Tryptophan is an essential amino acid which is, together with other amino acids, a constituent of most proteins. Tryptophan is fluorescent, and its fluorescence is the dominating part of protein fluorescence. Tryptophane fluorescence is dependent on biological parameters, such as protein constitution, protein folding, and presence of other amino acids in the closer molecular environment. This dependence makes tryptophan a potential fluorescence marker for molecular parameters in biological systems. In particular, this is the case when tryptophan fluorescence is detected by fluorescence lifetime imaging (FLIM). Problem is, the excitation wavelength of tryptophane is deep in the UV, and cannot be reached in a normal microscope. In this article we show how tryptophane lifetime images can be obtained by TCSPC FLIM in combination with two-photon excitation by a new green femtosecond laser. We demonstrate the use of the system by recording TRP images of E. coli cells and yeast cells.

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Two-Photon FLIM of Tryptophan with a Green Femtosecond Laser

W. Becker, J. Heitz

Becker & Hickl GmbH, Berlin, Germany

Abstract: Tryptophan is an essential amino acid which is, together with other amino acids, a constituent of most proteins. Tryptophan is fluorescent, and its fluorescence is the dominating part of protein fluorescence. Tryptophane fluorescence is dependent on biological parameters, such as protein constitution, protein folding, and presence of other amino acids in the closer molecular environment. This dependence makes tryptophan a potential fluorescence marker for molecular parameters in biological systems. In particular, this is the case when tryptophan fluorescence is detected by fluorescence lifetime imaging (FLIM). Problem is, the excitation wavelength of tryptophane is deep in the UV, and cannot be reached in a normal microscope. In this article we show how tryptophane lifetime images can be obtained by TCSPC FLIM in combination with two-photon excitation by a new green femtosecond laser.

Fig. 1: FLIM image of yeast cells showing tryptophane fluorescence. Two-photon excitation at 540 nm, detection from 320 to 380 nm.

Tryptophan Fluorescence

The problem of tryptophane (TRP) fluorescence imaging is that the excitation and emission wavelengths are in the UV. The excitation maximum is around 280 nm, the emission band extends from about 300 nm up to 400 nm [9]. Tryptophan absorption and emission spectra are shown in Fig. 2.

Fig. 2: Absorption (left) and emission spectra (right) of tryptophan (from [9])

By conventional fluorescence techniques, tryptophan fluorescence can only be recorded in a conventional fluorescence spectrometer or a similar setup with a special UV objective lens. This way, fluorescence decay curves can be obtained [10]. In a normal microscope, direct excitation of tryptophan is not possible because the required excitation wavelength does not pass the microscope optics. In a laser scanning microscope the problem is even greater because the excitation has to pass also the scanner optics.

A promising way to avoid the UV problem is to use multiphoton excitation. It has been shown that useful results can be obtained with three-photon (3-p) excitation. For 3-p excitation a wavelength in the range from 750 nm to 840 nm is used. Appropriate wavelengths can easily obtained from a Ti:Sa laser. An early result for 3p-excited tryptophan imaging has been published by Jyontikumar et al. [8]. The authors used a Zeiss LSM 780 with a bh SPC-152 FLIM system. They could show a decrease in the TRP lifetime at increased concentration of bound NADH when the cells were treated with glucose. Alam et al. [1] used a similar system to record lifetime images of the NAD(P)H, FAD, and TRP fluorescence from prostate cancer (PCa) cells and to quantify the metabolic response to treatment with Doxorubicin.

Unfortunately, 3p excitation of tryptophane has a potential problem. 3p excitation needs high laser power, typically more than 10 mW in the focal plane. This is no problem for a tryptophane solution, but it is a problem in cells. Cells contain NADH and FAD. At 740 nm to 840 nm, the laser hits the absorption band of these co-enzymes with the full power of two-photon excitation, see Fig. 3. [1, 8] used 7 mW to 8 mW at 740 nm. This is significantly more than normally used for NADH and FAD imaging, and almost certainly causes damage to the cells. The count rates obtained from the cells in the TRP emission band were on the order of less than 10,000 counts per second.

Another possibility is to excite tryptophan by two-photon (2p) excitation. The optimum excitation wavelength for tryptophan would then be in the range from 530 to 560 nm. There is no significant absorption of NADH or FAD at this wavelength, see Fig. 3. It can therefore be expected that photodamage will remain at an acceptable level. Until recently, the problem was that no suitable fentosecond lasers with the required wavelength were available. Now, a femtosecond laser with 540 nm wavelength has been introduced [11]. This has prompted us to attempt two-photon FLIM of tryptophane with 2p excitation.

Fig. 3: Excitation of TRP by multiphoton excitation. Red: 825 nm laser. Green: 825 nm laser. It can be seen that 3p excitation at 825 nm excites also FAD. Two-photon excitation at 540 nm does not have this problem.

2-Photon Tryptophan FLIM System

The basis of our tryptophan system is a standard Becker & Hickl DCS-120 MP Multiphoton FLIM system. The principle is shown in Fig. 4.

The optical part consists of a bh DCS-120 scan head [4], a Nikon Ti 2E microscope, a 560 nm VALO Aalto laser with SHG option [11], and two HPM-100 hybrid detectors [3]. The principle is shown in Fig. 4. The laser delivers 50 mW of optical power at a wavelength of 540 nm. The laser beam passes a shutter and a variable attenuator. Scanning is performed by the DCS-120 MP scan head. The scan lens of the scan head projects the laser beam down the microscope beam path to the microscope lens. [3, 4]. To obtain high efficiency both for excitation and detection we used an x40 NA=1.3 oil immersion lens. The fluorescence light is collected back through the microscope lens, separated from the excitation light by a 520 nm dichroic beamsplitter in the filter carousel of the microscope, and sent out through the back port of the microscope. The light is transferred to the detectors via a normal NDD (non-descanned detection) beam path [3]. Scattered laser light is removed by a 500 nm short pass filter. The DCS system has two detectors. For tryptophan recording we used only one of them. The detected signal is further cleaned by a 360/40 band pass filter. The signal is recorded by bh's multi-dimensional TCSPC FLIM process [2, 3, 5] via one of the two SPC-180NX FLIM modules [3] of the DCS-120 system. Please see [3, 4] for further details.

Fig. 4: Optical principle of the 2-photon tryptophan FLIM system

Results

Fig. 5 shows a tryptophan lifetime image of live yeast cells. The excitation power was 2 mW in the plane of the sample, the acquisition time 60 seconds. With a count rate of 90×10³ photons per second, 5.5 million photons were acquired within this time. The number of photons is sufficient for double-exponential decay analysis. The decay components at the cursor position are 49 % of 632 ps and 51% of 3525 ps. The amplitude-weighted lifetime is 2108 ps.

Fig. 5: Tryptophan FLIM of Yeast cells. Left: Lifetime image. Amplitude-weighted lifetime of double-exponential decay. Right: Decay function at cursor position. Image format 512 x 512 pixels, 1024 time channels, two-photon excitation at 540 nm, lifetime range from 1000 ps to 4000 ps. Analysis by bh SPCImage NG 9.0 [6].

Fig. 6 shows a tryptophan lifetime image of live Escherichia coli (E. coli) bacteria. E. coli bacteria are not an easy target. They are small and move quickly. The image shown was recorded with an acquisition time of 90 seconds. The entire image contains about 10 million photons. The image was analysed with a double exponential decay model. The decay components at the cursor positions are 62.7 % of 1573 ps and 37.3 % of 4740 ps. The amplitude-weighted lifetime is 2762 ps.

Fig. 6: Tryptophan FLIM of E. coli bacteria. Left: Lifetime image. Amplitude-weighted lifetime of double-exponential decay. Right: Decay function at cursor position. 512 x 512 pixels, 1024 time channels, two-photon excitation at 540 nm, lifetime range from 1000 ps to 4000 ps. Analysis by bh SPCImage NG 9.0 [6].

For comparison, Fig. 7 shows a decay curve of tryptophan in aqueous solution. The data were acquired with the same system as the FLIM data above, the curve was created by combining the data of all pixels in a single decay curve [6].

Fig. 7: Decay curve of tryptophan in water. Recorded with the same system as the FLIM images. Acquisition time 160 seconds, analysis by bh SPCImage NG 9.0.

With a composition of 64.8 % of 2463 ps and 35.2% of 3192 ps, the decay is close to single-exponential. The amplitude-weighted lifetime is 2719 ps. These values are compatible with the lifetimes obtained from the yeast cells and the E. coli cells. This is a strong indication that the emission from the cells really comes from tryptophan. The fact that the decay function in the cells are more double exponential (a₁ higher and t₁ lower than in solution) can be explained by heterogeneity of the TRP environment and, possibly, by FRET from the TRP into NADH. FRET has been found in PCa cells [1], and it appears plausible that it also occurs in the yeast and the E. coli cells. An attempt to calculate a hypothetical FRET efficiency in the yeast cells delivers the FRET image shown in Fig. 8. A colour-coded image of the classic FRET efficiency [7] is shown on the left, the distribution of the FRET efficiency and the decay parameters at the cursor position are shown on the right. The most frequent FRET efficiency is 0.46, which is a plausible value for TRP-NADH FRET.

Fig. 8: FRET image for hypothetical TRP-NADH energy transfer. Left: Image showing colour-coded (classic) FRET efficiency. Right. Distribution of FRET efficiency over the pixels and decay parameters at cursor position. Analysis by bh SPCImage NG 9.0.

Summary

We present a FLIM system that records lifetime image of tryptophan (TRP) in live cells. The system is based on a standard bh DCS-120 MP multiphoton FLIM system [4]. In contrast to known 3-photon approaches, the system uses two-photon excitation by a new 540 nm femtosecond laser [11]. This way, photodamage by unwanted excitation of NADH and FAD is avoided. We demonstrated the use of the system by recording TRP images of E. coli cells and yeast cells. The excitation power in the sample plane was about 3 mW, the count rate in the TRP emission band about 90,000 to 100,000 photons per second. The decay functions were double exponential, with components of 632 ps and 3525 ps in case of the yeast cells and 1573 ps and 4740 ps in the case of the E. coli cells. The lifetime components of TRP in solution were 2463 ps and 3192 ps.