Spectrally resolved fluorescence lifetime imaging microscope using tunable bandpass filters

Abstract

A simple structure of spectral fluorescence lifetime imaging microscope (SLIM) is designed with the use of tunable bandpass filter, a kind of Fabry-perot filter that transmission wavelength is varying according to incident angle of light. Feasibility tests of this angle-tuned bandpass filter (ATBF) are performed and it shows high transmission and constant spectral bandwidth (20 nm) with respect to angle of incidence. Furthermore, using two ATBFs in series, spectral bandwidth can be adjustable down to 4 nm. In this paper, dual ATBFs are implemented to the detection part of fluorescence lifetime imaging microscope (FLIM) system so that we obtained spectrally resolved FLIM images. We compare these SLIM images with an original FLIM image and confirm that the former case provides high accuracy to analyze lifetime distribution as well as high contrast of images. The proposed SLIM microscope with good wavelength selectivity has many opportunities to utilize to other applications such as FLIM-Föster resonant energy transfer and autofluorescence imaging.

Bandwidth-variable tunable optical filter unit for illumination and spectral imaging systems using thin-film optical band-pass filters

“…However, when using different filter combinations, in principle the complete visible wavelength range can be covered. 5 The adjustable bandwidth range agrees well with the one reported by Jeong et al, 19 where a tuning range of 4-17.4 nm was achieved, however using a point-like source instead of the 1500 μm diameter fiber used in this study. Additionally, we showed in this study that the transmitted intensity decreases only linearly with the bandwidth for λ SB ≥ 6 nm, thus allowing optical filtering with a high transmission efficiency >75%, which indicates that the proposed arrangement of two tunable thin-film optical bandpass filters can be used without significant transmission efficiency loss compared to the one-filter setup.…”

“…Consequently, the total transmission efficiency is reduced if the filter pass-bands only overlap in the spectral range of the edges, thus limiting its use for very small spectral bandwidths. This problem cannot be overcome easily, as it requires either a source with smaller diameters, e.g., point-source like, 19 which may not be available as a high intensity non-coherent light source, or a larger focal width of the collimating lens, which is also not always applicable due to the usually high numerical aperture of the source, which would then result in a low transmission efficiency. Also, the filter dimensions restrict the beam diameter and therefore the collimating lens diameter, which limits the focal length at a given numerical aperture.…”

“…A single tunable thin-film filter shows a transmission efficiency of more than 90% in the pass-band, 5 whose central wavelength shifts upon rotation of the filter. The spectral bandwidth remains virtually constant and, in particular, is independent of the polarization of the transmitted light, 3,5,19 so that the transmission efficiency in the pass-band remains virtually constant as well. If two such filters are arranged successively, an independent rotation of the filters to different angles spectrally shifts the filter pass-bands against each other, and light is transmitted through the filter unit only in the spectral range where the filter pass-bands overlap.…”

“…In contrast to reflection filters, 15 no angular deviation occurs when rotating the filters, moreover preserving the fast tuning speed of the filter unit that is tuned by the angle of incidence. 15 A parallel beam shift occurs when light passes through the rotated filters, 19 which may be compensated at least partly by rotating the filters in opposite directions, so that the beam shifted by the first filter is partly shifted back by the second filter. The maximum transmission efficiency is found in the center of the spectral range where the filter pass-bands overlap.…”

“…Nonetheless, temperature adjustment is time consuming and therefore yields only a slow tuning speed of 0.39 s/nm. 16 An optical filter unit that overcomes some of these limitations uses the same concept of independent adjustment of overlapping filter pass-bands: 15,19 two commercially available thin-film optical band-pass filters in a transmission setup 12,19 are successively arranged for simultaneous adjustment of both the central wavelength and the transmission bandwidth. A single tunable thin-film filter shows a transmission efficiency of more than 90% in the pass-band, 5 whose central wavelength shifts upon rotation of the filter.…”