A multipurpose fiber fluorescenceâreflection spectrometer with multiwave excitation is intended for biomedical studies, including studies in autofluorescence ...
OPTICAL INSTRUMENTATION Fiber fluorescence–reflection spectrometer with multiwave excitation G. V. Papayana) and N. N. Petrishchev I. P. Pavlov St. Petersburg State Medical University, St. Petersburg, Russia
V. M. Zhurba and A. A. Kishalov VOLO Scientific-Manufacturing Enterprise, St. Petersburg, Russia
M. M. Galagudza V. A. Almazov Federal Heart, Blood, and Endocrinology Center, St. Petersburg, Russia
(Submitted November 5, 2013) Opticheskiı˘ Zhurnal 81, 38–43 (January 2014) A multipurpose fiber fluorescence–reflection spectrometer with multiwave excitation is intended for biomedical studies, including studies in autofluorescence light. For this use, it is equipped with an LED-based illuminating system possessing a number of technical features: fluorescence excitation by radiation with wavelengths 365, 405, and 450 nm, synchronized switching of blocking filters at the excitation wavelength, and rapid switching of illumination with white light and the exciting radiation. This device makes it possible to observe changes of living objects from diffuse reflection and fluorescence spectra recorded virtually simultaneously. The possibilities of the device are illustrated by using it to study how the autofluorescence-excitation conditions affect the metabolic status of myocardium. © 2014 Optical Society of America. OCIS codes: (170.0170) Medical optics and biotechnology; (170.6280) Spectroscopy, fluorescence and luminescence; (170.6510) Spectroscopy, tissue diagnostics; (170.3880) Medical and biological imaging; (170.1610) Clinical applications; (170.4580) Optical diagnostics for medicine. http://dx.doi.org/10.1364/JOT.81.000029
Multichannel spectrometers equipped with fiber probes1–6 are ordinarily used to carry out local measurements of the reflectance and fluorescence spectra in biomedicine at the organohistological level under in vivo and ex vivo conditions. Excitation with radiation of some single wavelength from a laser source is used in most cases. The variation of the excitation wavelength is made possible in Ref. 4 by using a tunable dye laser and in Ref. 5 by using a xenon illuminator with a monochromator. Reference 6 describes the Skin-AGE fluorescence– reflection fiber spectrometer, in which fluorescence is excited by radiation with a wavelength of 365 nm. It makes it possible to efficiently excite autofluorescence of the tissue, as well as a number of exogenous substances that are of interest for diagnosis. At the same time, the presence of several excitation wavelengths is required to solve certain problems—for example, those connected with choosing the optimum conditions for estimating the functional state of an organ under conditions of ischemic damage. It is necessary in this case to provide the possibility of efficiently going from one excitation wavelength to another without tuning the device and to maintain the 29
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possibility of rapidly switching between recording the fluorescence spectra and the diffuse-reflection spectra in order to study the kinetics of their variations. The FOS-M multipurpose fluorescence-reflection spectrometer with multiwave excitation described below possesses such properties. The device is equipped with a special illuminator system. Its optical layout is shown in Fig. 1. Monochromatic LEDs with central wavelengths 365, 405, and 450 nm (1–3) serve as fluorescence-excitation illuminators, and white-light LED 4 serves to record the reflection spectra. The radiations of LEDs 1–3 are collimated by three identical lenses 5, whose foci are occupied by the emitting areas of the LEDs. One of these radiations, depending on the position of movable mirror 6 and using fixed mirror 7 and lens 8, is focused on the end of illuminator bundle 9. Clean-up filters 10–12 are mounted in each of the illuminator excitation channels in the gap between lenses 5 and 8. Their task is to eliminate stray long-wavelength radiation of the LEDs that might hinder the recording of the weak fluorescence. The exciting radiation of the illuminator channel passes through the fiber probe and arrives at the object, with which
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© 2014 Optical Society of America
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FIG. 1. Optical layout of the illuminator of the FOS-M multiwave fiber fluorescence–reflection spectrometer (see text for explanation).
it is in contact. The bundle consists of seven quartz fibers 400 μm in diameter. Six of them lie on the periphery and serve for illumination, while the seventh fiber, located at the center of the probe, serves to collect the secondary radiation from the object. This radiation, after passing through fibers 13 and 14 and conjugating lenses 15 and 16, arrives at the input of the multichannel spectrometer. One of the movable filters 17, 18, or 19 is introduced between lenses 15 and 16. Its task is to attenuate the exciting radiation to the level of the fluorescence radiation, and this makes it possible to form the reference signal in reflected light. Blocking filters 17–19 are mounted along with auxiliary mirror 6 on a common platform, which is put into motion by a stepper motor. As the wavelength of the exciting radiation varies, mirror 6, mounted on the platform, is brought opposite the necessary photodiode, so that the corresponding barrier filter is brought into the receiver channel. Thus each position of the platform corresponds to its own emitting LED, as well as its own pair of filters—one cleanup filter and one barrier filter. This synchronously switches the radiation sources and the barrier filters by means of one common motor, simplifying the switching system and making it more reliable. The radiation of white LED 4 is introduced into illuminator bundle 9 through auxiliary lightguide 20, which is fabricated from a polymeric fiber 1.5 mm in diameter. Its input end is located in the immediate vicinity of LED 4, and this makes it possible to introduce the radiation without matching the optics. The output end of lightguide 20, made in the form of a total-internal-reflection prism (Fig. 2), is mounted in an SMA receptacle of the illuminator bundle in such a way that it does not prevent it from being illuminated with exciting rays. The layout of the multiwave illuminator of the FOS-M device is shown in Fig. 3, and its technical characteristics are listed in Table 1. As far as the spectrometric module of the FOS-M device is concerned, as in the Skin-AGE device, it uses the commercially available multichannel AvaSpec-2048 optical-fiber 30
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spectrometer (Avantes, Inc.), which records the spectrum in the 350–750 nm region. The device is controlled from a built-in controller by means of a personal computer attached to the device through a USB 2.0 port.
FIG. 2. Input of the radiation of the white LED into the illuminator bundle.
FIG. 3. Layout of the multiwave illuminator (designations the same as in Fig. 1). Papayan et al.
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TABLE 1. Technical characteristics of the FOS-M device Power on output probe
365 nm illumination channel 405 nm illumination channel 450 nm illumination channel White illumination channel Switched-on time 365 nm/405 nm 405 nm/450 nm 365 nm/450 nm 365 nm/white Spectral recording range of reflection spectra Spectral recording range of fluorescence spectra Connection bus with computer Overall dimensions of V. Sh. G. Supply Mass of device
3 mW 6 mW 9 mW 50 μW 3s 3s 6s
