Broad and safe access to ultrafast laser technology has been hindered by the absence of optical fiber-delivered pulses with tunable central wavelength, pulse repetition rate, and pulse width in the picosecond–femtosecond regime. To address this long-standing obstacle, we developed a reliable accessory for femtosecond ytterbium fiber chirped pulse amplifiers, termed a fiber-optic nonlinear wavelength converter (FNWC), as an adaptive optical source for the emergent field of femtosecond biophotonics. This accessory empowers the fixed-wavelength laser to produce fiber-delivered ∼20 nJ pulses with central wavelength across 950 to 1150 nm, repetition rate across 1 to 10 MHz, and pulse width across 40 to 400 fs, with a long-term stability of >2000 h. As a prototypical label-free application in biology and medicine, we demonstrate the utility of FNWC in real-time intravital imaging synergistically integrated with modern machine learning and large-scale fluorescence lifetime imaging microscopy.
SignificanceLabel-free nonlinear optical microscopy has become a powerful tool for biomedical research. However, the possible photodamage risk hinders further clinical applications.AimTo reduce these adverse effects, we constructed a new platform of simultaneous label-free autofluorescence multi-harmonic (SLAM) microscopy, featuring four-channel multimodal imaging, inline photodamage monitoring, and pulse repetition-rate tuning.ApproachUsing a large-core birefringent photonic crystal fiber for spectral broadening and a prism compressor for pulse pre-chirping, this system allows users to independently adjust pulse width, repetition rate, and energy, which is useful for optimizing imaging conditions towards no/minimal photodamage.ResultsIt demonstrates label-free multichannel imaging at one excitation pulse per image pixel and thus paves the way for improving the imaging speed by a faster optical scanner with a low risk of nonlinear photodamage. Moreover, the system grants users the flexibility to autonomously fine-tune repetition rate, pulse width, and average power, free from interference, ensuring the discovery of optimal imaging conditions with high SNR and minimal phototoxicity across various applications.ConclusionsThe combination of a stable laser source, independently tunable ultrashort pulse, photodamage monitoring features, and a compact design makes this new system a robust, powerful, and user-friendly imaging platform.
Quality control in molecular optical sectioning microscopy is indispensable for transforming acquired digital images from qualitative descriptions to quantitative data. Although numerous tools, metrics, and phantoms have been developed, accurate quantitative comparisons of data from different microscopy systems with diverse acquisition conditions remains a challenge. Here, we develop a simple tool based on an absolute measurement of bulk fluorophore solutions with related Poisson photon statistics, to overcome this obstacle. Demonstrated in a prototypical multiphoton microscope, our tool unifies the unit of pixelated measurement to enable objective comparison of imaging performance across different modalities, microscopes, components/settings, and molecular targets. The application of this tool in live specimens identifies an attractive methodology for quantitative imaging, which rapidly acquires low signal-to-noise frames with either gentle illumination or low-concentration fluorescence labeling.
KEYWORDS: Tumors, Cell death, In vivo imaging, Fluorescence lifetime imaging, Fluorescence intensity, Photon counting, Fluorescence, Time correlated single photon counting, In vitro testing, Single photon detectors
Multiphoton fluorescence lifetime imaging microscopy (FLIM) is used to collect label-free metabolic information from biological samples via autofluorescence lifetime imaging of reduced nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate (NAD(P)H). However, FLIM has traditionally been limited by slow acquisition due to the limited bandwidth of analog electronics that perform photon counting and time-tagging. This slow acquisition has restricted the applicability of multiphoton FLIM of NAD(P)H by impeding the ability to accurately study biological problems that require characterization of fast dynamics. Faster image acquisition can be achieved by directly digitizing the amplified output of a hybrid photodetector and computationally determining photon counts via the Single- and multi-photon PEak Event Detection (SPEED) algorithm. This method, bypassing the limited-bandwidth analog electronics used for photon counting and time-tagging of photons in traditional FLIM, enables fast photon counting capabilities which are well suited for fast, high-dynamic range biological processes such as metabolic changes during apoptosis. Here, we utilize this technology to examine fast dynamics of apoptosis in 2D culture of normal and cancerous human breast cell lines, rat mammary tumor tissue-derived organoids, and in vivo rat mammary tumors. Results indicate that apoptosis-related metabolic dynamics are biological model-dependent and based on local pharmacokinetics, with tumor derived organoids in Matrigel showing a significantly slower response than in vivo or in vitro 2D cell models. Future work should carefully consider these implications when determining which tumor model to use for experimentation and should improve tumor models to better represent in vivo tumor apoptosis dynamics.
It is challenging to image sparse samples with fast FLIM since the laser clock is not directly sampled. Instead, it is inferred from the cumulative photon statistics of an entire line. We present a method for registering the photon arrival times to the excitation using time-domain multiplexing of the laser clock to ensure accuracy for fewer photons for fast FLIM. Our technique also does not add to the existing bottleneck of data throughput and eliminates the errors in registering the photons from different frames, yielding more accurate images at faster frame rates.
For high-signal-rate nonlinear microscopy using an analog PMT to detect multiple photons per excitation pulse, the conversion of analog output to photon counts is required to realize quantitative imaging. To enable this ability, we propose a method for photon counting with analog photodetection based on “imaging” of standard dye solutions and a mathematical model of Poisson photon statistics. Using this method, we not only enable the quantification across different types of PMTs from different setups, but also quantify the channel leakage for multichannel or multispectral imaging systems. This method paves the way for analog PMTs in high-signal-rate quantitative imaging of either label-free or labeled samples.
Label-free nonlinear optical microscopy has become a powerful tool for biomedical research. However, the low imaging speed and the accompanying photodamage risk hinder further clinical applications. To reduce these adverse effects, in this study, we constructed a new generation of simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy, featuring high-speed, multimodal imaging, monitorable-photodamage, and tunable ultrashort pulses. By using birefringent photonic crystal fiber and a pulse shaper, this system has the ability to allow users to independently adjust repetition rate, pulse width, and average power without overlapping interference, and can realize multiphoton generation in each channel from a single excitation pulse. These outstanding advantages represent a powerful and user-friendly imaging platform.
The goal towards developing faster and minimally damaging quantitative MPM involves keeping the exposure time per pixel as low as possible. Both the excitation and detection parameters affect the number of measurements required for quantification and therefore, the overall efficiency of the setup. We present our estimation for the minimum number of photons needed and our ability to resolve them to quantify label-free MPM of NAD(P)H and FAD using their intensity, fluorescence lifetime, and optical redox ratio. We not only utilize these results to guide the imaging parameters for in vitro and ex vivo studies, but also to normalize the results from different setups.
Pulsatile signals from the cutaneous blood flow could be informative for evaluating the health condition of an individual. One of the popular optical measuring devices, photoplethysmogram (PPG) is often used to detect the pulse signal from skin. However, the origin of the PPG signal still remains controversial. Benefiting from the non-invasive, label-free, 3D imaging tool, optical coherence tomography (OCT) is able to capture the intrinsic tissue signals at different penetration depth in high spatial and temporal resolution. Periodic pulse signal was observed by taking advantage of the optical microangiography (OMAG) algorithm which is sensitive to the motion of blood flow. The pulsatile pattern from the capillary and arteriole was successfully differentiated and their morphology showed distinct property at different local blood pressure. The pulse signal from the arteriole is more consistent and has similar waveform as the PPG signals. The result indicated that the PPG signal could be deceive by the mixing signal from the capillary bed and arterioles since it measures the total blood volume change in the plexuses. This study may shed some new light on understanding the mechanical property of how blood travel through different types of vasculature networks and elucidate its potential application in disease assessments.
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.