Slide-free virtual histochemistry ( Part I ) : development via nonlinear optics

Histochemistry is a microscopy-based technology widely used to visualize the molecular distribution in biological tissue. Recent developments in label-free optical imaging has demonstrated the potential to replace the conventional histochemical labels/markers (fluorescent antibodies, organic dyes, nucleic acid probes, and other contrast agents) with diverse optical interactions to generate histochemical contrasts, allowing “virtual” histochemistry in three spatial dimensions without preparing a microscope slide (i.e. laborintensive sample preparation). However, the histochemical information in a label-free optical image has often been rather limited due to the difficulty in simultaneously generating multiple histochemical contrasts with strict spatial co-registration. Here, in the first part (Part I) of this two-part series study, we develop a technique of slide-free virtual histochemistry based on label-free multimodal multiphoton microscopy, and simultaneously generate up to four histochemical contrasts from in vivo animal and ex vivo human tissue. To enable this functionality, we construct and demonstrate a robust fiber-based laser source for clinical translation and phenotype a wide variety of vital cells in unperturbed mammary tissue. © 2018 Optical Society of America under the terms of the OSA Open Access Publishing Agreement


Introduction
A long-sought goal in biomedical engineering is to perform tissue molecular imaging in a clinical setting, in real-time, in vivo, without stains, without slides, and in three-dimensions at optical resolutions.To this purpose, histochemical imaging contrasts have been generated from fresh unlabeled tissue specimens by varying the incident light that interacts with the specimen, rather than by treating (e.g., histologically processing and labeling) the specimen and illuminating it with fixed light.Versatile approaches of this type of imaging include tuning the optical frequency difference in stimulated Raman scattering microscopy (SRS) to obtain molecular vibration contrasts [1], varying the absorption wavelength in photoacoustic microscopy to obtain absorption contrasts [2], and programming light excitation and detection in multimodal multiphoton microscopy to obtain diverse nonlinear optical contrasts [3].However, for fast in vivo imaging, only one contrast (black-and-white image) is generated in one raster scan of the specimen, typically.It is possible to obtain additional types of contrast by scanning the same tissue section with different conditions of light excitation and/or detection, but this rescanning does not guarantee the co-localization of the two contrasts in in vivo imaging and increases the risk of sample photo-damage.Temporal multiplexing of different excitation conditions at each imaging pixel has been implemented for both SRS [4] and photoacoustic microscopy (e.g. in oxygen-metabolic vasculature imaging [5]) to simultaneously acquire two spectroscopic contrasts in one scan.This methodology guarantees co-localization of image data, but for imaging at a given signal-to-noise ratio, retains the risk of sample photo-damage.
With no such risk, spectral multiplexing of different detection channels has been realized in simultaneous multimodal multiphoton imaging of plant cells at 1230-nm excitation [6].We envision that this technique should allow multicolor "virtual" histochemistry of animal/human tissue at 1110-nm excitation via two-photon excited auto-fluorescence (2PAF, yellow), threephoton excited auto-fluorescence (3PAF, cyan), second-harmonic generation (SHG, green), and third-harmonic generation (THG, magenta) (Fig. 1(a)).The shift of the excitation wavelength from 1230-nm to 1110-nm allows for spectrally-separated detection of the four colored contrasts, in which the 3PAF and 2PAF contrasts correspond, respectively, to the emission bands of NADH and FAD with minimum spectral overlap (Fig. 1(b), upper panel) [7][8][9].A recent study demonstrated the power of simultaneous label-free autofluorescencemultiharmonic (SLAM) microscopy for intravital imaging based on a solid-state laserinduced fiber supercontinuum [8].To further the clinical applicability of this methodology, this paper leverages the recent advances in the fiber-laser industry by building the SLAM imaging platform on a fiber-laser-induced supercontinuum for better stability and ease of operation.Further, the applicability of this new imaging platform is evaluated with a broad array of biological specimens.
This paper forms the first part (Part I) of our two-part series study on the envisioned labelfree multiphoton histochemical imaging, with a focus on the technological development of slide-free virtual histochemistry via nonlinear optics in both free-space and fiber configuration.The second part (Part II) [10] of this two-part series study will focus on the application and unique capability of the developed slide-free virtual histochemistry to detect field cancerization in peri-tumoral fields.

Laser and microscope
The desired excitation band centered at 1110-nm is not directly available from femtosecond fiber lasers with limited wavelength tunability [11].We therefore sought to replace the solidstate laser with a femtosecond fiber laser as the master laser for coherent fiber supercontinuum generation [12,13], which has been employed in multiphoton imaging of human skin [14] (Table 1).All these prior studies chose to custom build the master fiber laser.It is unclear whether this choice is due to the noise or decoherence mechanism [15] that might be associated widely available commercial femtosecond fiber lasers.Taking the hint from the master fiber laser that was custom built to generate near transform-limited pulses [12,13], we selected a commercial laser that produced a small time-bandwidth product (Table 1).
This industrial fiber laser (Satsuma, Amplitude Systemes) is widely used in laser-assisted in situ keratomileusis (LASIK) eye surgery [16], but has not been actively pursued for biomedical imaging.The free-space coupling of 2.3 W laser power to the fiber generated a supercontinuum output of 1.8 W (Fig. 1(c)).The multiphoton microscope in this study largely followed the setup reported in a prior study [8].The band of 1110 ± 30 nm in the supercontinuum was spectrally selected by a pulse shaper (MIIPS Box640, Biophotonics Solutions) with an average output power of 50 mW.Then the near-transform-limited pulses ~35 fs (FWHM) were raster scanned by a galvanometer mirror pair (6215H, Cambridge Technology) (Fig. 1(a), upper panel).
An objective with high UV transmission (UAPON 40XW340, N.A. 1.15, Olympus) was used to produce a field-of-view of 350 × 350 µm 2 .The loss along the excitation beam path resulted in an average focusing power of ~20 mW on the tissue (well below the ANSI standard of ~100 mW at this wavelength).The reflected 4-channel multiphoton signals were then spectrally separated by appropriate dichroic mirrors and optical filters (Semrock, Inc.) (Fig. 1(a), upper panel) and simultaneously collected by 4 photomultipliers (H7421-40, Hamamatsu).By directly comparing the pulse-shaped fiber supercontinuum generated from this fiber laser with that from a similar solid-state laser (femtoTrain, Spectra-Physics Inc.) (Table 1) (Fig. 1(c)), we obtained comparable imaging performance from the same sample.The additional noise or decoherence associated with the fiber laser-induced supercontinuum, if present, has no appreciable effect on our tetra-modal imaging.Also, the feasibility to simultaneously image NADH and FAD by 1110-nm excitation via the 3PAF and 2PAF detection channels was demonstrated by imaging the NADH and FAD solutions (Fig. 1

(b), lower panel).
There are several practical benefits associated with this replacement of the master laser.First, the bulky, water cooled, and frequently unstable solid-state laser is replaced with a compact air-cooled robust fiber laser suitable for portable applications [1].Second, the deterministic supercontinuum generation in the normal dispersion regime of the photonic crystal fiber, and subsequent pulse shaping, are both passive optical processes (in comparison to the active processes of lasing) stable against mechanical and thermal disturbance [15].This stability, along with the excellent beam pointing stability of the fiber laser, enables long-term stable operation (hundreds of hours over 6 months) of the supercontinuum source free of realignment or service.Third, daily operation of the supercontinuum source resembles its turn-key master fiber laser, allowing a user with minimum laser training to begin imaging after only a 5-min warmup.

Human a
Female rats Institutional Champaign.F by intraperito d by the Urbanak old rats centration of 55 mg/kg.The first injection was performed on the left abdominal side, and a second injection was performed one week later on the right abdominal side.The lesions were first palpable from the abdominal surface about 4 weeks after the second injection, and were allowed to grow up to 1.5 cm afterward.For the control group, an equal amount of saline was injected at each time point to account for plausible effects from the injection.Small surgeries were performed to expose the mammary glands of the rats from both groups 6-16 weeks after the second injection.The rats underwent in vivo imaging of the mammary tissue and were then euthanized.
Human breast cancer tissue (from tumor resection surgery) and normal tissue (from breast reduction surgery) were obtained under a protocol approved by the Institutional Review Boards at the University of Illinois at Urbana-Champaign and Carle Foundation Hospital.The permission for investigational use of all tissue specimens was obtained from subjects who preoperatively signed an informed consent.The tissue specimens were stored in saline-filled conical tubes and transported on ice for imaging within 12 hours after surgery.

In vivo animal imaging
An experimental rat with palpable lesion(s) or a control rat was anesthetized with isoflurane, and a small incision was made in the abdominal skin to expose a tumor (~1 cm) or normal mammary gland.The surrounding skin was flipped on a microscope coverslip so that the field-of-view could be arbitrarily placed at the visually detected lesion site.The rat itself was positioned on a three-dimensional motorized piezoelectric stage to allow depth-resolved imaging and large-field imaging with a mosaic of high-resolution fields-of-view.The imaging focal plane was placed 5-100 µm below the sample surface.Anesthesia was maintained throughout the imaging session while the rat was kept warm at physiologic temperature with a heating pad and blanket.The galvanometer mirror-based scanning along with the unique optical excitation allowed acquisition of multi-contrast multiphoton images (512 × 512 pixelated frame with 350 × 350 µm 2 standard field-of-view) at a frame rate of 0.5 Hz, corresponding to a pixel dwell time of ~8 µs.The full "field-of-view" image presented in the figures were a result of mosaic acquisition (3x3, or 5x5 depending on the final image size).Raw data from the photomultipliers were used to produce all images without additional processing such as deconvolution or maximum intensity projection.This in vivo imaging of rat mammary tissue was also employed in ex vivo imaging of human breast tissue without the in vivo aspects.

Characterizing slide-free virtual histochemistry
To demonstrate that our fiber laser-based source retained the capability of the programmable light multiphoton imaging [3], we first performed the tetra-modal imaging on ex vivo mouse kidney and readily differentiated the NADH-rich (cyan-colored) epithelial cells from FADrich (yellow-colored) epithelial cells in kidney tubules and collecting ducts (Fig. 1(d), left two panels) [17].We then reprogramed the excitation to generate 30-fs 920-nm pulses [3], and conducted FAD imaging through the SHG detection channel of the tetra-modal imaging, according to the established excitation/detection wavelengths of this endogenous compound [7].As expected, only the FAD-rich epithelial cells were observed after the reprogramming (Fig. 1(d), right panel).Similarly, we first performed the tetra-modal imaging of ex vivo human mammary tissue (control) and easily differentiated the 2PAF-visible elastin fibers from SHG-visible collagen fibers in the extracellular matrix (Fig. 1(e), left two panels).We then carried out the same reprogramming of excitation and selectively imaged these elastin fibers through the SHG detection channel of the tetra-modal imaging (Fig. 1(e), right panel).Thus, endogenous elastin fibers can be revealed by 2PAF imaging using the excitation/detection wavelengths of (720/440 nm) [9], (880/515 nm) [18], (940/562 nm) [   l mammary tissue lidated with green yellow contrast) as ers may be bio dshifted excita aging "labels" is also applicab maging (Fig. 1( er than their nu but nontrivial i ndicates the pre , upper panel) hondria) of so used acridine o etra-modal ima ar inside the etra-modal ima Fig. 1

Phenotyping vital cells from rat to human
We then conducted systematic in vivo tetra-modal imaging of various cells in rat mammary tissue under the same excitation conditions and at similar imaging depths (20-40 µm).The imaging revealed locations in the normal mammary tissue of a control rat, or in the tumor center, vicinity of the tumor boundary or margin, and in the peri-tumoral field of specimens from a pre-clinical carcinogen-injected mammary tumor rat model.All tetra-modal images were directly comparable after plotting the four pseudo-color contrasts of magenta, cyan, green, and yellow with the same set of dynamic ranges.A distinct cell phenotype was recognized by the visibility of yellow, cyan, and magenta optical markers from both the nucleus and cytoplasm, termed as the optical phenotype (Table 2).Comparison of histological images of the same sample has been provided as Fig. 2 in Part II of this study [10].
Combining the optical phenotype with the local context of the extracellular matrix, we recognized a wide variety of cells including stromal cells or fibroblasts, erythrocytes, immune cells, endothelial cells, and tumor cells within the authentic tumor microenvironment (Fig. 3).All these cells observed in vivo were also observed with the same optical phenotypes in fresh ex vivo mammary tissue <12 hrs after resection, allowing a time window for consistent and comparative imaging and analysis.The endothelial cells were recognized by their unique elongated shape as well as their context in which they were well-aligned along the direction of the vessel, which highly correlates with their typical histologic morphology.The different optical signatures could be attributed to their active metabolic status.Endothelial cells with cyan outlines indicated high NADH concentrations, which are associated with glycolysis as well as hypoxia.Endothelial cells with strong THG signals indicated the local presence of water around the cells [25].Similar phenotypical heterogeneity is found in human breast tissue <12 hrs after resection (Figs.3(o), 3(s)), with heterogeneous carcinogenesis similar to the animal model (Figs. 4, 5).Unique correspondences can be established between some rat and human cells according to their similar optical phenotypes and organization of their extracellular matrices (bidirectional arrows; Fig. 3).The magenta-yellow-colored rat immune cells with a round-like shape (arrowhead; Fig. 3(a)) have been validated by their wide presence in lymph nodes, and may be correlated with similar human cells in ex vivo tissue (arrowhead; Fig. 3(p)) that could be difficult to image in vivo.As expected, in both rat and human specimens, abundant yellowcolored normal stromal cells (Figs. 3(f), 3(k)) are widely present in the stromal regions of control and cancer-associated specimens (e.g., see Visualization 1 corresponding to Fig. 3(b)).However, the unique presence of cyan-or magenta-colored cancer-associated cells in the latter distinguishes a tumorous stroma (Figs.3(h), 3(m)) from a normal stroma (Figs.3(f), 3(k)).Our cell phenotyping cannot only differentiate normal and cancer-associated stroma and mammary ducts, but also normal (or angiogenic) and cancer-associated blood vessels.In normal blood 3(b), 3(q)) w corresponding vessels/ducts  important biological implications (Visualizations 1-4), which are not routinely available from conventional histochemistry due to the additional time needed for serial sectioning and threedimensional reconstruction of digitized slide images.It is to be noted that the 2PAF channel is not entirely specific to FAD, as multiple endogenous fluorophores (e.g.lipofuscin and porphyrin) share similar spectral characteristics in the spectral window of 600-650 nm [26].It is by the combination of the 2PAF signal intensity and the cellular morphology, as well as the microenvironmental context, that we assigned the 2PAF channel largely to FAD, and subsequently analyzed the optical redox ratio for the identified cells.With the multimodal optical signatures collected with this system, there will likely be a continuum of values that represent a continuum of metabolic and molecular states.Along with the morphological features, these will combine to form the "optical phenotypes" we observe.Further investigations will be performed in the future to systematically establish the link between the optical phenotypes and different cell types.The focus areas of this paper are to demonstrate the clinical potential of our proposed imaging platform, to seek clues on the underlying carcinogenesis, and to reveal potential diagnostic markers.
Cahill et al developed another type of virtual H&E histology via high-speed multiphoton imaging, which demonstrated strong potential for margin assessment for breast cancer with the aid of stains [27].In contrast, the system proposed in this work compromised imaging speed (pixel dwelling time 5-20 µs) for label-free molecular profiling capability and highlights stain-free, slide-free, 3D, structural and functional imaging of cells and cellular dynamics in the authentic microenvironment.Although the relatively small fields-of-view make full tissue/margin assessment impractical, the high-dimensional molecular and functional changes revealed in the tumor micro-and macro-environments demonstrate the importance and the feasibility of characterizing the peri-tumor microenvironment for the underlying carcinogenesis as well as for potential diagnostic markers.The advantage of this system over existing label-free multimodal multiphoton microscopy systems is that it enables simultaneous and efficient excitation and detection of auto-fluorescence and harmonic generation from a vast array of cellular and stromal components in living tissue by using a single-excitation fiber-based source.We anticipate this technology and methodology will be an attractive complementary approach to existing clinical tissue assessment methods thanks to its label-free nature, technical simplicity, real-time functionality, versatility, and rich molecular/metabolic content [8].
Our demonstrated slide-free virtual histochemistry also complements the label-free histochemical imaging by SRS microscopy [1,4] or photoacoustic microscopy [2,5], which may achieve similar cell phenotyping by rapidly tuning the molecular vibration frequency or absorptive wavelength during the imaging.Our technique is based on well-established multiphoton microscopy with a long history of development.The compatibility of our technique with commercial multiphoton microscopy (Fig. 1(a)) may enable its more widespread use by simply replacing the standard Ti:sapphire laser in the microscope with our fiber laser-induced supercontinuum source.The latter is compact, reliable, and suitable for users without extensive laser training.The histochemical contrasts of the tetra-modal imaging (Fig. 1(a), lower panel) using 1110-nm single-band excitation can be expanded by reprogramming the fiber laser-induced supercontinuum to generate additional tailored histochemical contrasts [3].Thus, we believe the demonstrated slide-free virtual histochemistry in this paper will provide an attractive way to conduct clinical histochemistry in comparison to various alternative techniques.As a prototypical demonstration, in the second part (Part II) of this two-part series study, we show how this slide-free virtual histochemistry is used to detect field cancerization in peri-tumoral fields [10].

1 .
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