Transient absorption microscopy (TAM) provides imaging contrast from absorptive pigments such as hemeproteins and melanin, based on femtosecond to picosecond-timescale relaxation dynamics. TAM operates by exciting the sample with a short pump pulse, then measuring the time-dependent change in optical absorption, after excitation, with a probe pulse. Here we show that a 520nm pump and 620nm probe provides label-free imaging contrast for hemoglobin, myoglobin, and respiratory chain hemes of mitochondria with sensitivity to redox. We also introduce a simple convolutional neural network for analysis of TAM stacks. Finally, we will discuss future clinical applications to mitochondrial disease.
We present label-free transient absorption microscopy (TAM) of mitochondrial redox in live muscle fibers. Image stacks consisting of TAM images at different pump-probe delays, processed with principal component analysis (PCA) reveal picosecond timescale differences between reduced and oxidized mitochondria, in addition to heterogeneity among mitochondria within individual muscle fibers. We will discuss these findings in light of the photoinduced transient conformational and vibronic states of mitochondrial respiratory chain hemeproteins and relaxation pathways.
Cytochrome c is a small heme protein located within the inner membrane of the mitochondrion. It is an essential component of the electron transport chain. Its redox state is closely related to mitochondrion functions, such as ATP generation and oxygen utilization. Therefore, distinguishing the redox state of cytochrome c represents a potentially novel method for monitoring mitochondrial function in conditions where alterations in electron transport are implicated, such as diabetes, cancer and mitochondrial disease.
In our experiment, a pair of pump and probe ultrafast laser pulses, with duration of 750 fs, have been focused at the same spot in a tissue slice. The difference in arrival time of the two pulses can be adjusted with picosecond resolution. It is known that a pump pulse readily dissociates one of the amino acids attached to the central iron ion, but only in the reduced form of cytochrome. This alters the absorption spectrum of reduced cytochrome until the ligand reattaches in about 5ps. That absorption change can be detected by pump-probe microscopy and allows distinction between redox states.
By using 533nm pump and 490 nm probe, our system is able to distinguish redox state of cytochrome c in solution. We also have acquired pump-probe response from live insect muscle, selected for high cytochrome content and absence of hemoglobin and myoglobin, and are progressing towards redox imaging of tissue.
Pump-probe imaging obtains imaging contrast based on relaxation from a photoexcited state. This has been used as a label-free imaging technique for melanin, hemoglobin, carotenoids, and recently, mitochondrial respiratory chain components. Pump-probe provides a unique opportunity for imaging non-fluorescent molecules, which relax very rapidly (within ~ 200 femtoseconds) to a vibrationally-excited, electronic ground state. In practice, pump and probe wavelengths must be selected that provide good separation between molecules of interest. This is essentially an analytical chemistry application of a technique that is more commonly for physical chemistry. Because of this, much of the pump-probe literature on molecules of interest provide data focused on elucidating energy transfer pathways and molecular structure; predicting from these data which pump/probe wavelengths will work best in an analytical scenario is not straightforward. In this presentation, we discuss general features of pump-probe responses of non-fluorescent molecules, with an eye toward pump/probe wavelength selection. We will focus on spectral shifting and broadening that characterize the vibrationally-excited, electronic ground state, and building a simplified transient absorption model to inform imaging experiments. The end goal of this work is to separate respiratory chain heme proteins and their redox states in a label-free manner. This will lead to new methods for probing mitochondrial function with sub-cellular resolution.
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.
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