( 2017 2016)
CARS microscopy; Stain-free histopathology. — Basal cell carcinoma (BCC) is the most common malignancy in Caucasians. Nonlinear microscopy has been previously utilized for the imaging of BCC, but the captured images do not correlate with standard H&E staining. Last year, we have developed a novel algorithm to post-process images obtained from dual vibration resonance frequency (DVRF) CARS measurements to acquire high-quality pseudo H&E images of BCC samples. We adapted our CARS setup to utilize the distinct vibrational properties of CH3 (mainly in proteins) and CH2 bonds (primarily in lipids). As a result, we acquired two images: one for “lipids” and one for ”proteins” when we properly set a multiplication factor to minimize the non-specific background. By merging these images, we obtained high contrast H&E “stained” images of BBCs. Nonlinear microscope systems upgraded for real time DVRF CARS measurements providing pseudo H&E images can be suitable for in vivo assessment of BCC in the future.
This year, we introduced a fast and cost-efficient spectral modulation technique (IF-CARS) for sub-100 fs pulse Ti-sapphire lasers, which allows us to modulate the laser spectrum on a ms time scale with the use of a piezo-driven Michelson interferometer. Switching between the properly shaped “on-resonance” and “off-resonance” laser spectra can be synchronized either to the electronic “line” or to the “frame” signals of our laser scanning microscope, which allows us to perform real time non-resonant background suppression during CARS imaging. It might have applications in brain research, e.g., when investigating myelin breakdown in murine models with multiple sclerosis (MS). In an alternative setting, we modulate the laser spectrum in such a way that CARS imaging for “lipids” and “proteins” does not require the tuning of our pump laser or readjustment of the time delay, which paves the way for real-time stain-free histopathology.
We started from our CARS imaging setup referenced above, which received the Applied Research Prize of Wigner SZFI in 2015. For our IF-CARS experiments, we constructed a small size Michelson interferometer (see Fig. 1). In the beam path of the Ti:sapphire laser, we replaced one of the 45-degree folding mirrors by our small-size interferometer. One of its mirrors (M2) was placed on a piezo-electric linear actuator. The optical path difference (2*ΔL) had an offset value of ~0.1 mm, which resulted in different modulated laser spectra at the interferometer output, depending on the phase difference of the two arms. For our stain-free histopathology imaging experiments on in vitro brain slices, we used different interferometer settings: we switched the phase difference between π/2 and - π/2 at the laser central wavelength of 975 nm. As a result, we obtained two wavelength-shifted laser spectra with maxima at 793 and 796 nm corresponding to the vibrational resonance of CH3 (mainly in proteins) and CH2 bonds (primarily in lipids).
Figure 1 A Michelson-interferometer is used for spectral modulation of the pump pulses. Depending on the electronically controlled phase difference of the two arms, different spectra can be generated for CARS imaging. ΔL has a properly set offset value depending on the spectral bandwidth of the laser applied. The phase difference is electronically controlled and synchronized to the frame signal of our microscope.
In the following, we show representative images recorded from in vitro brain slices using this settings.
Myelin sheaths – wrapping around the axons of neurons – are rich in lipid; therefore we can record high-quality 3D CARS images. Degradation of the myelin sheath is the cause of neurodegenerative diseases such as multiple sclerosis (MS), but a clear mechanistic understanding of myelin loss is missing. Previously, we studied myelin breakdown in murine models with multiple sclerosis (MS) using the toxin cuprizone. We found that myelin debris form lipid droplets alongside myelinated axon fibers. For automatic lipid reconstruction, a strong and specific lipid signal is needed. Therefore, to exclude CH3 signal originating primarily from proteins and non-resonant background, we introduced spectral modulation with our interferometer. Our results are summarized in Fig. 2.
Figure 2. IF-CARS imaging of in vitro brain slices with a Mai Tai pump laser tuned to 795 nm. A-B) Images of somatosensory cortex coronal slices of layer 6 and white matter were recorded for two different, spectrally modulated pump pulses with spectral maxima at 793 nm (“CH3”) and 796 nm (“CH2”). C) Subtraction of images (CH2 - CH3) highlights more lipid structures. D) Inverse image subtraction (CH3- CH2) reveals protein rich background and somata of neurons. E) Composit image of lipid (red, CH2- CH3) and protein (green, CH3-CH2). Arrows show somata of neurons in layer 6, sidelong lines show the border of layer 6 and the white matter.
Analysis of quantitative parameters in images of the collagen structure of basal cell carcinoma from SHG microscopy. — BCCs often have poorly defined borders, the clinical assessment of the tumor margins can be challenging. Therefore, there is an emerging demand for efficient in vivo imaging techniques for the evaluation of the tumor borders of BCC prior to and during surgeries. This demand might be met in the near future by nonlinear microscope techniques (such as second-harmonic generation (SHG) mosaic imaging) utilizing our novel, fiber-laser based, hand-held 3D nonlinear microscope system (FiberScope), which received the Applied Research Prize of Wigner SZFI in 2017. This year, we compared various quantitative parameters and algorithms for the analysis of SHG images of collagen in ex vivo human BCC and healthy skin samples to evaluate the utility of these methods in the detection of BCC.
Among others, we performed Fast Fourier Transformation (FFT) on the SHG images and converted the output of FFT into power plots. We fitted an ellipse to the power plots and collagen orientation index (COI) was calculated by COI = [1-(short axis/long axis)]. A COI value close to 0 reflects a normal sample with isotropic behavior, while a value close to 1 suggests parallel oriented fibers. Collagen bundle packing (CBP) was expressed as CPB = 512*(1/h), where h is the distance between the centers of gravity of two first-order maxima of FFT plots. FFT analysis was performed by ImageJ software (NIH, USA). FFT images of BCC displayed significantly higher COI, indicating that the collagen fibers are less randomly arranged, while no difference was found in the CBP value (Fig. 3). In the future, our novel image analysis methods (such as the FFT method briefly introduced here) could be integrated in our hand-held non-linear microscope system for sensitive and specific identification of BCC that, in longer term, can result in different applications such as surgery by robots.
Figure 3. A-B panels: Power plots of fast-Fourier-transformed second-harmonic generation images. A: basal cell carcinoma (BCC), B: control skin; C: collagen orientation index; D: collagen bundle packing. Error bars represent standard deviation, *p<0.05.
Characterization of DHEA-induced PCOS-model by CARS microscopy. — Polycystic ovary syndrome (PCOS) is one of most frequent female endocrine disorder, affecting 5%–10% of women, causing infertility, disfunctional follicular maturation and ovulation, multicystic ovaries, hyperandrogenism. PCOS play a role in the enhancement of the risk of cardiovascular diseases and the development of diabetes. Postnatal treatment of rodents with DHEA (Dehydroepiandrosterone) induced human PCOS characteristics of acyclicity, anovulation, polycystic ovaries, and hyperandrogenism: DHEA induces ovarian cysts and causes abnormal hormon level (increased serum testosterone, androstenedione and 5-α-dihydrotestosterone) similar to the women with PCOS. Development of cysts causes an alteration of ovarian function and an imbalance in the oxidant–antioxidant balance. Increased reactive oxygen species (ROS) within ovarian cells is associated with the impaired ovarian function. DHEA transformed into potent estrogens such as estradiol and produces estrogenic effects of female sex hormone. Estradiol is involved in the regulation of the female reproductive cycles and responsible for the development of female secondary sexual characteristics such as the breasts, widening of the hips, and a feminine pattern of fat distribution in women. Mouse cumulus oocyte complexes (COCs) exhibit lipotoxicity responses in association with obesity or following treatment with high levels of lipids in vitro. Traditional medicine, marjoram herb (Origanum majorana) tea was found to improve insulin sensitivity and reduce the levels of adrenal androgens in the hormonal profile of PCOS women in a randomised, double-blind, placebo-controlled trial. Spearmint (Mentha spicata) has treatment potential on PCOS through inhibition of testosterone and restoration of follicular development in ovarian tissue. In our recent study, we have investigated the effects of pure 100 % natural essential oil mix of Origanum majorana and Mentha piperita in DHEA-induced PCOS-model by nonlinear microscopy, the results of which are summarized below.
4 week-old (~18 g) female C57 bl/6 mice were kept at 22 ± 2 °C under a 12 h light/12 h darkness cycle. The animals were fed normal diet and water was available ad libidum. C57BL/6 female mice were treated with dehydroepiandrosteron (DHEA) daily (6 mg/100 g body weight in 0.1 ml oil subcutan) for 20 consecutive days. The DHEA treated animals were randomized into different treatment groups (n=6): DHEA-K group and DHEA + Essential oil mix group. The latter group, after a DHEA treatment, was subsequently treated per os for 10 consecutive days with water solution of Origanum majorana (150 mg/kgbody mass, CAS 84082-58-6) and Mentha piperita (20 mg/kgbody mass, CAS 8006-90-4) essential oils. After 20 days DHEA and 10 days Essential oil mix treatment, the mice were injected intraperitoneally (i.p.) with PMSG (pregnant mare’s serum) at 5 IU/12 g of body weight, followed 48 h later by i.p. injection of hCG (human chorionic gonadotropin) at 5 IU/12 g of body weight. The ovaries were dissected and COCs were isolated from the oviducts at 16 h after hCG injection, placed in HEPES-buffered α-MEM (5 % FBS) and counted under microscope. The COCs were stained by Bodipy for lipid content or by MitoSOX Red, which is a mitochondrial superoxide indicator. During the staining procedure manufacturer’s instructions was followed. After washing steps, the COCs were fixed by paraformaldehide. We used a 2PEF and CARS imaging setup similar to that described above.
Figure 4 Bodipy staining is concentrated mainly into the cytosol, which shows a clear co-localization with the detected CARS-lipid signal. Interestingly, the CARS image shows slightly higher spatial resolution than the corresponding 2PEF image.
As a first step, we checked the co-localization of Bodipy fluorescent labelling and the detected CARS-lipid signal (see Fig.4). We found that even the small lipid droplets in the cells are clearly seen in both pictures. Interestingly, the CARS image shows slightly higher spatial resolution than the 2PEF image. As a next step, we compared the DHEA-induced changes of lipid content and ROS-level in COCs of murine PCOS-model by label-free CARS and MitoSox Red-labelled 2PE fluorescence microscopy (see Fig. 5). We found that DHEA treatment of female mice results in elevated lipid concentration of COCs parallel with increased mitochondrial ROS-production. Essential oil-mix treatment of mice decreased lipid and mitochondrial ROS-level in COCs. According to these results, we dare to say that CARS imaging might be a plausible approach to examine the effects of drugs on murine PCOS DHEA-model.
Figure 5. Comparision of DHEA-induced changes of lipid content and ROS level in COCs of murine PCOS-model by label-free CARS and MitoSox Red-labelled 2PE fluorescence microscopy.
Coherent anti-Stokes Raman Spectroscopy (CARS) microscopy; Stain-free histopathology. — Basal cell carcinoma (BCC) is the most common malignancy in Caucasians. Non-linear microscopy has been previously utilized for the imaging of BCC, but the captured images do not correlate with standard hematoxylin and eosin (H&E) staining. This year we have developed a novel algorithm to post-process images obtained from dual vibration resonance frequency (DVRF) CARS measurements to acquire high-quality pseudo H&E images of BCC samples (Fig. 1). We adapted our CARS setup to utilize the distinct vibrational properties of CH3 (mainly in proteins) and CH2 bonds (primarily in lipids). In a narrow-band setup, the central wavelength of the pump laser is set to 791 nm and 796 nm to obtain optimal excitation. Due to the partial overlap of the excitation spectra and the 5-10 nm FWHM spectral bandwidth of our lasers, we set the wavelengths to 790 nm (proteins) and 800 nm (lipids). Non-resonant background from water molecules also reduces the chemical selectivity which can be significantly improved if we subtract the DVRF images from each other. As a result, we acquired two images: one for “lipids” and one for ”proteins” when we properly set a multiplication factor to minimize the non-specific background. By merging these images, we obtained high contrast H&E “stained” images of BBCs. Non-linear microscope systems upgraded for real time DVRF CARS measurements, providing pseudo H&E images can be suitable for in vivo assessment of BCC in the future.
Figure 1. CARS images of mouse epidermis (A-C) and human basal cell carcinoma (D-F). A, D: “CH2“ images; B, E: “CH3“ images; C, F: merged color DVRF-CARS images (red: CH2-CH3, blue: CH3-CH2). Scale bar: 50 µm.
Multimodal stain-free mosaic imaging of malignant tumour in the skin – BCCs often have poorly defined borders, the clinical assessment of the tumor margins can be challenging. Therefore, there is an emerging demand for efficient in vivo imaging techniques for the evaluation of the tumor borders of BCC prior to and during surgeries. This demand might be met in the near future by non-linear microscopy techniques (such as auto-fluorescence (AF) and second harmonic generation (SHG) mosaic imaging) utilizing our novel, fibre-laser based, hand-held 3D nonlinear microscope system (FiberScope).
Figure 2. Multimodal (AF+SHG) mosaic image of human basal cell carcinoma comprising 6x10 microscope images with an overall area of 2,5x 4.2 mm2. Yellow: detected AF signal, purple: SHG signal of collagen.
This year, we have measured AF and SHG signal of collagen on 10 ex vivo healthy control and BCC skin samples and compared the images by different quantitative image analysis methods. These included integrated optical density (IOD) measurements on AF and SHG images and application of fast Fourier transform (FFT), CT-FIRE and CurveAlign algorithms on SHG images to evaluate collagen structure. In the BCC samples, we found significantly lower IOD of both the AF and SHG signals and higher collagen orientation index utilizing FFT. CT-FIRE algorithm revealed increased collagen fiber length and decreased fiber angle while CurveAlign detected significantly higher fiber alignment of collagen fibers in BCC. These results are in line with previous findings which describe pronounced changes in the collagen structure of BCC. In the future, these novel image analysis methods could be integrated in our FiberScope imaging system for sensitive and specific identification of BCC.
Figure 3. Normal human skin (A, C) and basal cell carcinoma (B, D) collagen SHG images processed by different numerical algorithms. Image size: 420x420 μm2.
Femtosecond fiber lasers for nonlinear microscopy. — During the last couple of years, we have been developing a pulsed Yb-fiber oscillator and amplifier system with a variable repetition rate in the 1 to 36 MHz range in collaboration with our industrial partner, R&D Ultrafast Laser Ltd. First we used this laser in our novel, hand-held 3D nonlinear microscope system (FiberScope) for applications in dermatology and nanomedicine. The laser delivers ~0.4 ps pulses at around ~1030 nm, which makes it an excellent candidate for two-photon imaging of GCaMP in neuroscience as well. GCaMP is a genetically encoded calcium indicator, and its main advantage is that it can be genetically specified for studies in living organisms. In the absence of calcium, this protein is in a poorly fluorescent state, but after Ca2+-binding, it is brightly fluorescent. In Figure 1, we show 3D reconstructions of 2PE auto-fluorescence images of brain slices comprising GCaMP in its certain neurons in the “poorly fluorescent state”. The high quality of the images results from the fact that our fiber laser was operated at a relatively low, ~6 MHz repetition rate.
Figure 1. 2PE auto-fluorescence images of GCaMP expressing neurons excited by our mode-locked Yb-fiber laser oscillator - amplifier system operating at ~6 MHz repetition rate (central wavelength ~1030 nm, pulse duration: ~0.4 ps, average power on sample: ~14 mW). Left: 3D reconstruction of z-stack images with dimensions of 600 µm x 600 µm. Right: 3D reconstruction of z-stack images with dimensions of 50 µm x 50 µm.
Coherent anti-Stokes Raman spectroscopy (CARS) microscopy. — Last year, CARS imaging of the CH2 bonds of lipids was successfully used to perform a quantitative analysis of the myelin loss in a cuprizone model of sclerosis multiplex (SM) depending on the drug treatment. This year, we have aimed at label-free imaging of proteins as well as NO in living tissues, such as in murine skin or in the brain. To this end, we optimized our CARS imaging system for protein and NO detection, which required several modifications and improvement in our optics and the laser system. Note that the concentration (and hence the CARS signal) of NO is orders of magnitude smaller than that of the lipids in myelin, that is why NO imaging is a challenging task. For demonstrative purposes, we show two CARS images of murine skin recorded for “lipid” and “protein” settings (Fig. 2, left) and one CARS image of an artificial sample containing NO in a gaseous state (Fig. 2, right). In the latter experiment, NO was generated from an aqueous solvent of sodium nitroprusside (SNP) after exposing it to white light for a few tens of seconds before the imaging.
Figure 2. Left: CARS images of the stratum corneum of murine skin for “lipid” (green) and “protein” (blue) settings. Right: CARS image of NO distribution in a SNP solvent containing artificial tissue.
Currently, we are working on the development of more sophisticated, non-resonant background-free imaging techniques for 3D distribution detection of low-concentration molecules (e.g. NO), such as FM-CARS or our newly developed TD-CARS method.
Broadband beam steering mirrors in nonlinear microscopy. — Broadband dielectric beam steering mirrors are key components for nonlinear microscopes comprising tunable Ti:sapphire lasers. This year we characterized dispersion of a few mirror samples by spectral interferometry and showed how they affected our imaging quality. As an example, we show the measured group delay of a BB1-E03 mirror (product Thorlabs Inc.) for s- and p-polarized light (Fig. 3, up), and how it affects the pulse shape after four reflections at around a resonance wavelength (Fig. 3, down). In practice, one obtains considerably lower signal in a nonlinear microscope when the peak intensity of the optical pulses is reduced by such unexpected dispersive effects (see Fig. 3, down).
Figure 3. Up: Measured group-delay vs. wavelength functions of an ultra-broadband dielectric mirror (two reflection, AOI: 45 degrees, Thorlabs Inc. BB1-E03). Down: Calculated intensity vs. time functions of transform limited 130 fs pulses being reflected on the same dielectric mirrors for s-polarized light at 45 degrees of AOI at different central wavelenghts.