Spatial-spectral coupling in hyperspectral CARS microscopy image formation

Coherent anti-Stokes Raman scattering microscopy.

This thesis describes a new technique that improves specificity, selectivity and sensitivity in coherent anti-Stokes Raman scattering (CARS) microscopy. CARS microscopy is a nonlinear optical technique that utilizes specific bonds of molecules, sometimes referred to as the `fingerprint' of a molecule, to obtain chemically specific imaging of a sample. A single `unique' vibrational resonance within a molecule can be addressed and the appearance of this resonance within a sample can be imaged. This nonlinear vibrational microscopy technique provides a noninvasive method to identify structures of a specific chemical composition. CARS features high collection efficiency and absence of interference from one-photon autofluorescence. However, CARS suffers from a frequency-independent background signal, the `non-resonant background'. In mixtures, the resonant CARS signal of less abundant constituents may be overwhelmed by the non-resonant background, preventing detection of the resonant molecules. A cascaded phase preserving chain is used for heterodyne detection and yields amplitude and phase. The vibrational phase of the oscillators in the focal volume can be obtained by the subtraction of the local excitation phase from the heterodyne phase. We call this technique ``Vibrational Phase Contrast CARS'' and can be regarded as a vibrational extension of the linear (refractive index) phase contrast microscopy introduced by Zernike in 1933. The phase detection allows for rejection of the non-resonant background signal, without reduction of the resonant signal. CARS microscopy is applied to biology, pharmacology and food, where chemical selectivity and label-free detection are essential. Other nonlinear signals, generated simultaneously, can be used to obtain more information from the sample; CARS, second harmonic generation microscopy and spontaneous Raman spectroscopy are used side-by-side. Two in-depth studies are presented on the dissolution behavior of a drug and on early bone growth in cells.

Coherent anti-Stokes Raman scattering (CARS) spectroscopy and microscopy have many potential applications in biology and medicine. Among many variants of the technique itself, the method of single-pulse CARS spectroscopy and microscopy is attractive for its simplicity and quick implementation. Single-pulse CARS microscopy can be performed by shaping the excitation spectrum using a notch filter, yet the resonant signal rides on a large background caused by a non-resonant signal, a background which is usually removed by lock-in detection. Here, we show that the background can be reduced significantly by adding a small chirp to the pulse and can even be made smaller than the resonant signal. In order to enhance the CARS signal and thus the contrast further, double-notch shaping is introduced. The double-notch induces two sets of CARS features shifted by the frequency difference between the two notches, thereby coherently enhancing a particular CARS feature. The experimental results agree well with theoretical simulations. We applied this scheme to perform lock-in free CARS microscopy of bone tissue with enhanced contrast.

CARS spectra of polystyrene sheet at excitation of fs pulses and chirped pulses

A new vibrational imaging method based on coherent anti-Stokes Raman scattering (CARS) has been used for high-speed, selective imaging of neutral lipid droplets (LDs) in unstained live fibroblast cells. LDs have a high density of C-H bonds and show a high contrast in laser-scanning CARS images taken at 2,845 cm-1, the frequency for aliphatic C-H vibrations. The contrast from LDs was confirmed by comparing CARS and Oil Red O (ORO)-stained fluorescence images. The fluorescent labeling processes were examined with CARS microscopy. It was found that ORO staining of fixed cells caused aggregation of LDs, whereas fixing with formaldehyde or staining with Nile Red did not affect LDs. CARS microscopy was also used to monitor the 3T3-L1 cell differentiation process, revealing that there was an obvious clearance of LDs at the early stage of differentiation. After that, the cells started to differentiate and reaccumulate LDs in the cytoplasm in a largely unsynchronized manner. Differentiated cells formed small colonies surrounded by undifferentiated cells that were devoid of LDs. These observations demonstrate that CARS microscopy can follow dynamic changes in live cells with chemical selectivity and noninvasiveness. CARS microscopy, in tandem with other techniques, provides exciting possibilities for studying LD dynamics under physiological conditions without perturbation of cell functions.

We report the retrieval of the Raman signal from coherent anti-Stokes Raman scattering (CARS) spectra using a convolutional neural network (CNN) model. Three different types of non-resonant backgrounds (NRBs) were explored to simulate the CARS spectra viz (1) product of two sigmoids following the original SpecNet model, (2) Single Sigmoid, and (3) fourth-order polynomial function. Later, 50 000 CARS spectra were separately synthesized using each NRB type to train the CNN model and, after training, we tested its performance on 300 simulated test spectra. The results have shown that imaginary part extraction capability is superior for the model trained with Polynomial NRB, and the extracted line shapes are in good agreement with the ground truth. Moreover, correlation analysis was carried out to compare the retrieved Raman signals to real ones, and a higher correlation coefficient was obtained for the model trained with the Polynomial NRB (on average, ∼0.95 for 300 test spectra), whereas it was ∼0.89 for the other NRBs. Finally, the predictive capability is evaluated on three complex experimental CARS spectra (DMPC, ADP, and yeast), where the Polynomial NRB model performance is found to stand out from the rest. This approach has a strong potential to simplify the analysis of complex CARS spectroscopy and can be helpful in real-time microscopy imaging applications.

Lipid droplets (LDs) of mammalian cells are coated with one or more members of the PAT protein family (e.g. Perilipin), which serve important functions in regulating fat storage and mobilization by interacting with hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). However, the interplay of lipid-associated proteins (LAP) that regulate lipolysis is not fully understood. PURPOSE: In this study, we examined the colocalization, trafficking, and interactions of key proteins involved in lipolysis as well as dynamic morphological change affecting LDs during lipolytic activation in differentiated 3T3-L1 adipocytes. METHODS: Time-lapse images of 3T3-L1 cells stably expressing GFP- and/or RFP-fused LAP were acquired during 2 hrs of cAMP-dependent PKA activation. In addition, dynamic morphological changes of LDs during lipolytic activation were obtained using coherent anti-Stokes Raman Scattering (CARS) microscopy, which visualizes LDs in living cells. RESULTS: During the course of stimulated lipolysis, GFP-perilipin (PLIN) and RFP-CGI58, which interacts with PLIN on LDs, began to disperse from central large LDs into the cytoplasm. In addition, using CARS microscopy, we observed numerous micro-LDs (mLDs) coated with CGI58 emerging after lipolytic stimulation. Further we showed that mLDs might be derived from a large central LDs coated with PLIN and CGI58. GFP-HSL and GFP-ATGL were found in the cytoplasm in basal condition. Upon lipolytic stimulation, GFP-HSL was targeted to various sized LDs, where it colocalized with PLIN. On the other hand, lipolytic stimulation did not alter the location of GFP-ATGL. Following subcellular fractionation, we observed that PLIN and CGI58 were predominantly fractionated with LDs in lysates from both basal and 1 hr PKA-stimulated adipocytes, but CGI58 also detected in membrane fraction, where organelles are involved, after 1 hr lipolysis. ATGL were found in membrane fraction as well as cytosolic fraction of both basal and PKA-stimulated adipocytes. CONCLUSION: These results suggest that mLDs are important physiologically to enhance the efficiency of lipolysis by increasing the total LD surface area where PLIN and HSL can interact. As well, it is possible that active lipolysis occurs by means of interplays of CGI58 and ATGL even in the organelles.

Multiple emulsions consisting of water droplets dispersed in an oil phase containing emulsifier which is emulsified in an outer water phase (W/O/W) are of great interest in pharmacology for developing new drugs, in the nutrition sciences for designing functional food, and in biology as model systems for cell organelles such as liposomes. In the food industry multiple emulsions with high sugar content in the aqueous phase can be used for the production of sweets, because the high sugar content prevents deterioration. However, for these emulsions the refractive indexes of oil and aqueous phase are very similar. This seriously impedes the analysis of these emulsions, e.g., for process monitoring, because microscopic techniques based on transmission or reflection do not provide sufficient contrast. We have characterized the inner dispersed phase of concentrated W/O/W emulsions with the same refractive index of the three phases by micro Raman spectroscopy and investigated the composition and molecular distribution in water-oil-water emulsions by means of three-dimensional laser scanning CARS (coherent anti-Stokes Raman scattering) microscopy. CARS microscopy has been used to study water droplets dispersed in oil droplets at different Raman resonances to visualize different molecular species. Water droplets with a diameter of about 700 nm could clearly be visualized. The advantages of CARS microscopy for studying this particular system are emphasized by comparing this microscopic technique with conventional confocal reflection and transmission microscopies.

BackgroundCoherent anti-Stokes Raman scattering (CARS) microscopy provides fine resolution imaging and displays morphochemical properties of unstained tissue. Here, we evaluated this technique to delineate and identify brain tumors.MethodsDifferent human tumors (glioblastoma, brain metastases of melanoma and breast cancer) were induced in an orthotopic mouse model. Cryosections were investigated by CARS imaging tuned to probe C-H molecular vibrations, thereby addressing the lipid content of the sample. Raman microspectroscopy was used as reference. Histopathology provided information about the tumor's localization, cell proliferation and vascularization.ResultsThe morphochemical contrast of CARS images enabled identifying brain tumors irrespective of the tumor type and properties: All tumors were characterized by a lower CARS signal intensity than the normal parenchyma. On this basis, tumor borders and infiltrations could be identified with cellular resolution. Quantitative analysis revealed that the tumor-related reduction of CARS signal intensity was more pronounced in glioblastoma than in metastases. Raman spectroscopy enabled relating the CARS intensity variation to the decline of total lipid content in the tumors. The analysis of the immunohistochemical stainings revealed no correlation between tumor-induced cytological changes and the extent of CARS signal intensity reductions. The results were confirmed on samples of human glioblastoma.ConclusionsCARS imaging enables label-free, rapid and objective identification of primary and secondary brain tumors. Therefore, it is a potential tool for diagnostic neuropathology as well as for intraoperative tumor delineation.

A picosecond time-resolved multiplex coherent anti-Stokes Raman scattering (CARS) spectrometer based on a new principle has been constructed using a femtosecond laser, nanosecond lasers, and a streak camera. The second harmonic of the output from the femtosecond Ti:sapphire oscillator/regenerative-amplifier system is used for the photoexcitation. The second harmonic of a nanosecond Nd:YLF laser is used as CARS probing ω1 radiation, and the output of a nanosecond broadband dye laser is used as ω2 radiation. CARS signals, which are generated from the sample during the nanosecond probing pulses, are analyzed in the frequency domain by a spectrograph and are resolved in the time domain by a streak camera. This enables us multiplex CARS measurements not only in the frequency domain but also in the time domain (termed two-dimensional multiplex CARS). Consequently, CARS spectra at various delay times are obtainable with a single measurement. Owing to the short duration and high stability of the femtosecond pumping pulses, time resolution as high as 15 ps has been achieved. Picosecond time-resolved CARS spectra of all-trans retinal and β-carotene are presented to demonstrate the high capability of the constructed system. The factors which determine the time resolution of this CARS spectrometer are discussed.

The nematode Caenorhabditis elegans has been employed as a model organism to study human obesity due to the conservation of the pathways that regulate energy metabolism. To assay for fat storage in C. elegans, a number of fat-soluble dyes have been employed including BODIPY, Nile Red, Oil Red O, and Sudan Black. However, dye-labeled assays produce results that often do not correlate with fat stores in C. elegans. An alternative label-free approach to analyze fat storage in C. elegans has recently been described with coherent anti-Stokes Raman scattering (CARS) microscopy. Here, we compare the performance of CARS microscopy with standard dye-labeled techniques and biochemical quantification to analyze fat storage in wild type C. elegans and with genetic mutations in the insulin/IGF-1 signaling pathway including the genes daf-2 (insulin/IGF-1 receptor), rict-1 (rictor) and sgk-1 (serum glucocorticoid kinase). CARS imaging provides a direct measure of fat storage with unprecedented details including total fat stores as well as the size, number, and lipid-chain unsaturation of individual lipid droplets. In addition, CARS/TPEF imaging reveals a neutral lipid species that resides in both the hypodermis and the intestinal cells and an autofluorescent organelle that resides exclusively in the intestinal cells. Importantly, coherent addition of the CARS fields from the C-H abundant neutral lipid permits selective CARS imaging of the fat store, and further coupling of spontaneous Raman analysis provides unprecedented details including lipid-chain unsaturation of individual lipid droplets. We observe that although daf-2, rict-1, and sgk-1 mutants affect insulin/IGF-1 signaling, they exhibit vastly different phenotypes in terms of neutral lipid and autofluorescent species. We find that CARS imaging gives quantification similar to standard biochemical triglyceride quantification. Further, we independently confirm that feeding worms with vital dyes does not lead to the staining of fat stores, but rather the sequestration of dyes in lysosome-related organelles. In contrast, fixative staining methods provide reproducible data but are prone to errors due to the interference of autofluorescent species and the non-specific staining of cellular structures other than fat stores. Importantly, both growth conditions and developmental stage should be considered when comparing methods of C. elegans lipid storage. Taken together, we confirm that CARS microscopy provides a direct, non-invasive, and label-free means to quantitatively analyze fat storage in living C. elegans.

Microalgae have great prospects as a sustainable resource of lipids for refinement into nutraceuticals and biodiesel, which increases the need for detailed insights into their intracellular lipid synthesis/storage mechanisms. As an alternative strategy to solvent- and label-based lipid quantification techniques, we introduce time-gated coherent anti-Stokes Raman scattering (CARS) microscopy for monitoring lipid contents in living algae, despite strong autofluorescence from the chloroplasts, at approximately picogram and subcellular levels by probing inherent molecular vibrations. Intracellular lipid droplet synthesis was followed in Phaeodactylum tricornutum algae grown under (1) light/nutrient-replete (control [Ctrl]), (2) light-limited (LL), and (3) nitrogen-starved (NS) conditions. Good correlation (r(2) = 0.924) was found between lipid volume data yielded by CARS microscopy and total fatty acid content obtained from gas chromatography-mass spectrometry analysis. In Ctrl and LL cells, micron-sized lipid droplets were found to increase in number throughout the growth phases, particularly in the stationary phase. During more excessive lipid accumulation, as observed in NS cells, promising commercial harvest as biofuels and nutritional lipids, several micron-sized droplets were present already initially during cultivation, which then fused into a single giant droplet toward stationary phase alongside with new droplets emerging. CARS microspectroscopy further indicated lower lipid fluidity in NS cells than in Ctrl and LL cells, potentially due to higher fatty acid saturation. This agreed with the fatty acid profiles gathered by gas chromatography-mass spectrometry. CARS microscopy could thus provide quantitative and semiqualitative data at the single-cell level along with important insights into lipid-accumulating mechanisms, here revealing two different modes for normal and excessive lipid accumulation.

By simultaneous coherent anti-Stokes Raman scattering (CARS) and 2-photon fluorescence microscopy of Thioflavin-S stained Alzheimer´s diseased human brain tissues, we show evidence of lipid deposits co-localizing with fibrillar β-amyloid (Aβ) plaques. Two lipid morphologies can be observed; lamellar structures and coalescing macro-aggregates of sub-micron sizes to ~25 μm. No significant lipid deposits were observed in non-fibrillar, diffuse plaques identified by Aβ immuno-staining. CARS microscopy of unlabeled samples confirms the lamellar and macro-aggregate lipid morphologies. The composition of the plaques was analyzed by CARS microspectroscopy and Raman microscopy; vibrational signatures of lipids with long acyl chains co-localize with the β-sheet vibrations. The lipid fluidity was evaluated from the CARS spectra, illustrating that the lipid composition/organization varies throughout the plaques. Altogether this indicates close amyloid-lipid interplay in fibrillar Aβ plaques, rendering them more dynamic compositions than previously believed and, hence, potential sources of toxic oligomers.

Label-free imaging of lipid dynamics using Coherent Anti-stokes Raman Scattering (CARS) and Stimulated Raman Scattering (SRS) microscopy

Since the first introduction of Raman microscope in 1973, optical and laser technology has made a tremendous step forward. However, despite of the obvious advantages of being a very informative and nondestructive method of studying biological samples, spontaneous Raman scattering suffers from a series of limitations such as a fluorescent background and a low signal level. Nonlinear Raman spectroscopy and, in particular, spectroscopy of coherent anti-Stokes Raman scattering (CARS) can resolve most of the problems associated with conventional Raman spectroscopy. In this report, the most critical issues of the CARS microspectroscopy setup design are reviewed and several exciting potential applications of the broadband CARS microspectroscopy, where the CARS microscopy has an advantage with respect to Raman microscopy, are outlined.