Ultra-deep imaging of turbid samples by enhanced photon harvesting

We constructed an advanced detection system for two-photon fluorescence microscopy that allows us to image in biological tissue and tissue phantoms up to the depth of a few mm with micron resolution. The innovation lies in the detection system which is much more sensitive to low level fluorescence signals than the fluorescence detection configuration used in conventional two-photon fluorescence microscopes. A wide area photocathode photomultiplier tube (PMT) was used to detect fluorescence photons directly from a wide (1 inch diameter) area of the turbid sample, as opposed to the photon collection by the microscope objective which can only collect light from a relatively small area of the sample. The optical path between the sample and the photocathode is refractive index matched to curtail losses at the boundaries due to reflections. The system has been successfully employed in the imaging of tissue phantoms simulating brain optical properties and in biological tissues, such as murine small intestine, colon, tumors, and other samples. The system has in-depth fluorescence lifetime imaging (FLIM) capabilities and is also highly suitable for SHG signal detection, such as collagen fibers and muscles, due to the intrinsically forward-directed propagation of SHG photons.


INTRODUCTION
The ability to visualize features in deep layers of biological tissue with high resolution is a very sought-after feature of an imaging system employed in medical diagnostics and in clinical and biological applications. Deep-tissue imaging has many applications, all based on the necessity of exploring cells and molecules in the intact organism, a sort of "optical pathology" that removes the need of biopsies and fixing, and grants access to structures and functions in the native physiological environment.
Both the geometrical and optical properties of the sample and the characteristics of the microscope system affect the achievable imaging depth. While transparent specimens can be easily imaged with a traditional microscope, biological tissue is an intrinsically turbid medium, which produces a strong multiple scattering, absorption and exhibits inhomogeneity of the refractive index. These features make traditional light and fluorescence microscopy, even with the aid of staining and the addition of fluorescent markers to improve contrast, ineffective past the 100-200 μm surface layer of the sample [1].
In order to access deeper layer of turbid samples, high power, short pulse emitting lasers in the near infrared (NIR) are required. This allows taking advantage of reduced absorption and scattering at longer wavelengths. The advent of multi-photon microscopy has improved the achievable imaging depth, but the current microscope technology is still limited by an imaging depth of about 1 mm [2,3,4,5].
We have constructed a two-photon fluorescence microscope capable of imaging in turbid media simulating brain optical properties up to the depth of a few mm. Such an increased penetration depth is the result of a novel photon harvesting system which is able to collect emission photons from a wide area of the sample in contrast to the conventional detection scheme that collects photons only from a relatively small area, while most photons are lost. Light losses are also reduced by means of refractive index matching throughout the optical path. The system has proven extremely effective in imaging of many biological and artificial samples, such as murine colon and small intestine, vasculature in the skin, subcutaneous xenograft tumors in mice and various tissue phantoms.  A tunable femtosecond pulsed Ti:Sa laser (Mai Tai, Spectra Physics) is used for two-photon fluorescence excitation and SHG. The laser is coupled to a group velocity dispersion compensator (DeepSee, Spectra Physics) to achieve maximum fluorescence excitation efficiency in the sample. An acousto-optic modulator (AOM, AA Opto-Electronics MT 110-B50A1) is used to adjust the power of the excitation beam. The beam is then directed to a x-y galvanometric scanner (ISS) coupled with an Olympus BX illumination module equipped with long working distance objectives (Olympus LCPlanFl 20x/0.4 and Olympus LUMPlanFl 40x/0.80 W). The sample is directly placed on the detection system in a transmission configuration (described below) attached to a motorized x-y-z stage used for positioning of the specimen and focusing.
The detector used in the transmission configuration is the key feature of the imaging system that allows us to image in turbid samples up to the depth of a few millimeters [16,17]. As it will be shown below, this system has also proven extremely efficient in the detection of SHG photons, due to their intrinsic forward directed propagation. The detector here described was modified by adding a filter wheel to separate the emission photons by wavelength. The detector comprises of a large photocathode area head-on PMT (Hamamatsu R7600P-300), working in photon counting mode, coupled to the shutter. The shutter is custom made of a sealed aluminum case that houses the filter wheel rotated by a stepper motor. The filter case is filled with index matching fluid to keep the optical path between the sample and the PMT window completely index matched. This minimizes losses of fluorescence and SHG photons caused by multiple reflections at the boundaries. This detection scheme allows the efficient collection of emission photons directly from the wide surface area of the sample, which makes it extremely sensitive to very low signal levels. The system is equipped with a FLIMBox (ISS) that allows fluorescence lifetime imaging microscopy (FLIM). The system is also equipped with a second PMT (Hamamtsu H7422P-40) that works in the epi-fluorescence configuration to compare the results between the conventional 2-photon fluorescence microscope configuration and our system.

Sample preparation
Silicone tissue phantoms (reduced scattering coefficient μ s '=1.13 mm -1 at 800nm excitation wavelength) were prepared according to [18] to which yellow-green (2 μm and 15μm) and red (10 μm) fluorescent beads (Invitrogen) were added. Samples were also prepared in the same way with the addition of urea crystals (Aldrich) for SHG experiments. Before being mixed with the polymer the urea crystals were powderized in a mortar. Two types of samples containing urea crystals were prepared: one type with a scattering agent (TiO 2 ) and the other without scattering agent (clear). The sample dimensions are 35 mm in diameter by 8 mm thickness.
Samples of cells embedded in collagen matrix were prepared as following. Type I collagen was purchased from BD Biosciences (Franklin Lakes, NJ), with original concentration of 3.75mg/ml. Collagen was diluted with 10X PBS with phenol red and water to a final concentration of 1X PBS and 2mg/ml collagen solution. NaOH was added to neutralize the collagen solution before mixing it with cells. Fluorescent labeled paxillin MDA-MB-231 cells in serum free DMEM were mixed with 2mg/ml collagen solution, with the final concentration of 5 x 10 4 cells/ml. The collagen/cell mixture was polymerized at 20°C for 1 hr and then at 37°C for 20 min. Full medium was applied after polymerization. Measurements were performed 2 to 4 days after the cells were cultured in the collagen matrix.
Samples of murine colon and small intestine were prepared according to [19]. The rodent dorsal chamber procedures can be found in [20]. Xenograft tumors grown subcutaneously in immune-deficient mice were excised from a skin flap and immediately imaged. The mouse was euthanized right after the removal of the tumor.

Imaging of turbid samples
We have previously [16,17] demonstrated that by employing the described detection scheme images of fluorescent beads in turbid samples could be acquired up to the depth of 3 mm, while a commercial Zeiss LSM 710 microscope could image the same samples only to a depth of 500 μm. Our system has been ameliorated by the addition of a more  al tissue is intri dex, which va th that will als nd ability to co ch tissues than         (Fig. 6b)  in the epi-and ering) silicone ght (Fig. 5a)

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