Photothermal Confocal Spectromicroscopy of Multiple Cellular Chromophores and Fluorophores

Abstract

Confocal fluorescence microscopy is a powerful biological tool providing high-resolution, three-dimensional (3D) imaging of fluorescent molecules. Many cellular components are weakly fluorescent, however, and thus their imaging requires additional labeling. As an alternative, label-free imaging can be performed by photothermal (PT) microscopy (PTM), based on nonradiative relaxation of absorbed energy into heat. Previously, little progress has been made in PT spectral identification of cellular chromophores at the 3D microscopic scale. Here, we introduce PTM integrating confocal thermal-lens scanning schematic, time-resolved detection, PT spectral identification, and nonlinear nanobubble-induced signal amplification with a tunable pulsed nanosecond laser. The capabilities of this confocal PTM were demonstrated for high-resolution 3D imaging and spectral identification of up to four chromophores and fluorophores in live cells and Caenorhabditis elegans. Examples include cytochrome c, green fluorescent protein, Mito-Tracker Red, Alexa-488, and natural drug-enhanced or genetically engineered melanin as a PT contrast agent. PTM was able to guide spectral burning of strong absorption background, which masked weakly absorbing chromophores (e.g., cytochromes in the melanin background). PTM provided label-free monitoring of stress-related changes to cytochrome c distribution, in C. elegans at the single-cell level. In nonlinear mode ultrasharp PT spectra from cyt c and the lateral resolution of 120 nm during calibration with 10-nm gold film were observed, suggesting a potential of PTM to break through the spectral and diffraction limits, respectively. Confocal PT spectromicroscopy could provide a valuable alternative or supplement to fluorescence microscopy for imaging of nonfluorescent chromophores and certain fluorophores.

Multimodal label-free microscopy

“…70 The heating e®ects of the excitation light can also be measured through photothermal imaging, which detects the local modi¯cations of the refractive index. 71 While this approach has often beem used with nanoparticles to enhance the heating e®ects, 72 measurement have also been applied to living organisms 73 and cells, making it possible for example to image mitochondria 74 or intracellular heme. 75 …”

Photothermal Microscopy for High Sensitivity and High Resolution Absorption Contrast Imaging of Biological Tissues

“…PT spectral images were acquired by using a wavelength-tunable optical parametric oscillator as a pump beam with switching the laser wavelength and repeating scan [4,7]. However, this scheme would cause an error in performing spectral unmixing because of mechanical drift of the tissue or cell migration.…”

“…If we know the number of species in the sample and their absorption spectra, the abundance of species may be determined from spectral images (images measured with different pump wavelengths) through the spectral unmixing procedure. PT spectral images were acquired by using a wavelength-tunable optical parametric oscillator as a pump beam with switching the laser wavelength and repeating scan [4,7]. However, this scheme would cause an error in performing spectral unmixing because of mechanical drift of the tissue or cell migration.…”

“…It has been applied to visualize the distribution of endogenous chromoproteins in biological specimens such as cytochromes in mitochondria [1][2][3][4], hemoglobin in red blood cells in blood vessels [5,6], and melanin pigments in skin cancer [7]. Furthermore, gold nanoparticles have been used to identify biomolecules in antibody labelling techniques applied in cellular imaging [8][9][10][11][12].…”

Reduction of distortion in photothermal microscopy and its application to the high-resolution three-dimensional imaging of nonfluorescent tissues

“…Thus far, it has been applied to visualizing the distribution of cytochrome in mitochondria [1][2][3], the three-dimensional (3D) imaging of haemoglobin in blood vessels [4], and the high-resolution 3D imaging of melanin pigments in skin cancer [5]. Furthermore, 5-nm gold nanoparticles have been used to identify biomolecules with antibody labelling techniques that have been applied to cellular imaging by means of PT microscopy [6][7][8].…”