Abstract: We report FLIM results obtained from tissue stained with ICG, DTTCC, and Methylene Blue. The results were obtained by bh FLIM systems connected to bh DCS‑120 WB confocal scanning system and to the Zeiss LSM 710 NLO and Intune microscopes. All the dyes investigated showed easily detectable lifetime variations. Whereas the variations observed for ICG and DTTCC appear to be simple aggregation effects Methylene Blue appears to report true variations in the tissue composition or tissue state.
Near-infrared dyes are used as contrast agents and as fluorophores in diffuse optical imaging applications . Diffuse optical imaging (or diffuse optical tomography, DOT) reconstructs internal structures and tissue parameters from photons diffusing through thick biological tissue. DOT uses the fact that the scattering and absorption coefficients decrease with increasing wavelength. By using wavelengths between 650 and 900 nm information about the tissue condition can therefore be obtained up to a depths of about 5 cm. For these applications it is important to have information about binding of the dyes to proteins, DNA, collagen, and other cell constituents available. It is also important to know whether the dyes change their fluorescence lifetimes on binding, and whether these lifetime changes depend on the binding targets . Possible lifetime changes may interfere with the reconstruction of the tissue structure and tissue parameters from time-resolved data, but may also be exploitable to gain additional biological information.
Moreover, the penetration depth - even for a confocal system - with NIR excitation and NIR detection can be expected to be noticeable higher than for visible excitation and detection.
At first glance, NIR wavelengths should neither be a problem on the detection nor on the excitation side: FLIM detection in the 700 to 900 nm range can be achieved by bh HPM‑100‑50 hybrid detectors or by PMC‑100-20 PMT modules. Picosecond diode lasers for the red and near-infrared range are available with 640 nm, 685 nm, and 785 nm, the Intune of the Zeiss LSM 710 can be tuned up to 645 nm, and super-continuum lasers with acousto-optical filters deliver any wavelength from the visible range to more than 1000 nm. Multiphoton microscopes have a Ti:Sapphire laser that works from about 700m to 1000 nm. The wavelengths available from these light sources are compatible with the (one-photon) excitation spectra of a variety of NIR dyes.
Unfortunately, there are a few pitfalls in the microscope optics: The internal beamsplitter of the scan head must reflect the excitation light towards the microscope lens, and transmit the fluorescence back from the lens to the detectors (Fig. 1, second right). Neither a normal confocal nor a multiphoton microscope has the right dichroic beamsplitter: The transition wavelengths of the beamsplitters of the confocals are too short (Fig. 1, left), and the transmission range of the multiphoton beamsplitter is at the wrong side of the excitation wavelength (Fig. 1, second left).
Fig. 1: Main dichroic beamsplitter of normal confocal, of a multiphoton microscope, and dichroic beamsplitter required for NIR FLIM
The problem of the internal beamsplitter is avoided by using two-photon excitation and non-descanned detection. In that case, the fluorescence is split off by a beamsplitter cube in the filter carousel directly behind the microscope lens. The cube in this place can easily be replaced and configured for the desired transition wavelength. Two-photon excitation of NIR dyes and FLIM detection by an SPC‑730 FLIM module and a PMC‑100-20 NIR detector has indeed been demonstrated . It requires, however, a laser or an OPO hat delivers wavelengths between 1400 and 1600 nm.
The problems can easily be solved in confocal systems that have a wideband beamsplitter available, see Fig. 1, right. A wideband beamsplitter loses some light but works at any combination of laser and fluorescence wavelength. Wideband beamsplitters are available in the bh DCS‑120 system and in the Zeiss LSM 710 family microscopes.
In the DCS‑120 WB system NIR excitation is obtained by connecting a BDL‑SMC 640nm, 685nm, or 785nm ps diode laser to the scanner. Any other laser of suitable wavelength and with Point-Source compatible fibre output a can be used as well [3, 4.
The Zeiss LSM 710 family microscopes have an 80/20 beam splitter in their main beam splitter wheel. There is also a near perfect solution to the excitation source: The LSM 710/780 NLO multiphoton microscopes have a Ti:Saphire laser that delivers tuneable pulsed excitation in the NIR. The laser is used as a one-photon excitation source. The microscope must have a confocal output from the scan head to which an HPM‑100-50 detector is attached .
Indocyanin Green (ICG) has an absorption maximum around 780 nm. At concentrations higher than about 50 µm/l aggregates form which have an absorption maximum at 690 nm. The fluorescence is emitted around 820 nm .
The images shown in Fig. 2 were recorded by a DCS‑120 WB system. The samples were immersed for 30 minutes in a 30 µm ICG solution. The small stokes shift between the absorption of the monomers and the emission makes it difficult to suppress scattered laser light and wideband spectral background of the laser. Fortunately, ICG has sufficient absorption down to 630 nm. We therefore used a 640 nm BDL-SMC ps diode laser for the experiments. This provides convenient spacing between the excitation and emission wavelengths, and avoids any filter leakage problems.
The image shown in Fig. 2, left is from a focal plane about 20 µm below the top of the skin. The image on the right was taken from the back of the skin sample. Both images show the intensity-weighted lifetime of a double-exponential fit (ti in SPCImage).
Fig. 2: Pig skin sample stained with ICG. Left: Image from the top of the skin, about 20 µm from the highest structures. Right: Image taken from the back of the skin sample. Fit with double-exponential model, intensity-weighted lifetime. DCS‑120 WB system, HPM‑100-50 detector. FLIM data format 512 x 512 pixels, 256 time channels.
There are surprisingly large changes in the fluorescence lifetime throughout the sample. It is known that ICG has a lifetime of about 200 ps in water, and about 600 ps when bound to serum albumin. Typical fluorescence decay functions and the corresponding decay parameters are shown in Fig. 3.
Fig. 3: Decay functions from selected 15x15 pixel areas of Fig. 2. Left: Short-lifetime areas (yellow orange). Right: Long-lifetime areas (green)
In the short-lifetime areas (yellow-orange in the images) the lifetimes of the decay components are 231 ps and 576 ps. This is marginally compatible with a mixture of bound and unbound ICG. In the long-lifetime areas (green in the images) the fit delivers two decay components, with lifetimes of 470 ps and 833 ps. This is clearly incompatible with a simple mixture of bound and unbound ICG.
The results show that the assumption of an essentially invariable fluorescence lifetime (and thus quantum efficiency) of ICG in biological systems is not correct. The variability may have an impact on the reconstruction of tissue parameters in diffuse optical imaging experiments.
A pig skin sample stained with DTTCC is shown in Fig. 4. The image was recorded by a Zeiss LSM 710 NLO system. The Ti:Sapphire laser was used as a one-photon excitation source. The laser wavelength was 780 nm. The 80/20 beamsplitter of the LSM 710 scan head was used, and the fluorescence selected by a 800 nm long-pass filter in front of the HPM‑100‑50 detector. The general behaviour of the DTTCC is similar to that of ICG: The lifetime is short where the tissue was exposed to high dye concentration, and longer inside the tissue. This may be due to aggregation of the dye. The lifetime may therefore not reflect any biologically relevant parameters. Even so, the long excitation and detection wavelength results in high contrast images as they are not normally obtained by confocal detection in thick tissue.
Fig. 4: Pig skin samples stained with DTTCC. Zeiss LSM 710 NLO, Ti:Sa laser used for one-photon excitation. Excitation wavelength 780 nm, detection wavelength 800 nm to 900 nm. HPM‑100‑50 hybrid detector, Simple-Tau 152 FLIM system. Lateral size of the images 212x212 µm, depth about 30 µm from surface. Note the high contrast of the images.
Methylene Blue has an absorption band from 550 to 690 nm, with a maximum at 660 nm. Fluorescence is emitted from 650 nm to 750 nm, with a maximum at 680 nm. Methylene Blue is a biomedically interesting compound. It has anti-viral and anti-bacterial effects, and it has been used as a drug against malaria . It has been used to induce apoptosis in cancer cells , and to treat psoriatic skin lesions by photodynamic therapy . The use to treat Alzheimer disease is under clinical trial . Fig. 5, left, shows a pig skin sample stained with Methylene Blue. As can be seen from the lifetime image and from the decay curves (Fig. 5, right) Methylene Blue delivers distinctly different decay times depending on the tissue structures it binds to. Both the lifetimes of the decay components and the amplitudes change. The exact mechanism of the lifetime changes is not known. Methylen blue shows changes in its absorption spectrum on pH variation. It can be expected that these are acompanied by lifetime changes. More important, it is known that Methylene Blue is a redox indicator. It is thus possible that the changes originate from variation in the redox state of the tissue. If this speculation is correct Methylene Blue would be a highly potent marker for the tissue state.
Fig. 5: Pig skin stained with methylene blue. Left: Lifetime image, double-exponential model, amplitude-weighted lifetime. Right: Decay curves in 3x3 pixel areas. Top: From red spots (fast decay). Bottom: From green areas (slow decay). DCS‑120 WB system, 640 nm ps diode laser.
The absorption wavelength of Methylene Blue can be reached by the Zeiss LSM 710 Intune (tuneable excitation) system. Results obtained by this system are shown in Fig. 6. The Methylene Blue was excited at 645 nm, the fluorescence was detected from 660 to about 750 nm. The results confirm the lifetime variations seen in the DCS‑120 images.
Fig. 6: Pig skin stained with methylene blue. LSM 710 Intune system, excitation 645 nm, detection from 660 nm up. Simple-Tau 152 FLIM system, HPM‑100‑50 hybrid detector. Image sizes 212x212µm (left) and 90x90µm (right)
The results show that even dyes known for decades may hold surprises when used for fluorescence lifetime imaging. New NIR fluorophores are constantly under development [1, 2, 7]. These should be tested for environment-dependent lifetime changes and for use as possible probes for molecular environment parameters.
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