405 nm diode laser, descarmed detection, 512 x 512 pixel scan, plant sample. Left Colour represents the mean lifetime of the donhle exponential decay, hlne to red = 200 ps to 2 ns. Right Colour represents the ratio of the amphtudes of the fast and slow decay component, Blue to red = 1 to 10...

The detectors, especially MCP-PMTs, can be severely overloaded by daylight leaking into the detection path. Moreover, the halogen or mercury lamp of the microscope may be a source of detector damage. Therefore, an NDD FLIM system must protect the detectors from overload. Detector protection by suitably controlled shutters is described under Sect. 7.3, page 302. 

A potential application of multimodule systems is high-speed two-photon multibeam scaiming systems [53, 77]. FLIM systems with 4, 8 or even 16 beams and the same number of parallel TCSPC channels appear feasible. The problem is to direct the fluorescence signals from the individual beams to separate PMTs or separate charmels of a multianode PMT. If this problem is solved, two-photon lifetime images can be recorded with unprecedented speed and resolution. 

The term microfluorometer or microspectrofluorometer is used for systems that excite a small, usually diffraction-limited volume of a sample under a microscope and record the fluorescence, either with wavelength resolution or without. The borderline with FLIM techniques is not clearly defined. A TCSPC FLIM system can be used to record the fluorescence of a single point, and a microspec-trofluorometer combined with a scanning stage can be used as a FLIM system. Some typical principles of microfluorometry are shown in Fig. 5.97. 

R.V. Krishnan, H. Saitoh, H. Terada, V.E. Centonze, B. Herman, Development of a multiphoton fluorescence lifetime imaging microscopy (FLIM) system using a streak camera. Rev. Sci. Instrum. 74, 2714-2721 (2003)... 

Ideally, the choice for a specific FLIM system should be based on the specific application and its requirements in terms of speed, spatial resolution and lifetimes to be resolved. In practice, however, this choice is often dictated by financial considerations, equipment already available, and the expertise available. Typically, FLIM instruments are custom-build by the users, although a number of companies nowadays make products that are specifically made to help transform an existing microscope into a FLIM system [35-37]. 

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