Multimodal 3D photoacoustic remote sensing and confocal fluorescence microscopy imaging

Abstract

. Significance: Complementary absorption and fluorescence contrast could prove useful for a wide range of biomedical applications. However, current absorption-based photoacoustic microscopy systems require the ultrasound transducers to physically touch the samples, thereby increasing contamination and limiting strong optical focusing in reflection mode. Aim : We sought to develop an all-optical system for imaging cells and tissues using the three combined imaging modalities: photoacoustic remote sensing (PARS), epifluorescence, and confocal laser scanning microscopy (CLSM). Approach: A PARS subsystem with ultraviolet excitation was used to obtain label-free absorption-contrast images of nucleic acids in ex vivo tissue samples. Co-integrated epifluorescence and CLSM subsystems were used to verify the 2D and 3D nuclei distribution. Results : Complementary absorption and fluorescence contrast were demonstrated in phantom imaging experiments and subsequent cell and tissue imaging experiments. Lateral and axial resolution of ultraviolet-PARS (UV-PARS) is shown to be 0.39 and , respectively, with 266-nm light. CLSM lateral and axial resolution was measured as 0.97 and , respectively. This resolution is sufficient to image individual cell layers with fine optical sectioning. UV-PARS images of cell nuclei are validated in thick tissue using CLSM. Conclusions : Multimodal absorption and fluorescence contrast are obtained with a non-contact all-optical microscopy system for the first time and utilized to obtain images of cells and tissues with subcellular resolution.

Photoacoustic imaging plus X: a review

“…In addition to combining with ultrasound imaging, PAI has also been integrated with other optical imaging modalities, such as optical coherence tomography (OCT) and fluorescence imaging. In a study by Brendon et al., 133 an innovative all-optical microscopy system was introduced; it combined PA remote sensing (PARS), fluorescence microscopy, and confocal laser scanning microscopy. The system overcame the limitation of physical tissue contact required by ultrasound probes.…”

Common methods in mitochondrial research (Review)

“…In most cases, microscopy can be used to observe and analyze two-dimensional mitochondrial morphologies and quantities. However, although this method is suitable for analyzing adherent cells with flat morphology, it is not suitable for thicker cells (77)(78)(79)(80)(81)(82)(83). Three-dimensional confocal microscopy can be used to observe mitochondrial morphology by observing specifically labeled mitochondrial proteins at the 3d level (84)(85)(86)(87).…”

Imaging approaches for monitoring three‐dimensional cell and tissue culture systems

“…NAD(P)H FLIM data of label-free samples has also been demonstrated for detection of Microglia in a heterogeneous sample [256]. Similar approaches had been implemented for classification, such as healthy or diseased cells from image flow cytometry data [257], nuclei detection from phase information [258] and cell s confocal [246,247] ultrasound [248] Raman [249] OCT [243], PTI Raman [221,222] PAM [223] OCT [224,225] a Typical values, excluding super-resolution techniques. Values dependent on NA, and illumination wavelengths.…”