Fluorescence imaging is a powerful technique for the analysis of biological samples. In the simplest case, an imaging system such as an epifluorescence microscope equipped with an excitation lamp and a filter-cube is used to illuminate the sample at one wavelength while imaging it at another.
However, significantly more information can be obtained from the same sample if the more advanced techniques of Hyper-Spectral Imaging (HSI) or Fluorescence Lifetime Imaging (FLIM) are employed. In a HSI setup the complete fluorescence spectrum is acquired for every point of the object. It is a particularly useful method to clearly discriminate different structural features within a sample when multiple fluorescent dyes are employed whose spectra overlap (T. Zimmermann, “Spectral imaging and linear unmixing in light microscopy,” Adv. Biochem. Eng. Biotechnol. 95, 245-265 (2005)). Rather than measuring (spectral) intensity, FLIM involves the mapping of the decay times of fluorescence (J. R. Lakowicz, Principles of fluorescence spectroscopy (Kluwer Academics, 1999)), which can vary depending on the local environment of exogenous or endogenous fluorophores. Such a functional imaging approach is well suited to the study of biochemical and biophysical processes in tissues and cells, often in a non-destructive manner (D. Elson, J. Requejo-Isidro, I. Munro et al. “Time domain fluorescence lifetime imaging applied to biological tissue,” Photochem. Photobiol. Sci. 3, 795-801 (2004)).
Wide-field spectral imaging is usually performed using a set of optical emission filters mounted on a filter wheel or rotary filter-cube holder. This approach is straightforward and relatively cheap, but is usually rather slow and cumbersome due to the mechanical switching of the filters. Typically only a limited number of spectral bands can be acquired which allows only partial separation of overlapping fluorescence spectra. More versatile HSI systems employ electro-optic devices such as Liquid Crystal Tunable Filters (LCTF), Linearly Variable band-pass Dielectric Filters (LVDF) or Acousto-Optical Tunable Filters (AOTF) (M. Bouhifd, M. P. Whelan, M. Aprahamian, “Fluorescence imaging spectroscopy utilizing acouto-optic tuneable filter”, 5826A-23 (OptoIreland, 2005)). The LCTF solution offers approximately 30% of passband transmission efficiency, which is often unacceptable for low light-level applications. The LVDF resolution is roughly 15 nm, its spectral range covers 400-700 nm, and its transmission efficiency is around 40%. While these parameters are good enough for static imaging, using a LCTF for HSI to study dynamic behavior in low-light level conditions can be problematic. The AOTF is an electronically controllable, variable bandwidth optical filter, which provides significant versatility and performance in comparison to other tunable filters. It supports random access to any transmission-band or continuous spectral tuning and thus is very suitable for HSI when combined with a sensitive camera. However, all these wide-field HSI systems based on tunable filters can suffer from relatively poor image quality due to the scattering present in most biological samples. This can result in a decrease in image contrast and the loss of quantitative information, such as the concentration of a fluorophore.
As an alternative, reconstruction of a fluorescence map by making point-by-point measurements has proven to deliver images of superior quality (B. W. Pogue, S. L. Gibbs, B. Chen, M. Savellano, “Fluorescence Imaging in Vivo: Raster Scanned Point Source Imaging Provides More accurate Quantification then Broad Beam Geometries” Technology in Cancer Research and Treatment 3, 15-21 (2004)), which reveal more accurately the localization and concentration of fluorescent markers. However, since this approach usually requires raster scanning carried out by intricate electro-mechanical systems it is difficult and costly to implement.
Fluorescence lifetime imaging in the time-domain can generally be approached in two ways, namely wide-field fluorescence lifetime imaging or point-by-point raster scanning. Wide-field (or broad beam) imaging systems employ a Gated and Optical Intensified (GOI) camera in combination with a high-power pulsed laser. Typically, excitation light is irradiated on the area to be detected and fluorescence from the area is captured all at once by a CCD array sensor to thereby obtain fluorescence information of a two-dimensional region. Such a FLIM system is fast (J. Requejo-Isidro, J. McGinty, I. Munro, D. S. Elson et al. “High-speed wide-field time-gated endoscopic fluorescence-lifetime imaging,” Optics Letters 29, 2249-2251 (2004)) but demonstrates lower temporal and spatial resolution in comparison to scanning measurements. The high cost and lack of portability are also issues, which have limited its uptake. On the other hand the successful demonstration of GOI-based FLIM using picosecond laser diodes has helped matters somewhat (D. S. Elson et. al., “Fluorescence lifetime system for microscopy and multiwell plate imaging with blue picosecond diode laser,” Optics Letters 12, 1409-1411 (2002)).
Still with respect to wide-field imaging, since the fluorescence information of the image-taken area is detected as a whole at the same time—problems of image blurring due to light scattering may arise. Indeed, since the image pickup area is illuminated as a whole by a light source, detection and separation of weak fluorescence is difficult where there are multiple fluorescence spots around the weak point, because fluorescence light is scattered over the neighbourhood area, increasing background noise and overlapping the fluorescence to be measured. To avoid these problems, excitation light pattern generating devices have been developed that permit to irradiate different intensities and locations on a sample in sequence at relatively high frame rate. A fluorescence detecting apparatus using such excitation light pattern generating device, based on a digital micromirror device (DMD) or reflection-type liquid crystal device, is e.g. described in US 2006/0226375.
The second approach (scan-type) to time-domain FLIM exploits Time Correlated Single Photon Spectroscopy (TCSPS) method combined with point-by-point scanning (Y. Zhang, S. A. Soper, L. R. Middendorf, J. A. Wurm, R. Erdmann, M. Wahl, “Simple Near-Infrared Time-Correlated Single Photon Counting Instrument with a Pulsed Diode Laser and Avalanche Photodiode for Time-Resolved Measurements in Scanning Applications,” Applied Spectroscopy 53, 497-504 (1999)) or laser confocal scanning (M. Kress, T. Meier, R. Steiner, F. Dolp, R. Erdmann, U. Ortmann, A. Rück, “Time-resolved microspectrofluorometry and fluorescence lifetime imaging of photosensitizers using picosecond pulsed diode lasers in laser scanning microscopes,” Journal of Biomedical Optics 8, 26-32 (2003)). Typically a punctual light beam of excitation light irradiates the sample so that the fluorescence from the irradiated spot is detected by e.g. a Photo-Multiplier Tube (PMT), and this irradiation and detection scanning throughout the object to be observed allows to obtain fluorescence information of a two-dimensional area. Picosecond laser diodes are the most popular excitation source because of their compactness and reasonable cost, while a PMT or an Avalanche Photo-Diode (APD) may be used to detect the emitted fluorescence photons. With respect to wide-field FLIM, scanning-TCSPS is more straightforward and less expensive to implement. It also offers higher temporal resolution, but at the price of much slower imaging rates. To obtain FLIM images, either the sample is moved on a translation stage under the excitation light spot or the sample is held steady while the excitation spot is raster-scanned using galvano-mirrors. Although a number of attractive commercial solutions exist based on both scanning methods, the disadvantages of point scanning in general are its low imaging rate and the lack of flexibility in how an image is formed. Normally a minimum sampling time of 100 μs required per measurement point (pixel), thus leading to a duration of several seconds to tens of minutes to acquire a complete image. The actual duration depends on a number of factors including the strength of the fluorescence signal and the desired field of view, temporal and spatial resolution. Most scanning systems are analogue by nature and thus neither offer random access to any point or region of interest on the sample, nor the possibility to bin or combine the photons emitted from different locations.
An important issue in FLIM/HIS imaging is of course also the accuracy of data analysis. To reduce the acquisition time and full image frame reconstruction, data acquisition time per single pixel is typically reduced, which in turn reduces signal to noise ratio and further decreases the accuracy and reliability of data processing. By the extreme or complex nature of the phenomena observed the data analysis itself is also complex and time consuming. Namely, to adjust phenomenological parameters in the photo-physical model to reach; satisfying agreement between experimental dataset and the model, one needs to perform iterative data fitting. To keep high spectral/temporal resolution of measurements, large dataset/pixel are produced and large number of pixels; need to be analysed to reconstruct the FLIM/HSI image. Thus the fitting algorithm is extremely time consuming. Number of methods has been proposed to decrease the computational time, which are based on the so-called “Global Analysis Approach”. Most commonly the fluorescence signals from all the pixels are post-processed by summing all together to increase S/N ratio. The result, i.e. the phenomenological parameters of the model are then used as initial parameters for data analysis algorithm performed off-line (i.e. after fluorescence data acquisition) for every single spatial location on the sample. Modifications of the Global Analysis include dataset summation from quarters of the image or from manually chosen regions based on white light anatomical image of the sample [S. Pelet, M. J. R. Previte, L. H. Laiho, P. T. C. So, “A fast global fitting algorithm for fluorescence lifetime imaging microscopy based on image segmentation”, Biophysical Jourlan, vol. 87, 2807-2817 (2004)]. These methods suffer however either from loosing details (weak signal and its impact to the overall fluorescence is weak) or the need for high experience in manual marking the desired regions of interest and their use is limited to specific cases. Moreover the data analysis is performed off-line, having the dataset per every pixel already acquired.
JP 2006 171024 A describes a multi-point fluorescence spectrophotometry method using a DMD, wherein a sample is illuminated to excite its fluorescence and an image thereof is acquired; image segmentation is based on the existence of fluorescence (e.g. using a luminance threshold); and the fluorescence of each Region of Interest is acquired one by one.