KEYWORDS: Digital holography, 3D image reconstruction, Reverse modeling, Holograms, Deep learning, Mathematical optimization, Image restoration, Education and training
Untrained Physics-based Deep Learning (DL) methods for digital holography have gained significant attention due to their benefits, such as not requiring an annotated training dataset, and providing interpretability since utilizing the governing laws of hologram formation. However, they are sensitive to the hard-to-obtain precise object distance from the imaging plane, posing the Autofocusing challenge. Conventional solutions involve reconstructing image stacks for different potential distances and applying focus metrics to select the best results, which apparently is computationally inefficient. In contrast, recently developed DL-based methods treat it as a supervised task, which again needs annotated data and lacks generalizability. To address this issue, we propose reverse-attention loss, a weighted sum of losses for all possible candidates with learnable weights. This is a pioneering approach to addressing the Autofocusing challenge in untrained deep-learning methods. Both theoretical analysis and experiments demonstrate its superiority in efficiency and accuracy. Interestingly, our method presents a significant reconstruction performance over rival methods (i.e. alternating descent-like optimization, non-weighted loss integration, and random distance assignment) and even is almost equal to that achieved with a precisely known object distance. For example, the difference is less than 1dB in PSNR and 0.002 in SSIM for the target sample in our experiment.
KEYWORDS: Atomic force microscopy, Modeling, Polystyrene, Mechanics, Equipment, Composites, Beam analyzers, Analog electronics, 3D modeling, 3D metrology
This study explores Atomic Force Microscopy's (AFM) applicability for 3D analysis by determining its "mechanical focal plane" in soft composites. The investigators determined the AFM's ability to measure mechanics from a distance when target components are immersed in a secondary medium. Using the Kelvin-Voigt model under quasi-static and dynamic conditions, a sample material with polystyrene beads embedded in agarose gel is analyzed at varying scanning parameters. The results include a model of the effective depth and the effect that a secondary medium has on the ability to measure an embedded component's properties.
Histologic examination of skin biopsies is currently the gold standard to definitively diagnose malignant skin lesions; however, biopsies are minor, invasive procedures with potential risks. With the advancement of imaging techniques such as laser speckle contrast imaging (LSCI), it is now possible to evaluate neoplastic skin lesions in real-time and noninvasively. LSCI has been widely used to image surface blood flow in tissues, such as skin, retina, and brain. In this preliminary study, we hypothesized that blood flow within microvessels differs between neoplastic and non-neoplastic skin. This study presents a descriptive demonstration of LSCI application in dermatology. LSCI was utilized to assess surface blood flow in potentially neoplastic skin lesions at our institution’s dermatology clinics. Preliminary data demonstrated decreased contrast within speckle contrast images of malignant and premalignant skin lesions, suggesting increased blood flow to these areas of interest. LSCI may show utility as a noninvasive technique to evaluate neoplastic skin lesions prior to biopsy; however, further systematic optimization is required.
Articular cartilage in the tibiofemoral joint contains unique tissue microstructures that serve specific functions, including reduction of friction and distributing the dynamic and static cyclic loading at the ends of diarthrodial joints. A proficient understanding of these microstructures can lead to significant clinical advances in diagnosing orthopedic diseases such as osteoarthritis and improving cartilage repairs. The surface of tibiofemoral condyles can be roughly separated into loadbearing and meniscus-covered areas. Due to the difference in mechanical loading between the two regions, we hypothesize that their microstructures differ. To test this hypothesis, we used cartilage punches harvested from the tibial condyle of porcine knee joints as an example tissue and a custom nonlinear optical microscope for performing a dye-free imaging study. The custom nonlinear optical microscope could simultaneously acquire Two-Photon excitation Auto-Fluorescence (TPAF) and Second Harmonic Generation (SHG) images. Through the TPAF channel, elastin fibers are visible along with chondrocytes. The SHG channel was utilized for observing the vast collagen network and its evident orientation throughout the tibial condyle. Images were analyzed by ImageJ to reveal alignment angles of the collagen network and elastin fibers. The load-bearing region exhibits a denser uniform collagen network with minimum elastin fibers. In contrast, the meniscuscovered areas have a distinctive collagen orientation with a greater magnitude of co-localized elastin fibers. The biological differences are likely derived from their different biomechanical environments in the tibiofemoral joint.
Maintaining the heart's health is one of the largest challenges in medicine due to the proclivity of life-threatening cardiovascular diseases, such as myocardial infarctions. When the heart experiences an infarction, a scar begins forming within an hour of the event, which will continue to grow and weaken the heart’s ability to contract. The myocardium after an infarction will increase in stiffness as the tissue becomes fibrotic; the influx of collagen dampens the flexion of the ventricles and reduces the cardiac output. The nature of the tissue stiffness is vital to understand not only at the tissue level but also at the mesoscopic domain. It is necessary to specify how the primary structural tissue components at the subcellular level contribute to the mechanical behavior of the muscle. To investigate this, we produced a procedure for mapping the mechanical nature of fresh myocardium: using atomic force microscopy to measure the mechanical properties of each structural component imaging determined by our second harmonic generation (SHG) microscopy. To coregistered AFM and SHG image, which has not been accomplished previously, we developed a convenient means of marking PDMS to be visible in SHG at 830 nm. Our research draws the line between the macroscopic mechanical behavior of the tissue to the nanoscopic structures.
Visualization of collagen fibers in cardiac tissues is essential for clinical diagnosis and pathological analysis of cardiac fibrosis. Selecting a proper imaging method is still challenging for researchers and clinicians who want to determine specific information about the collagen network in cardiac tissues. We examined fibrillar collagen network from mouse ventricular myocardium by commonly available light microscopy techniques using our home-built multimodal microscope. Myocardial slices were unstained or stained with either Picrosirius red or collagen type I antibody/dye conjugation, then imaged by polarized light, confocal fluorescence, second harmonic generation (SHG), two-photon excited fluorescence (TPEF), and stimulated emission depletion (STED) microscopy techniques. This study is intended to serve as a reference for basic research and clinical evaluation of fibrillar collage network in cardiac tissues.
Mechanical signaling in vascular tissue can have major effects on remodeling outcomes and the viability of bypass grafts. When a vein is placed into an environment matching that of an artery, the vein begins to remodel to act like an artery. This change is dependent on mechanotransduction pathways that sense stress from the blood flow. To properly study these pathways, vessels need to be studied ex vivo to control the stress patterns vessels will experience within a patient. The mechanical properties of the vessel will then need to be analyzed at a cellular level to correlate the strain of the environment to the cell response. Optical coherence tomography (OCT) is able to capture b-scans of the entire vessel wall to observe changes at different lateral and axial positions. Multiple b-scans can be captured as the vessel experiences a pressure waveform mimicking physiological pressures and digital image correlation (DIC) can be performed to quantify the mechanical response of the tissue at each spot in the b-scan. A custom-built optical coherence tomography system was used to record images of a porcine carotid artery undergoing pressure changes to observe movements inside the vessel wall. DIC was performed to correlate the strain of the tissue to the experienced stress as a means of testing the system. This imaging method will provide valuable mechanical information as a vein is remodeled in a perfusion bioreactor.
Understanding cardiomyocyte-extracellular matrix (ECM) interactions at the molecular level is essential for deeper insights into their mechanical signaling function for cardiac development, homeostasis and remodeling. We report a lab-built microscope integrating two-color STED microscopy with second harmonic generation (SHG) microscopy to investigate the detailed architecture of cardiomyocyte-ECM interactions in murine myocardium at a subdiffractive level. SHG microscopy is used to locate possible interaction sites at the cell-ECM interface through the intrinsic SHG signal generated by collagen assemblies and myosin filaments. Two-color STED microscopy is used to obtain a subdiffractive view of proteins at sites of interest registered by SHG microscopy. Because large field-of-view (FOV) STED microscopy is still challenging, with photobleaching often a major concern, imaging only SHG-registered sites is advantageous. Further, using intrinsic contrast in the study reduces the number of biomarkers for fluorescent staining and thereby the number of detection channels for fluorescent imaging, simplifying sample preparation procedures and STED microscopy architectures. For purpose of demonstration, we show images of immunostained type I collagen, type Ⅳ collagen and laminin as ECM structures of interest in rat ventricular sections without counterstaining.
Chondrocyte viability is an important measure to consider when assessing cartilage health. Dye-based cell viability assays are not suitable for in vivo or long-term studies. We have introduced a non-labeling viability assay based on the assessment of high-resolution images of cells and collagen structure using two-photon stimulated autofluorescence and second harmonic generation microscopy. By either the visual or quantitative assessment, we were able to differentiate living from dead chondrocytes in those images. However, both techniques require human participation and have limited throughputs. Throughput can be increased by using methods for automated cell-based image processing. Due to the poor image contrast, traditional image processing methods are ineffective on autofluorescence images produced by nonlinear microscopes. In this work, we examined chondrocyte segmentation and classification using Mask R-CNN, a deep learning approach to implement automated viability analysis. It has been demonstrated an 85% accuracy in chondrocyte viability assessment with proper training. This study demonstrates that automated and highly accurate image analysis is achievable with the use of deep learning methods. This image processing approach can be helpful to other imaging applications in clinical medicine and biological research.
The non-selective beta-blocker timolol has shown promising evidence for healing chronic, recalcitrant wounds, improving scar cosmesis, and expediting the completion of secondary intention. The purpose of our pilot study is to use clinical imaging, two-photon excitement fluorescence (TPF) and second harmonic generation (SHG) microscopy to evaluate the temporal and molecular effects of timolol vs. normal saline in Sprague-Dawley rats traumatized by 5-millimeter dermal punch biopsy. Initial findings suggest timolol delays wound contraction, but advanced imaging techniques may reveal novel collagenous or vascular mechanisms by which timolol is affecting acute wound healing.
In the recent studies of cartilage imaging with nonlinear optical microscopy, we discovered that autofluorescence of chondrocytes provided useful information for the viability assessment of articular cartilage. However, one of the hurdles to apply this technology in research or clinical applications is the lack of image processing tools that can perform automated and cell-based analysis. In this report, we present our recent effort in the cell segmentation using deep learning algorithms with the second harmonic generation images. Two traditional segmentation methods, adaptive threshold, and watershed, were used to compare the outcomes of different methods. We found that deep learning algorithms did not show a significant advantage over the traditional methods. Once the cellular area is determined, the viability index is calculated as the intensity ratio between two autofluorescence channels in the cellular area. We found the viability index correlated well with the chondrocyte viability. Again, deep learning segmentation did not show a significant difference from the traditional segmentation methods in terms of the correlation.
Using our lab built two-photon excitation fluorescence (TPEF) and second harmonic generation (SHG) hybrid confocal imaging system, we observed, for the first time, the dynamic sarcomeric addition process in a rat cardiomyocyte cell culture system; this culture system expressed in vivo-like myofibril structure and mimicked mechanical overload experienced in a heart muscle tissue. Micro-grooved topographic patterned substrates com- bined with electrical stimulation are used to achieve the in vivo-like myofibril structure. After cardiomyocytes aligned, longitudinal and transverse mechanical stretch was applied to cardiomyocytes in parallel or perpendicu- lar, respectively, to the direction of alignment via stretching the substrates to mimic mechanical overload. Z discs, in which alpha-actinin expressed, have been proposed to involve in the process. TPEF detected alpha-actinin that labeled with enhanced yellow florescent protein via plasmid transfection. SHG is intrinsic to noncentrosym- metric structures, thus was used to detect myosin, a polar molecule expressed in myofibril. Pulse splitter system and synchronized recording system was introduced on TPEF-SHG imaging system to reduce the photodamage during live cell imaging. In our study, TPEF-SHG imaging system was used to study the dynamic process of sarcomeric addition in in vivo-like culture model under mechanical overload. This microscopic technique is ideal for tracking sarcomeric components to successively assemble onto pre-exist myofibrils and for revealing the role of Z discs played in sarcomeric addition. Transition of Z discs from continuous to broadened striation and from broadened to uniform striation under stretch has been observed. We concluded that continuous Z discs is the place of new sarcomeric addition.
Covering the ends of long bones, articular cartilage provides a smooth, lubricated surface to absorb impact and distribute loads during movement so that underlying bone is protected. This function is facilitated by a complex and well-organized extracellular matrix (ECM). Being the only cell type in articular cartilage, chondrocytes are responsible for maintaining the homeostasis of the cartilage ECM; as such, the viability of chondrocytes is a critical parameter to reflect the quality of the cartilage. Most prevalent cell viability assays rely on dye staining and thereby cannot be performed for longitudinal monitoring or in-vivo assessment. Here we demonstrate that two-photon autofluorescence (TPAF) microscopy distinguishes live cells from dead cells in intact ex-vivo cartilage tissues, which provides a non-invasive method to assess cell viability. In our study, the endogenous fluorophores such as nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were used to image chondrocytes in cartilages on rat tibia condyles immediately after harvesting. Second harmonic generation (SHG) imaging was also performed to examine the integrity of the extracellular and pericellular matrix. On the same tissue, the cell viability assay with Calcein-AM and Ethidium homodimer-1 (EthD-1) labeling was used as a gold standard to identify live or dead cells. We found that live cells presented stronger NAD(P)H fluorescence than dead cells in general and were readily identified if pseudo colors were used for showing two-channel images. Owing to its non-destructive nature, this method holds the potential value in assessing cell viability of engineered or living tissues without dye labeling.
The Bessel beam belongs to a typical class of non-diffractive optical fields that are characterized by their invariant focal profiles along the propagation direction. However, ideal Bessel beams only rigorously exist in theory; Bessel beams generated in the lab are quasi-Bessel beams with finite focal extensions and varying intensity profiles along the propagation axis. The ability to engineer the on-axis intensity profile to the desired shape is essential for many applications. Here we demonstrate an iterative optimization-based approach to engineering the on-axis intensity of Bessel beams. The genetic algorithm is used to demonstrate this approach. Starting with a traditional axicon phase mask, in the design process, the computed on-axis beam profile is fed into a feedback tuning loop of an iterative optimization process, which searches for an optimal radial phase distribution that can generate a generalized Bessel beam with the desired onaxis intensity profile. The experimental implementation involves a fine-tuning process that adjusts the originally targeted profile so that the optimization process can optimize the phase mask to yield an improved on-axis profile. Our proposed method has been demonstrated in engineering several zeroth-order Bessel beams with customized on-axis profiles. High accuracy and high energy throughput merit its use in many applications.
Stimulated emission depletion microscopy (STED) has become one of the powerful research tools in the field of superresolution microscopy. As its spatial resolution is gained by phase modulation of the light field, the aberrations produced by optical systems and specimens may have negative impact on the focusing properties of two beams, especially the STED beam, resulting in reduced spatial resolution. In thick samples, the aberration effect may play an even more critical role in affecting spatial resolution. Here, we report our recent effort in correcting the aberration in STED microscopy by using coherent optical adaptive technique (COAT) so that the resolution can be improved.
With the rapid development of life sciences, there is an increasing demand for intravital fluorescence imaging of small animals. However, large dimensions and limited working distances of objective lenses in traditional fluorescence microscopes have limited the imaging applications mostly to superficial tissues. To overcome this disadvantage, researchers have developed the graded-index (GRIN) probes with small diameters for imaging internal organs of small animals in a minimally invasive fashion. Here, we present the development of a fluorescence endoscopic imaging system based on a GRIN lens using two-photon excitation. Experimental results showed that this system could perform dynamic fluorescence microendoscopic imaging and monitor the blood flow in anesthetized living mice using two-photon excitation.
Bessel beams have been used in many applications due to their unique optical properties of maintaining their intensity profiles unchanged during propagation. In imaging applications, Bessel beams have been successfully used to provide extended focuses for volumetric imaging and uniformed illumination plane in light-sheet microscopy. Coupled with two-photon excitation, Bessel beams have been successfully used in realizing fluorescence projected volumetric imaging. We demonstrated previously a stereoscopic solution–two-photon fluorescence stereomicroscopy (TPFSM)–for recovering the depth information in volumetric imaging with Bessel beams. In TPFSM, tilted Bessel beams were used to generate stereoscopic images on a laser scanning two-photon fluorescence microscope; upon post image processing we could successfully provide 3D perception of acquired volume images by wearing anaglyph 3D glasses. However, tilted Bessel beams were generated by shifting either an axicon or an objective laterally; the slow imaging speed and severe aberrations made it hard to use in real-time volume imaging. In this article, we report recent improvements of TPFSM with newly designed scanner and imaging software, which allows 3D stereoscopic imaging without moving any of the optical components on the setup. This improvement has dramatically improved focusing qualities and imaging speed so that the TPFSM can be performed potentially in real-time to provide 3D visualization in scattering media without post image processing.
Bessel beams have been proved to have the ability to extend the depth of focus in fluorescence microscopy. But the depth discrimination was not investigated thoroughly. Following our previous work2, we investigated focal fields of Bessel-Gauss beams at different scanning angles. We found that the central focusing lines were tilted differently at different scanning angles. This effect manifests the ability of the true perspective view in the fluorescence stereomicroscopy.
Structured-illumination microscopy (SIM) is an efficacious tool to decrease the contribution of the out-of-focus light to images of specimens. However, in SIM, the frequency of the spatial modulation applied to specimens should be adjustable according to the optical properties of the specimens to reach the optimal contrasts. Hence, a common theme in SIM is how the flexibility and quality of modulations at different frequencies can be improved. Digital scanned laser light-sheet microscopy with structured illumination (DSLM-SI) has been the most flexible means for generating modulation and optical sectioning. The complexity of synchronization between the temporal modulation and the beam scanning makes it hard to use and less stable; it also takes more time to acquire images for one plane than selective plane illumination microscopy (SPIM). In this report, we present a recent effort to use a spatial light modulator (SLM) to provide spatial modulation in SPIM. With the SLM, both of the frequency and phase of lateral modulation can be changed rapidly; moreover, this SLM-based SPIM can achieve fast imaging without mechanical moving parts.
Three dimensional distributions of cells can be usually acquired by optical sectioning methods, such as multiphoton
excitation and confocal fluorescence laser scanning microscopy. Though the lateral scan rates can reach up to
several kHz, the relatively slow axial scan comprises the speed of real-time imaging of a volume. Here we propose a
three dimensional imaging method that uses Bessel beams as excitation in multiphoton fluorescence microscopy.
The extended focus of the Bessel beam allows recording a volume of cells without scanning the depth. The depth
information can be retrieved by recording a pair of parallax views of the same volume. We have demonstrated the
stereoscope capability on a homebuilt two-photon fluorescence microscope.
We developed a hybrid two-photon excitation fluorescence-second harmonic generation (TPEF-SHG) imaging system with an on-stage incubator for long-term live-cell imaging. Using the imaging system, we observed the addition of new sarcomeres during myofibrillogenesis while a cardiomyocyte was spreading on the substrate. The results suggest that the TPEF-SHG imaging system with an on-stage incubator is an effective tool for investigation of dynamic myofibrillogenesis.
We developed a hybrid SHG-TPEF polarization imaging system that allowed the excitation beam from an fs Ti:Sappire
laser being bi-directionally raster scanned across the focal plane using a pair of orthogonal galvanometers. To implement
high-speed scanning, the turning regions of the triangular waves were smoothed by a custom-designed waveform. The
SHG and TPEF signals from samples were recorded by two PMTs in the forward and backward direction. Using this
imaging system, we obtained 3D images of the sarcomere structure via SHG and DiO-stained lipid membrane via TPEF
in live cardiomyocytes isolated from neonatal and adult rats. The results demonstrated the potential applications of SHG
and TPEF in the research of myofibrillogensis.
As some of the most ubiquitous and biologically important natural pigments, melanins play essential roles in the photoprotection of skin. Changes in melanin production could potentially be useful for clinical diagnosis of the progression stage of melanoma. Previously we demonstrated a new method for imaging melanin distribution in tissue with two-color transient absorption microscopy. Here we extend this study to longer wavelengths and show that we are able to image melanin in fixed thin skin slices with higher signal-to-noise ratios (SNRs) and demonstrate epimode imaging. We show that both photothermal effects and long-lived excited states can contribute to the long-lived signal. Eumelanin and pheomelanin exhibit markedly different long-lived excited state absorption. This difference should enable us to map out their respective distribution in tissue samples with subcellular resolution. This technique could provide valuable information in diagnosing the malignant transformation of melanocytes.
The ability to perform high-resolution imaging of microvasculature and its oxygenation is very important in studying early tumor development. Toward this goal, we improved upon our excited state absorption (ESA)–based imaging technique to allow us to not only image hemoglobin directly but also differentiate between oxy- and deoxyhemoglobin in tissue. We demonstrate the separation of arterioles from venules in a live nude mouse ear using our imaging technique.
We have demonstrated a new optical microscopy technique for imaging microvasculature without any labeling. With a
very sensitive two-color excited state absorption (ESA) measurement method, we demonstrated that oxy-hemoglobin
and deoxy-hemoglobin show distinct excited state dynamics. Since this is a collinear measurement, we can readily apply
it to the microscopic study of biological tissue. We have already demonstrated in vivo imaging of blood vessels in the
nude mouse ear. Here we optimized the excitation and detection pulse train toward longer wavelengths, where tissue
scatters less and greater penetration depth can be obtained. More importantly, we are able to separate arterioles from
venules by employing different pump and probe wavelength combinations. This provides a powerful method to image
blood vessels and their oxygenation level at the same time with micrometer resolution.
As a main pigment in skin tissues, melanin plays an important role in photo-protecting skin from UV radiation. However, melanogenesis may be altered due to disease or environmental factors; for example, sun exposure may cause damage and mutation of melanocytes and induce melanoma. Imaging pigmentation changes may provide invaluable information to catch the malignant transformation in its early stage and in turn improve the prognosis of patients. We have demonstrated previously that transmission mode, two-photon, one- or two-color absorption microscopy could provide remarkable contrast in imaging melanin in skin. In this report we demonstrate significantly
improved sensitivity, so that we are now able to image in epi-mode (or back reflection) in two-photon absorption. This
improvement makes possible for us to characterize the different types of pigmentation on the skin in vivo at virtually any location. Another finding is that we can also image transient photothermal dynamics due to the light absorption of melanin. By carefully choosing excitation and probe wavelengths, we might be able to image melanin in different structures under different micro-environments in skin, which could provide useful photochemical and photophysical insights in understanding how pigments are involved in photoprotection and photodamage of cells.
We develop a new approach in imaging nonfluorescent species with two-color two-photon and excited state absorption microscopy. If one of two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the other pulse train when nonlinear absorption takes places in the medium. We can easily measure 10−6 absorption changes caused by either two-photon absorption or excited-state absorption with a RF lock-in amplifier. Sepia melanin is studied in detail as a model system. Spectroscopy studies on the instantaneous two-photon absorption (TPA) and the relatively long-lived excited-state absorption (ESA) of melanin are carried out in solution, and imaging capability is demonstrated in B16 cells. It is found that sepia melanin exhibits two distinct excited states with different lifetimes (one at 3 ps, one lasting hundreds of nanoseconds) when pumped at 775 nm. Its characteristic TPA/ESA enables us to image its distribution in cell samples with high resolution comparable to two-photon fluorescence microscopy (TPFM). This new technique could potentially provide valuable information in diagnosing melanoma.
We have demonstrated a new method for imaging melanin with two-color excited state absorption microscopy. If one of
two synchronized mode-locked pulse trains at different colors is intensity modulated, the modulation transfers to the
other pulse train when nonlinear absorption takes place in the medium. We can easily measure 10-6 absorption changes
caused by either instantaneous two-photon absorption or relatively long lived excited state absorption with a RF lock-in
amplifier. Eumelanin and pheomelanin exhibit similar excited state dynamics. However, their difference in excited state
absorption and ground state absorption leads to change in the phase of the transient absorption signal. Scanning
microscopic imaging is performed with B16 cells, melanoma tissue to demonstrate the 3D high resolution imaging
capability. Different melanosome samples are also imaged to illustrate the differences between eumelanin and
pheomelanin signals. These differences could enable us to image their respective distribution in tissue samples and
provide us with valuable information in diagnosing malignant transformation of melanocytes.
Even though multi-photon fluorescence microscopy offers higher resolution and better penetration depth than traditional
fluorescence microscopy, its use is restricted to the detection of molecules that fluoresce. Two-photon absorption (TPA)
imaging can provide contrast in non-fluorescent molecules while retaining the high resolution and sectioning capabilities
of nonlinear imaging modalities. In the long-wavelength water window, tissue TPA is dominated by the endogenous
molecules melanin and hemoglobin with an almost complete absence of endogenous two-photon fluorescence. A
complementary nonlinear contrast mechanism is self-phase modulation (SPM), which can provide intrinsic signatures
that can depend on local tissue anisotropy, chemical environment, or other structural properties. We have developed a
spectral hole refilling measurement technique for TPA and SPM measurements using shaped ultrafast laser pulses. Here
we report on a microscopy setup to simultaneously acquire 3D, high-resolution TPA and SPM images. We have
acquired data in mounted B16 melanoma cells with very modest laser power levels. We will also discuss the possible
application of this measurement technique to neuronal imaging. Since SPM is sensitive to material structure we can
expect SPM properties of neurons to change during neuronal firing. Using our hole-refilling technique we have now
demonstrated strong novel intrinsic nonlinear signatures of neuronal activation in a hippocampal brain slice. The
observed changes in nonlinear signal upon collective activation were up to factors of two, unlike other intrinsic optical
signal changes on the percent level. These results show that TPA and SPM imaging can provide important novel
functional contrast in tissue using very modest power levels suitable for in vivo applications.
Multiphoton excitation fluorescence microscopy has proven to be a powerful method for non-invasive, in vivo, thick tissue imaging with molecular specificity. However, many important endogenous biomolecules do not fluoresce (NAD) or fluoresce with low efficiency (Melanin). In this report femtosecond pulse shaping methods are used to measure two-photon absorption (TPA) directly with very high sensitivity. Combining with the laser scanning microscope, this Two-photon Absorption Microscopy (TPAM) retains the penetration and localization advantages of two-photon fluorescence microscopy and permits direct observation of important endogenous molecular markers (melanin or hemoglobin) which are invisible in multiphoton fluorescence microscopy. We have demonstrated here for the first time that TPAM can successfully and more efficiently image melanoma cells and tissues and provide a good melanin contrast in optical sectioning of the melanoma lesions which are comparable to pathological histology. Combining with the two-photon fluorescence images acquired simultaneously, the distribution patterns of the melanocytes and their intratissue behavior could be studied without cutting the lesions from patients. TPAM will undoubtedly find the applications in the clinical diagnosis and biomedical research.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.