Frequency-encoded two-photon excited fluorescence microscopy.

Two-photon excited fluorescence (2PEF) microscopy is the most popular non-linear imaging method of biomedical samples. State-of-the art 2PEF microscopes use multiple detectors and spectral filter sets to discriminate different fluorophores based on their distinct emission behavior (emission discrimination). One drawback of 2PEF is that fluorescence photons outside the filter transmission range are inherently lost, thereby reducing the imaging efficiency and speed. Furthermore, emission discrimination of different fluorophores may fail if their emission profiles are too similar. Here, we present an alternative 2PEF method that discriminates fluorophores based on their excitation spectra (excitation discrimination). For excitation we use two lasers of different wavelengths (ω1, ω2) resulting in excitation energies at 2ω1, 2ω2, and the mixing energy ω1+ω2. Both lasers are frequency encoded (FE) by an intensity modulation at distinct frequencies while all 2PEF emission is collected on a single detector. The signal is fed into a lock-in-amplifier and demodulated at various frequencies simultaneously. A customized nonnegative matrix factorization (NNMF) then generates fluorescence images that are free of cross talk. Combining FE-2PEF with multiple detectors has the potential to enable the simultaneous imaging of an unprecedented number of fluorophores.

Coherent Stokes Raman scattering microscopy (CSRS).

We report the first implementation of laser scanning coherent Stokes Raman scattering (CSRS) microscopy. To overcome the major challenge in CSRS imaging, we show how to suppress the fluorescence background by narrow bandpass filter and a lock-in based demodulation. Near background free CSRS imaging of polymer beads, human skin, onion cells, avocado flesh and the wing disc of a drosphila larva are presented. Finally, we explain and demonstrate numerically that CSRS solves a major obstacle of other coherent Raman techniques by sending a significant part (up to 100%) of the CSRS photons into the backward direction under tight focusing conditions. We believe that this discovery will pave the way for numerous technological advances, e.g., in epi-detected coherent Raman multi-focus imaging, real-time laser scanning based spectroscopy or efficient endoscopy.

Live Stimulated Raman Histology for the Near-Instant Assessment of Central Nervous System Samples.

Central nervous system tumors encompass many heterogeneous neoplasms with different outcomes and treatment strategies. The current classification of these tumors is based on molecular parameters in addition to histopathology to define tumor entities. This genomic characterization of tumors is also becoming increasingly essential for physicians to identify targeted therapy options. The deployment of such genomic profiling relies on an efficient surgical sampling. To perform an appropriate tumor resection and a correct sampling of the tumor, the neurosurgeon may request an intraoperative pathological consultation. Stimulated Raman histology (SRH), an emerging nondestructive imaging technology, can address this challenge. SRH allows for a rapid and label-free microscopic examination of unprocessed tissues samples in near-perfect concordance with standard histology. In this study we showed that SRH enabled the near-instant microscopic examination of various central nervous system samples without any tissue processing such as labeling, freezing nor sectioning. Since SRH imaging is a nondestructive approach, we demonstrated that the tissue could be readily recovered after SRH imaging and reintroduced into the conventional pathology workflow including immunohistochemistry and genomic profiling to establish a definitive diagnosis.

Double-modulation stimulated Raman scattering: how to image up to 16-fold faster.

A stimulated Raman microscope is conventionally performed by modulating either the pump or Stokes beam and demodulating the other. Here, we propose a double modulation scheme that modulates both beams at fm and 2fm. Exploiting aliasing and reduction of the repetition rate, we show that the proposed double modulation scheme amplifies the signal amplitude by a factor of 1.5, 2, and 4 for different modulation frequencies and experimental realizations for the same average power at the sample. By deriving the noise power for different sources, we show that the double modulation scheme can perform stimulated Raman scattering (SRS) imaging with an up to 16-fold speed improvement as compared with single beam modulation.

Laser scanning dark-field coherent anti-Stokes Raman scattering (DF-CARS): a numerical study.

We present and model a dark-field illumination scheme for coherent anti-Stokes Raman scattering (DF-CARS) that highlights the interfaces of an object with chemical sensitivity. The proposed DF-CARS scheme uses dedicated arrangements of the pump kp1, Stokes kS and probe kp2 beams' k-wave-vectors to address the sample's interfaces along the x, y or z axis. The arrangements of the incident k-wave-vectors are derived from the Ewald sphere representation of the outgoing anti-Stokes radiation and the effective CARS excitation wave-vector keff = kp1 + kp2 - kS under the intention to avoid probing the object frequency K(0,0,0), i.e., the contribution of a homogeneous sample (dark-field configuration). We suggest a possible experimental realization using simple masks placed in the back pupil of the excitation microscope objective lens. Applying a full vectorial model, the proposed experimental implementation is numerically investigated on grounds of the Debye-Wolff integral and dynadic Green function to confirm the predicted chemical interface contrast.

3D-coherent anti-Stokes Raman scattering Fourier ptychography tomography (CARS-FPT).

Fourier ptychography tomography (FPT) is a novel computational technique for coherent imaging in which the sample is numerically reconstructed from images acquired under various illumination directions. FPT is able to provide three-dimensional (3D) reconstructions of the complex sample permittivity with an increased resolution compared to standard microscopy. In this work, FPT is applied to coherent anti-Stokes Raman scattering (CARS) imaging. We show on synthetic data that complex third-order susceptibilities can be reconstructed in 3D from a limited number of widefield CARS images. In addition, we observe that the non-linear interaction increases significantly the potential of CARS-FPT compared to linear FPT in terms of resolution. In particular, with a careful choice of the pump and Stokes beam directions, CARS-FPT is able to provide optical sectioning even in transmission configuration.

Shot-noise limited tunable dual-vibrational frequency stimulated Raman scattering microscopy.

We present a shot-noise limited SRS implementation providing a >200 mW per excitation wavelength that is optimized for addressing two molecular vibrations simultaneously. As the key to producing a 3 ps laser of different colors out of a single fs-laser (15 nm FWHM), we use ultra-steep angle-tunable optical filters to extract 2 narrow-band Stokes laser beams (1-2 nm & 1-2 ps), which are separated by 100 cm-1. The center part of the fs-laser is frequency doubled to pump an optical parametric oscillator (OPO). The temporal width of the OPO's output (1 ps) is matched to the Stokes beams and can be tuned from 650-980 nm to address simultaneously two Raman shifts separated by 100 cm-1 that are located between 500 cm-1 and 5000 cm-1. We demonstrate background-free SRS imaging of C-D labeled biological samples (bacteria and Drosophila). Furthermore, high quality virtual stimulated Raman histology imaging of a brain adenocarcinoma is shown for pixel dwell times of 16 µs.

Simultaneous stimulated Raman gain and loss detection (SRGAL).

The fidelity of stimulated Raman scattering (SRS) microscopy images is impaired by artifacts such as thermal lensing, cross-phase modulation and multi-photon absorption. These artifacts affect differently the stimulated Raman loss (SRL) and stimulated Raman gain (SRG) channels making SRL and SRG image comparisons attractive to identify and correct SRS image artifacts. To provide answer to the question: "Can I trust my SRS images?", we designed a novel, but straightforward SRS scheme that enables the dectection of the stimulated Raman gain and loss (SRGAL) simultaneously at the same pixel level. As an advantage over the conventional SRS imaging scheme, SRGAL doubles the SRS signal by acquiring both SRL as well as SRG and allows for the identification of SRS artifacts and their reduction via a balanced summation of the SRL and SRG images.

Background-suppressed SRS fingerprint imaging with a fully integrated system using a single optical parametric oscillator.

Stimulated Raman Scattering (SRS) imaging can be hampered by non-resonant parasitic signals that lead to imaging artifacts and eventually overwhelm the Raman signal of interest. Stimulated Raman gain opposite loss detection (SRGOLD) is a three-beam excitation scheme capable of suppressing this nonlinear background while enhancing the resonant Raman signal. We present here a compact electro-optical system for SRGOLD excitation which conveniently exploits the idler beam generated by an optical parametric oscillator (OPO). We demonstrate its successful application for background suppressed SRS imaging in the fingerprint region. This system constitutes a simple and valuable add-on for standard coherent Raman laser sources since it enables flexible excitation and background suppression in SRS imaging.

Spatial frequency modulated imaging in coherent anti-Stokes Raman microscopy.

For sparse samples or in the presence of ambient light, the signal-to-noise ratio (SNR) performance of single-point-scanning coherent anti-Stokes Raman scattering (CARS) images is not optimized. As an improvement, we propose replacing the conventional CARS focus-point illumination with a periodically structured focus line while continuing to collect the transmitted CARS intensity on a single detector. The object information along the illuminated line is obtained by numerically processing the CARS signal recorded for various periods of the structured focus line. We demonstrate experimentally the feasibility of this spatial frequency modulated imaging (SPIFI) in CARS (SPIFI-CARS) and SHG (SPIFI-SHG) and identify situations where its SNR is better than that of the single-point-scanning approach.

Stimulated Raman histology: one to one comparison with standard hematoxylin and eosin staining.

We present for the first time one-to-one correspondence between standard hematoxylin/eosin (H&E) stained tissue sections and stimulated Raman histology (SRH) - a label-free technique in which stimulated Raman scattering (SRS) and second harmonic generation (SHG) are combined to generate virtual H&E images. Experiments were performed on both human thin cryogenic slides from the gastrointestinal tract (GI) and thick freshly excised biopsies from endoscopic surgery. Results on cryogenic slides evidenced an excellent agreement between SRH and H&E images while the ones on biopsies established the relevance of SRH for rapid intraoperative histology to assist in surgical decision making.

Coherent anti-Stokes Raman Fourier ptychography.

We present a theoretical and numerical study of coherent anti-Stokes Raman scattering Fourier ptychography microscopy (CARS-FPM), a scheme that has not been considered so far in the previously reported CARS wide-field imaging schemes. In this approach, the distribution of the Raman scatterer density of the sample is reconstructed numerically from CARS images obtained under various angles of incidences of the pump or Stokes beam. Our inversion procedure is based on an accurate vectorial model linking the CARS image to the sample and yields both the real and imaginary parts of the susceptibility, the latter giving access to the Raman information, with an improved resolution.

Dual-focus stimulated Raman scattering microscopy: a concept for multi-focus scaling.

High-speed imaging is of the utmost importance for video-rate live cell investigations or to study extended sample areas at sufficient spatial resolution within reasonable time scales. Improving the speed of single-focus stimulated Raman scattering (SRS) microscopy is ultimately restricted by the sample's damage threshold and the shot noise of the demodulated laser source. To overcome this limitation, we present a dual-focus SRS approach modulating the pump laser for each focus at a distinct frequency. The corresponding probe beams are detected each by a photodiode and demodulated individually by two separate lock-in units to avoid inter-focal cross-talk. Two laterally or axially displaced images as well as hyperspectral SRS images can be obtained simultaneously within the field of view of the objective lens. The modular implementation presented here can be extended to multiple foci by using multi-channel acousto-optics modulators in combination with multi-channel lock-in amplifiers.

Simultaneous dual-channel stimulated Raman scattering microscopy demultiplexed at distinct modulation frequencies.

To increase the information per pixel in stimulated Raman scattering (SRS) microscopy as well as to correct from artifacts, it is valuable to acquire images at two different Raman shifts. We present a three-color SRS approach acquiring two perfectly registered SRS images where both pump beams are modulated at distinct frequencies while demodulating the Stokes beam. Our implementation uses two optical parametric oscillators that can be tuned to an almost arbitrary energy difference of Raman shifts, allowing investigation of fingerprint resonances simultaneously to CH-stretch vibrations.

Dual-focus coherent anti-Stokes Raman scattering microscopy using a compact two-beam fiber laser source.

We have developed a dual-focus coherent anti-Stokes Raman scattering (CARS) microscope based on a dual output, compact fiber laser source. The underlying concepts of time-multiplexed, two-beam scanning and demultiplexed detection that we already employed for second-harmonic generation are here naturally extended for CARS microscopy. The layout of a robust, all-fiber laser source was reconfigured to provide two outputs, each containing the two colors necessary for the CARS process. The utilization of the design for simultaneously imaging two laterally or axially separated fields of view and, thus, inherently speeding up the image acquisition process, is demonstrated on human artery tissue samples.

Hepatic Vitamin A Content Investigation Using Coherent Anti-Stokes Raman Scattering Microscopy.

Standard techniques for examining the distribution of vitamin A in liver either require staining or lead to rapid photobleaching of the molecule. A potentially better alternative approach is to use coherent anti-Stokes Raman scattering (CARS) microscopy; a fast, label-free, non-disruptive imaging method that provides contrast based on molecular vibrations. This contribution evaluates the viability of CARS microscopy for imaging vitamin A within thick hepatic tissue under physiological conditions by tuning into its characteristic vibrational band in the fingerprint region. Additional information about the morphology and architecture of the tissue was acquired using second harmonic generation (SHG) and multi-photon excited fluorescence (MPEF) to help mapping the intra-lobular positions of the vitamin A droplets. We demonstrate the capability of our multimodal imaging framework to selectively image lipid-soluble vitamin A droplets deep in bulk liver tissue with a high contrast while co-registering a complementary morphological background that clearly visualizes hepatic lobules. The results obtained envisage the good prospect of the technique for in vivo studies assessing vitamin A distribution heterogeneity and how it is affected by the progression of hepatic diseases.

Beyond endoscopic assessment in inflammatory bowel disease: real-time histology of disease activity by non-linear multimodal imaging.

Assessing disease activity is a prerequisite for an adequate treatment of inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis. In addition to endoscopic mucosal healing, histologic remission poses a promising end-point of IBD therapy. However, evaluating histological remission harbors the risk for complications due to the acquisition of biopsies and results in a delay of diagnosis because of tissue processing procedures. In this regard, non-linear multimodal imaging techniques might serve as an unparalleled technique that allows the real-time evaluation of microscopic IBD activity in the endoscopy unit. In this study, tissue sections were investigated using the non-linear multimodal microscopy combination of coherent anti-Stokes Raman scattering (CARS), two-photon excited auto fluorescence (TPEF) and second-harmonic generation (SHG). After the measurement a gold-standard assessment of histological indexes was carried out based on a conventional H&E stain. Subsequently, various geometry and intensity related features were extracted from the multimodal images. An optimized feature set was utilized to predict histological index levels based on a linear classifier. Based on the automated prediction, the diagnosis time interval is decreased. Therefore, non-linear multimodal imaging may provide a real-time diagnosis of IBD activity suited to assist clinical decision making within the endoscopy unit.

Pseudo-HE images derived from CARS/TPEF/SHG multimodal imaging in combination with Raman-spectroscopy as a pathological screening tool.

BACKGROUND: Due to the steadily increasing number of cancer patients worldwide the early diagnosis and treatment of cancer is a major field of research. The diagnosis of cancer is mostly performed by an experienced pathologist via the visual inspection of histo-pathological stained tissue sections. To save valuable time, low quality cryosections are frequently analyzed with diagnostic accuracies that are below those of high quality embedded tissue sections. Thus, alternative means have to be found that enable for fast and accurate diagnosis as the basis of following clinical decision making. METHODS: In this contribution we will show that the combination of the three label-free non-linear imaging modalities CARS (coherent anti-Stokes Raman-scattering), TPEF (two-photon excited autofluorescence) and SHG (second harmonic generation) yields information that can be translated into computational hematoxylin and eosin (HE) images by multivariate statistics. Thereby, a computational HE stain is generated resulting in pseudo-HE overview images that allow for identification of suspicious regions. The latter are analyzed further by Raman-spectroscopy retrieving the tissue's molecular fingerprint. RESULTS: The results suggest that the combination of non-linear multimodal imaging and Raman-spectroscopy possesses the potential as a precise and fast tool in routine histopathology. CONCLUSIONS: As the key advantage, both optical methods are non-invasive enabling for further pathological investigations of the same tissue section, e.g. a direct comparison with the current pathological gold-standard.

Multimodal nonlinear microscopy of head and neck carcinoma - toward surgery assisting frozen section analysis.

BACKGROUND: Treatment of early cancer stages is deeply connected to a good prognosis, a moderate reduction of the quality of life, and comparably low treatment costs. METHODS: Head and neck squamous cell carcinomas were investigated using the multimodal combination of coherent anti-Stokes Raman scattering (CARS), two-photon excited fluorescence (TPEF), and second-harmonic generation (SHG) microscopy. RESULTS: An increased median TPEF to CARS contrast was found comparing cancerous and healthy squamous epithelium with a p value of 1.8·10(-10) . A following comprehensive image analysis was able to predict the diagnosis of imaged tissue sections with an overall accuracy of 90% for a 4-class model. CONCLUSION: Nonlinear multimodal imaging is verified objectively as a valuable diagnostic tool that complements conventional staining protocols and can serve as filter in future clinical routine reducing the pathologist's workload. © 2016 Wiley Periodicals, Inc. Head Neck 38: First-1552, 2016.

Multimodal Imaging Spectroscopy of Tissue.

Advanced optical imaging technologies have experienced increased visibility in medical research, as they allow for a label-free and nondestructive investigation of tissue in either an excised state or living organisms. In addition to a multitude of ex vivo studies proving the applicability of these optical imaging approaches, a transfer of various modalities toward in vivo diagnosis is currently in progress as well. Furthermore, combining optical imaging techniques, referred to as multimodal imaging, allows for an improved diagnostic reliability due to the complementary nature of retrieved information. In this review, we provide a summary of ongoing multifold efforts in multimodal tissue imaging and focus in particular on in vivo applications for medical diagnosis. We also discuss the advantages and potential limitations of the imaging methods and outline opportunities for future developments.