Research ArticleOPTICS

Wide-field multiphoton imaging through scattering media without correction

See allHide authors and affiliations

Science Advances  12 Oct 2018:
Vol. 4, no. 10, eaau1338
DOI: 10.1126/sciadv.aau1338


Optical approaches to fluorescent, spectroscopic, and morphological imaging have made exceptional advances in the last decade. Super-resolution imaging and wide-field multiphoton imaging are now underpinning major advances across the biomedical sciences. While the advances have been startling, the key unmet challenge to date in all forms of optical imaging is to penetrate deeper. A number of schemes implement aberration correction or the use of complex photonics to address this need. In contrast, we approach this challenge by implementing a scheme that requires no a priori information about the medium nor its properties. Exploiting temporal focusing and single-pixel detection in our innovative scheme, we obtain wide-field two-photon images through various turbid media including a scattering phantom and tissue reaching a depth of up to seven scattering mean free path lengths. Our results show that it competes favorably with standard point-scanning two-photon imaging, with up to a fivefold improvement in signal-to-background ratio while showing significantly lower photobleaching.


A suite of powerful, disruptive optical imaging approaches across the physical and biomedical sciences has recently emerged. Super-resolution imaging led to new studies looking at nanometric features within cells that have revealed intricate aspects of subcellular processes (15). At the larger scale, methods such as optical coherence tomography (6) and light-sheet imaging (7) are taking hold in fields such as opthalmology, neuroscience, and developmental biology. In tandem with the requirement for a fast, wide-field visualization and super-resolved imaging across biomedicine, a grand challenge is to perform such imaging through highly scattering (turbid) media, namely, tissue. In particular, this is essential to move from superficial surface imaging to functional imaging at depth (811), which is crucial for biomedical areas including neuroscience. To address this area, aberration correction can be implemented (12). However, this does not readily take into account the properties of the medium, and actual retrieval of the emitted signal from depth in the medium can still be challenging. Key advances have emerged by a consideration of the propagation of light within a complex medium. In this field, a number of approaches use dynamic wavefront shaping for illumination of the sample with a calculated input complex wavefront (1316), which can focus light upon an embedded guide star. In essence, one determines the transmission matrix of the sample in this process (11, 17, 18). While this is powerful, the requirement of a guide star restricts the approach. Furthermore, it requires determination of the properties of the medium at one or more individual points, making it very challenging to implement for wide-field imaging.

An important advance would be the realization of a fast, wide-field imaging approach that would deliver and retrieve light from any given plane within a sample, even in the presence of turbidity. This would be without the requirement to characterize or even actively correct the aberrating effect of the turbid medium. Our approach to achieving this goal exploits temporal focusing (TF) microscopy (19, 20). By using the temporal rather than spatial degree of freedom, scanning of the optical axis for image reconstruction is avoided. Consequently, TF may record wide-field multiphoton images (19, 21). In addition, a little-recognized facet of TF is its ability to deliver light through scattering media. This ability has been used to project optical patterns for applications such as optogenetics, providing photostimulation at remarkable depths (9, 22, 23). Although TF can deliver light through a scattering medium very efficiently, collecting the emitted fluorescent light back through the same medium (i.e., truly achieving imaging) has not been accomplished to date. Separately, there has been the emergence of single-pixel detection, sometimes termed computational ghost imaging (24). In this form of imaging, known patterns illuminate an object, a single-element photodetector records the light intensity that is either transmitted or backscattered by the object, and images are reconstructed with the appropriate algorithm (25, 26).

However, while these studies in TF microscopy and in single-pixel detection have shown promise, none of them has addressed the challenge of correction-free wide-field imaging through turbid media. The scheme that we present here, which we call TempoRAl Focusing microscopy with single-pIXel detection (TRAFIX), uses a judicious combination of TF illumination with single-pixel imaging to obtain wide-field images of fluorescent microscopic samples within or even beyond biological tissues, in the presence of multiple scattering, without aberration correction or characterization of the turbid medium.


Principle of the technique

TF is based on decomposing an incident ultrashort pulsed light field into its constituent wavelengths with a diffraction grating. Each wavelength propagates along an individual path in the optical system, and these wavelengths constructively recombine to regain the original pulse duration only at the plane conjugate to the grating, generating axially confined mutiphoton excitation. In TRAFIX, orthonormal light patterns (in a Hadamard basis) are temporally focused through a turbid medium to illuminate a fluorescent microscopic sample of interest. The use of TF for this projection ensures the retention of the integrity of these patterns at any given plane within the turbid media (Fig. 1A). This can be regarded as due to the fact that ballistic photons remain unperturbed all the way to the object plane and arrive at the same time, contributing to the reconstitution of the pulse. In addition, the superposition of wavelets of slightly different wavelengths at the focal plane results in nearly speckle-free propagation through long distances in scattering media, as recognized by Papagiakoumou et al. (9). We confirm these aspects here with a numerical simulation. The same principle has been previously used for reducing out-of-focus excitation in line-scanning multiphoton microscopy (20). A scattering medium may affect the spatial and temporal degrees of freedom of an input field differently. In the time domain, the temporal profile of femtosecond pulses is not significantly distorted at substantial imaging depths such as 1-mm-thick brain tissue (27). As a consequence, TF may induce much more efficient multiphoton excitation when compared to standard point-focusing where spatial speckle greatly reduces the photon density at the focal spot. Consequently, TF is more robust than conventional focusing, resulting in a more intense fluorescence signal generated at large depths (28), which is a major attribute for our approach.

Fig. 1 Working principle of TRAFIX.

(A) A femtosecond laser beam is expanded onto a spatial light modulator (SLM) that generates Hadamard patterns. Subsequently, the beam is diffracted from a grating, and the Hadamard patterns are projected onto a fluorescent sample after propagating through a scattering medium. Fluorescent light emitted by the sample is collected by the same objective after passing through the scattering medium a second time (epifluorescence geometry), and the total intensity is measured by a single-pixel detector. (B) A TF beam propagates through a turbid medium with minimal distortion, retaining the integrity of illumination patterns in the sample plane. Emitted fluorescent photons scatter as they propagate back through the tissue. In contrast to standard TF microscopy, TRAFIX tolerates scrambling of back-propagating light since only an intensity measurement is performed. In a single-pixel measurement, the fluorescent target is sequentially illuminated with Hadamard patterns (ψn), and the total intensity detected is stored as a coefficient (ωn). Gray background in the second column denotes regions of zero intensity. By adding up the Hadamard patterns weighted by their respective coefficients, an image of the fluorescent sample is reconstructed.

The total intensity emitted by the fluorescent sample under each illumination pattern is collected by the same objective after passing a second time through the scattering material, in a configuration reminiscent of a single-pixel imaging. In this way, we remove the requirement for any spatial resolution on the imaging path, which, in turn, means that we can readily tolerate the scrambling of the emitted fluorescence through the scattering medium (Fig. 1B). We retain exact spatial information of where the sample is illuminated by virtue of using patterned illumination. This allows an original form of TF microscopy to be realized, enabling the use of the full penetration capabilities of TF beams for imaging at depth (29).

In the present experiments, an illumination laser with a central wavelength at 800 nm delivers 140-fs pulses (80-MHz repetition rate, average output power up to 4 W) onto the sample, and the emitted fluorescent photons are detected by an electron-multiplying charge-coupled device (EMCCD) camera, which is used as a single-pixel detector. The epifluorescence configuration of TRAFIX makes it readily suitable for a suite of biomedical applications. An additional microscope takes reference images of the fluorescent sample in a transmission geometry analogous to previous reports (9, 22). Reference images are taken with a CCD camera under uniform TF illumination across the field of view (FOV) (see Materials and Methods). We stress that this additional reference system is not required for imaging. Once all patterns have been sequentially projected and their intensity coefficients have been measured, images are reconstructed using an orthogonal matching pursuit (OMP) algorithm (30). The OMP algorithm determines which patterns contribute most effectively to the image reconstruction and sums them up to create an image (see the Supplementary Materials). The number of pixels in the retrieved image is determined by the size of the Hadamard basis used in the measurement. An n × n pixel image requires a Hadamard basis containing N = n2 patterns. Therefore, depending on the pixel resolution required, a different number of Hadamard patterns (typically 4096 or 1024) are encoded on an SLM. The acquisition time of the microscope is given by T = 2n2(texp + tSLM), where texp is the exposure time of the camera used as a single-pixel detector and tSLM is the time required to refresh the Hadamard patterns on the SLM (including data transmission).

As TRAFIX is based on patterned illumination, it lends itself to compressive sensing measurements (26, 31). One of the main advantages of compressive sensing is that sparse signals can be reconstructed with fewer samples than required by Nyquist sampling theory. In terms of microscopy, it means that one does not need to measure with the full set of Hadamard patterns to obtain a good-quality image. The compression ratio (CR) = N/M denotes how many patterns are used to reconstruct the image in relation to the total number of patterns in the Hadamard basis (26). Here, M is the number of patterns used in the reconstruction algorithm. For example, a 64-pixel by 64-pixel image requires a measurement with a Hadamard basis containing 4096 patterns. Consequently, a CR of 2 corresponds to using only half of the total patterns to reconstruct the image (i.e., 2048 patterns), a CR of 4 uses only 1024 patterns, and so on.

As we describe below, to demonstrate the performance of TRAFIX, we imaged various microscopic fluorescent samples making use of full Hadamard bases to obtain a high-quality image. Additional compressed images were obtained a posteriori to demonstrate that compressive sensing measurements are possible in this configuration (see the Supplementary Materials).

Imaging through scattering media

To begin with, 400-nm-diameter green fluorescent beads and fixed human embryonic kidney (HEK) 293T/17 cells labeled with green fluorescent protein (HEK293T/17-GFP) were imaged through scattering phantoms, designed to mimic the scattering properties of biological tissue, and through unfixed human colon tissue. A custom-made fluorescent microstructure was then imaged through fixed rat brain tissue. As scattering dominates over absorption in the range of wavelengths considered in our investigation (32, 33), we use scattering mean free path (ls) as a reference value to quantify imaging depth. The ls values for the scattering media used in the experiments presented here are approximately 140, 85, and 55 μm for the scattering phantom, colon tissue, and brain tissue, respectively (see Materials and Methods). Full Hadamard bases of either 32-pixel by 32-pixel or 64-pixel by 64-pixel resolution were projected onto the samples, and the resulting images were reconstructed with different CRs. The lateral resolution of the microscope is defined as twice the pixel size in the reconstructed images, and thus, it depends on the FOV. Using a Hadamard basis containing 32 pixels by 32 pixels and an FOV of 90 μm by 90 μm, the resolution is 5.6 μm, and for a larger basis of 64 pixels by 64 pixels, it is 2.8 μm (fig. S7). We measured the depth resolution in the absence of any scattering layer for a 40× numerical aperture (NA) = 0.8 objective to be 4.7 ± 0.5 μm (fig. S4) and initially tested the microscope’s performance in imaging beads and HEK cells without scattering (see the Supplementary Materials).

To quantify image quality, we measured the signal-to-background ratio (SBR) for all images presented in this work (see Materials and Methods). All values are summarized in table S1. To assess the effectiveness of this technique in photolocalization, we estimated the spacing between fluorescent beads and the size of cells in the reference image and in the reconstructed images and calculated their deviation from the reference value (see the Supplementary Materials).

Figure 2 shows images of the fluorescent beads and HEK cells obtained through 500 and 540 μm of scattering phantoms (3 to 4 ls), respectively. As a result of the strong scattering, very few illumination photons reach the sample plane, generating a very dim fluorescence signal; therefore, a long exposure time or large binning is required to even obtain a good reference image under uniform TF illumination in transmission. In standard TF microscopes, those few emitted fluorescence photons would need to travel back through the thick scattering medium, losing all their spatial information and hence resulting in uniform noise on the camera (29). Consequently, the SBR would essentially be unity, which means that no signal can be extracted from the background noise. However, in TRAFIX, there is no need to measure spatial information as total intensity coming from the sample under each illumination pattern is detected and used as a coefficient in the reconstruction algorithm. This results in a significant enhancement in signal detection and higher SBR values.

Fig. 2 Images of fluorescent microscopic samples through scattering phantoms.

Fluorescent beads of 400 nm in diameter and fixed HEK293T/17-GFP cells were imaged through 500- and 540-μm of scattering phantoms, respectively. (A and F) Images taken from the reference imaging system under uniform TF illumination across the FOV. Exposure time was set to 20 s, and camera binning was 4 × 4 (beads) and 2 × 2 (cells). (B to E, G, and H) Images obtained in epifluorescence configuration with TRAFIX using a Hadamard basis containing 4096 illumination patterns. They were reconstructed with different CRs corresponding to 100% (CR = 1), 50% (CR = 2), 25% (CR = 4), or 12.5% (CR = 8) of the total patterns. Each measurement under individual illumination patterns was taken with a binning of 64 × 64 and an exposure time of 0.5 s. The spacing between beads was measured in all five images obtaining deviations smaller than 3% from the reference image (table S3). The diameters of the cells in (F) were measured to be 20.7 and 14.3 μm, respectively, and their values in (G) and (H) differ less than 4 and 12% from the reference value (table S2). The SBR is shown for all reconstructed images. Scale bars, 10 μm.

The achievable level of compression depends on the sparsity of the image. If an image contains very little information, then high compression is possible with negligible information loss. As the images of fluorescent beads are sparse, image quality is preserved for large values of CR. A good image can be faithfully reconstructed with CR = 8 (12.5% of the total number of patterns). Similar CRs have been achieved in the literature, although with different optical imaging systems (26). Nevertheless, we did not achieve a high compression with cells in the present configuration. They covered a larger area of the image and thus cannot be compressed as much without a significant loss of information with the present algorithm and pixel resolution. In addition, the 800-nm excitation laser used in the experiments is not optimal for excitation of GFP, which has its highest absorption in the range of wavelengths between 870 and 920 nm (34). As a result, the cells appear very dim. Despite these issues, an SBR exceeding 4 was achieved at CR = 2 (i.e., using only 50% of the full Hadamard basis in the image reconstruction algorithm).

In the next stage, the performance of TRAFIX was tested with unfixed human colon tissue as a scattering medium. One of the main problems in imaging through biological tissue is autofluorescence. Coda et al. (35) showed the single-photon excitation spectrum of human colon tissue at different wavelengths. Under 435-nm single-photon excitation, colon tissue presents a very intense autofluorescence signal at the green part of the spectrum, overlapping with the light emitted from the beads or cells used in our experiment. Single-photon excitation at 435 nm is relatively similar to two-photon excitation at 800 nm, so it poses a big obstacle in the experiments because it reduces the number of photons that can reach the beads or HEK cells and it also generates undesired background light. The optical sectioning capability of TF would seem to circumvent this impediment; however, as temporally focused laser pulses propagate longer distances through scattering tissue, the resulting excitation plane becomes thicker (fig. S4) (36), and consequently, some autofluorescence is excited in the colon tissue, resulting in high noise levels even in the reference images taken in transmission. Despite the intense autofluorescence light emitted by the colon tissue, we succeeded in imaging both 400-nm fluorescent beads and HEK cells through 250 and 200 μm (~3 ls), respectively, obtaining high SBR values (Fig. 3). An additional image taken in a scattering phantom with fluorophores extending its entire volume confirms that TRAFIX can be used in presence of out-of-focus background fluorescence (fig. S8).

Fig. 3 Images of fluorescent microscopic samples through unfixed human colon tissue.

Fluorescent beads of 400 nm in diameter and fixed HEK293T/17-GFP cells were imaged through 250 and 200 μm of human colon tissue, respectively. (A and C) Images taken from the reference imaging system under uniform TF illumination across the FOV. Camera binning in (A) was set to 4 × 4, and exposure time was 5 s. No camera binning was used in (C), and exposure time was 15 s. (B and D) Images obtained with TRAFIX using a Hadamard basis containing 1024 and 4096 illumination patterns, respectively. All patterns were used for image reconstruction (CR = 1). Camera binning for each Hadamard pattern was set to 64 × 64, and exposure time values were (B) 1 s and (D) 0.75 s. The spacing between beads and the diameter of cells were measured to assess image quality (tables S3 and S2, respectively). The SBR is shown for all reconstructed images. Scale bars, 10 μm.

The photolocalization analysis looked at spacing between adjacent beads and cell size in a given image. It was satisfactory in the case of images through the scattering phantom obtaining deviations smaller than 3% for beads in any CR (table S3). We also obtained acceptable results in the case of cells for the image with no compression (table S2). In general, larger deviations were observed for the images through colon tissue presumably because of very low photon count reaching the detector and distortions caused by inhomogeneities in the tissue.

We generated fluorescent micropatterns having a more detailed structure than beads or cells on a thin fluorescent film (see Materials and Methods) and imaged them through a scattering phantom (figs. S7 and S9), colon tissue (fig. S10), and fixed rat brain tissue of different thicknesses (Fig. 4). The maximum imaging depth achieved through rat brain tissue was 400 μm, which corresponds to approximately 7 ls. The improvement of TRAFIX over conventional TF microscopy becomes evident by comparing Fig. 4B with Fig. 4 (C and D).

Fig. 4 Comparison of a hidden object and the retrieved images through fixed rat brain tissue.

(A) Reference image of a fluorescent micropattern without any scattering s