Doppler Raman (DR) spectroscopy is a coherent Raman technique that combines impulsive Raman excitation with novel frequency shift detection to enable high-sensitivity Raman spectroscopy in the biological fingerprint region (500cm-1-1500cm-1) and the low frequency regime from 10cm-1 – 500cm-1. Using DR, we demonstrate nonresonant Raman spectroscopy on a suite of biologically significant targets involved in cell respiration including cytochrome c, adenosine triphosphate (ATP), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NADH). High-sensitivity detection of low-to-medium frequency Raman vibrational modes may provide a tool to monitor states of cell respiration along with large molecular structural changes such as protein conformational dynamics.
Optical microscopy and spectroscopy are widely used in multiple research areas relating to biology. Label-free spectroscopy and imaging are valuable tools that permit interrogation of biological samples without the need for exogenous labels, allowing for investigation of unperturbed biological systems. We demonstrate a coherent Raman technique called Doppler Raman (DR) spectroscopy which combines impulsive excitation with a novel frequency shift detection scheme for rapid, high sensitivity detection of low to medium frequency vibrational modes from 10-1800cm-1. Briefly, the DR spectroscope is a pump-probe system where the pump beam generates a time-varying index of refraction proportional to the Raman response of the sample. The time-delayed probe beam undergoes a frequency shift in the sample due to the time-varying index of refraction that is resolved using a novel high-sensitivity detection scheme. Other coherent Raman techniques such as Stimulated Raman Scattering (SRS) and Coherent Anti-Stokes Raman Spectroscopy (CARS) have been used to provide sensitive, label-free contrast for an array of biological targets, but their ability to detect low frequency vibrational modes is limited. Biologically significant targets like cytochrome c (740-760cm-1), DNA (782, 788, 1095cm-1), hydroxyapatite, and numerous pharmaceutical drugs exhibit rich Raman spectra across a range of low frequency modes below the well-known “fingerprint region”. Additionally, many proteins like hemoglobin, insulin, and bovine serum albumin have breathing modes below 50cm-1. Sensitive detection of low-frequency Raman vibrational modes unlocks a suite of potential biological and chemical dynamics like protein conformational changes and protein super complex formation.
Ultrafast pump-probe microscopy enables visualization of non-fluorescent materials in biological tissue, such as melanin and hemoglobin. Whereas transient absorption has been primarily a physical chemistry technique, used to gain insight into molecular and electronic structure, pump-probe microscopy represents a paradigm shift in translating transient absorption into an analytical technique, which can clearly resolve pigments with nearly indistinguishable linear absorption spectra. Extending this technique to other important targets, such as mitochondrial respiratory chain hemes, will require new laser sources and new data processing techniques to estimate heme content from the pump-probe response. We will present recent developments on both of these fronts. The laser system we have developed to elicit a pump probe response of respiratory chain hemes is based on an amplified Yb:fiber ultrafast laser that uses modest spectral broadening followed by sum frequency generation to produce a tunable pulse pair in the visible region. Wavelength tuning is accomplished by changing quasi-phase matching conditions. We will present preliminary imaging data in addition to discussing management of sample heating problems that arise from performing transient absorption measurements at the high repetition rates needed for imaging microscopy. In the second part of the talk, we will present the use of regularized and non-negative least squares fitting, along with feature-preserving noise removal to estimate composition of a pixel from its pump-probe response.
Current label-free metabolic microscopy techniques are limited to obtaining contrast from fluorescent molecules NAD(P)H and FAD+, and are unable to determine redox state along the mitochondrial respiratory chain itself. The respiratory chain electron carriers do not fluoresce, but some are heme proteins that have redox-dependent absorption spectra. The most prominent of these, cytochrome c, has been extensively characterized by transient absorption spectroscopy, which suggests that pump-probe measurements in the vicinity of 450 - 600 nm can provide strong contrast between its redox states. Motivated by the success of pump-probe microscopy targeting another heme protein, hemoglobin, we seek to extend the technique to the cytochromes, with the ultimate goal of dissecting respiratory chain function of individual cells in live tissue. To that end, we have developed a new optical system producing ultrafast, visible, independently-tunable pulse pairs via sum-frequency generation of nonlinearly broadened pulses in periodically-poled lithium niobate. The system is pumped by a homebuilt fiber-based oscillator/amplifier emitting 1060 nm pulses at 1.3 W (63 MHz repetition rate), and produces tunable pulses in the vicinity of 488 and 532 nm. Pump-probe spectroscopy of cytochrome c with this source reveals differences in excited-state absorption relaxation times between redox states. Though redox contrast is weak with this setup, we argue that this can be improved with a resonant galvo-scanning microscope. Moreover, pump-probe images were acquired of brown adipose tissue (which contains dense mitochondria), demonstrating label-free contrast from excited-state absorption in respiratory chain hemes.
We discuss the theoretical framework of a new method of performing spectroscopy: labeling a unique intensity modulation frequency onto the optical frequencies of an incident or illumination power spectrum. In a manner similar to Fourier transform spectroscopy, we rescale the optical angular frequencies of the power spectrum down to readily measured frequencies on a square-law detector enabling rapid spectral update rates on a single element detector.
MultiPhoton SPatIal Frequency modulated Imaging (MP-SPIFI) has recently demonstrated the ability to simultaneously obtain super-resolved images in both coherent and incoherent scattering processes — namely, second harmonic generation and two-photon fluorescence, respectively.1 In our previous analysis, we considered image formation produced by the zero and first diffracted orders from the SPIFI modulator. However, the modulator is a binary amplitude mask, and therefore produces multiple diffracted orders. In this work, we extend our analysis to image formation in the presence of higher diffracted orders. We find that tuning the mask duty cycle offers a measure of control over the shape of super-resolved point spread functions in an MP-SPIFI microscope.
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