Microscopic optical inspection techniques have enabled a multitude of breakthroughs in biological research and clinical diagnostics over the past few centuries. From cytological assessment of mitotic cells in oncology 1, to structural imaging of neurons in biological research 2, optical microscopy has provided valuable insights into the composition, structure, and function of tissues and cells 3. Modern optical techniques broadly leverage scattering and absorption events to deliver visualizations in biological media, where each mechanism imparts different characteristics to the respective modalities.
Scattering-based modalities such as OCT 4, and darkfield microscopy 5, leverage scattering interactions to provide visualization of structural composition 6. Scattering contrast in biological media such as skin, brain, and fatty tissues tend to not exhibit significant variation with wavelength 6. This hampers the capabilities of label-free scattering-based microscopes in biological specimens. For this reason, many applications require ex-vivo sample preparation coupled with exogenous dyes to provide chromophore-specific visualizations in biological samples. A common example of this is hematoxylin and eosin (H&E) staining of tissues frequently used during histological assessment 7. Unfortunately, generating stained preparations may be undesirable as it can be time consuming and may alter biochemistry and biological structures. For example, when preparing tissues for H&E staining, lipids structures are removed completely 7.
Absorption based modalities such as photoacoustic microscopy (PAM) 8, fluorescence 9,10, and multiphoton fluorescence 10, leverage absorption interactions to provide visualizations of chromophores. Absorption contrast in biological tissues tends to be highly chromophore specific, where most molecules present unique absorption spectra 6,11. Therefore, optical absorption microscopy is particularly attractive for label-free imaging of biological samples, where endogenous absorption profiles can be leveraged to provide information on properties such as chemical bonding 12, sample composition 13, and temperature 14.
Absorbed energy may be dissipated by chromophores through either optical radiation (radiative) or non-radiative relaxation, most absorption imaging mechanisms may be broadly classified into these corresponding subcategories. During non-radiative relaxation, absorbed optical energy is converted into heat. If the excitation event is sufficiently rapid, this heating may cause thermoelastic expansion resulting in localized photoacoustic pressures 15. These temperature rises are leveraged in photothermal modalities 16, while the pressure rises are leveraged in photoacoustic imaging 8,11,15. In traditional PAM, pressure waves are allowed to propagate through the sample as ultrasound waves, which are then detected at the sample surface with ultrasound transducers 8,11,15. PAM has demonstrated label-free visualizations of a wide range of endogenous chromophores, including DNA 17,18, lipids 19–21, and hemeproteins 22,23. During radiative relaxation, absorbed optical energy is released through the emission of photons. Generally, emitted photons exhibit a different energy level compared to the absorbed photons. Radiative relaxation contrast encompasses a variety of mechanisms such as stimulated Raman scattering 24, fluorescence 9,10, and multiphoton fluorescence 10. For example, in multiphoton fluorescence imaging, the energy of two or more photons is absorbed then released as a higher energy fluorescent photon. In practice, a range of biomolecules including NADPH, collagen, and elastin, have been visualized label-free with such radiative absorption techniques 25.
To further provide label-free visualizations of biomolecules in complex media it would be desirable to have a technique which could capitalize on the advantages of scattering contrast and both radiative and non-radiative absorption modalities. Ideally, capturing all contrasts simultaneously. Here, a second-generation of Photoacoustic Remote Sensing (PARS) microscopy is presented, entitled total-absorption PARS (TA-PARS), which facilitates label-free non-contact capture of scattering, radiative absorption, and non-radiative absorption simultaneously. Unlike traditional radiative or non-radiative absorption modalities where contrast may be dictated by efficiency factors such as the photothermal conversion efficiency or fluorescence quantum yield, the TA-PARS may capture nearly all the optical properties of a chromophore, providing simultaneous sensitivity to most chromophores. By extension, capturing both radiative and non-radiative absorption fractions may also yield additional information. The ratio of the two absorption fractions is expected to provide an additional chromophore specific metric. This ratio of the radiative to non-radiative absorption fractions is proposed as the quantum efficiency ratio (QER). In biomolecules such as collagen, and DNA, the QER may enhance chromophore specific recovery. To the best of our knowledge, this array of imaging contrast (optical scattering, radiative absorption, non-radiative absorption) has not yet been provided by any other independent imaging modality (see more information in Supplementary Information Sect. 1: Table 1).
In TA-PARS a picosecond scale pulsed excitation laser elicits radiative and nonradiative (thermal and pressure) perturbations in a sample. The thermal and pressure perturbations generate corresponding modulations in the local optical properties. A secondary probe beam co-focused with the excitation captures the non-radiative absorption induced modulations to the local optical properties as changes in backscattering intensity (Fig. 1) 23,26. These backscatter modulations are then directly correlated to the local non-radiative absorption contrast 23,26. By the nature of the probe architecture, the unperturbed backscatter (pre-excitation event) also captures the scattering contrast as seen by the probe beam (Fig. 1) 27. Unlike traditional photoacoustic methods, rather than relying on the pressure waves to propagate through the sample before detection via acoustic transducer, the TA-PARS probe may instantaneously detect the induced modulations at the excited location 23,26. Therefore, TA-PARS offers non-contact operation, facilitating imaging of delicate, and sensitive samples, which would otherwise be impractical to image with traditional contact-based PAM methods. Since TA-PARS, like PAM, relies only on the generation of heat and subsequently pressure to provide contrast, the absorption mechanism is non-specific, and highly sensitive to small changes in relative absorption 23,26. This allows any variety of absorption mechanisms such as vibrational absorption 12, stimulated Raman absorption 28, and electronic absorption 15 to be detected with PARS and PAM. Previously, PARS has demonstrated label-free non-radiative absorption contrast of hemoglobin, DNA, RNA 18,29−33, lipids 20,21, and cytochromes 29,30, in specimens such as chicken embryo models 20, resected tissue specimens 29, and live murine models 23,34,35. In TA-PARS, a unique secondary detection pathway captures radiative relaxation contrast, in addition to the non-radiative absorption. The radiative absorption pathway was designed to broadly collect all optical emissions at any wavelength of light, excluding the excitation and detection. As a result, the radiative detection pathway captures non-specific optical emissions from the sample. Radiative relaxation signals may then be attributed to any number of radiative effects such as spontaneous Raman scattering, stimulated Raman scattering, autofluorescence, multiphoton autofluorescence, etc. The potential interactions captured during a TA-PARS excitation event are outlined in Fig. 1.
Previously, multimodal fluorescence microscopy and PAM have leveraged similar absorption mechanisms, capturing both radiative fluorescence contrast and non-radiative photoacoustic contrast 36–40. Such multimodal PAM and fluorescence microscopy systems have been inherently complicated, due to the acoustic detection mechanism of traditional PAM. In many cases, it may be challenging to guide the excitation pulses and emitted radiative relaxation light through or around the acoustic transducer. The need for acoustic coupling may also render these multimodal systems impractical for imaging delicate, and sensitive samples. Recently, Zhou et al. showed a non-contact dual-modality PARS and dye-based fluorescence microscope 41. The dual-modal system used Rhodamine B dye to generate fluorescence contrast to complement the PARS visualizations. In contrast, the TA-PARS captures simultaneous complementary radiative and non-radiative contrast label-free from a range of endogenous chromophores.
To improve the sensitivity of the TA-PARS and facilitate the detection of radiative absorption contrast, a variety of systematic changes are introduced compared to previously reported PARS systems. The presented TA-PARS features 266 nm and 515 nm excitation, providing sensitivity to DNA, heme proteins, NADPH, collagen, elastin, amino acids, and a variety of fluorescent dyes. The TA-PARS features a specific optical pathway with dichroic filters and avalanche photodiode, to isolate and detect the radiative absorption contrast. The TA-PARS probe beam was implemented with a 405 nm laser diode. This probe wavelength provides improved scattering resolution, which improves the confocal overlap between the PARS excitation and detection spots on the sample. Combined with a circulator-based probe beam pathway and avalanche photodetector, the TA-PARS provides improved sensitivity compared to previous implementations. The visible wavelength probe also provides improved compatibility between the visible and UV excitation wavelengths. The prevalence of chromatic aberrations was suppressed when using achromatic refractive optics by reducing the disparity in the excitation and detection wavelengths, as opposed to previous comparable NIR based PARS systems 18, 29–34,42.
The TA-PARS imaging contrast was explored in simple dye samples, unprocessed resected tissue specimens, and sections of preserved human tissues. The TA-PARS captures label-free features such as adipocytes, fibrin, connective tissues, neuron structures, and cell nuclei. Visualizations of intranuclear structures are captured with sufficient clarity and contrast to identify individual atypical nuclei. One potential proposed application of TA-PARS, label-free histological imaging, was explored in unstained sections of human tissues. TA-PARS visualization fidelity is assessed through one-to-one comparison against traditional H&E-stained images. The TA-PARS total-absorption and QER contrast mechanisms are also validated in a series of dye and tissue samples. Results show high correlation between radiative relaxation characteristics and TA-PARS-measured QER in a variety of fluorescent dyes, and tissues. These QER visualizations are used to extract regions of specific biomolecules such as collagen, elastin, and nuclei in tissue samples. This enables realization of a broadly applicable high resolution absorption contrast microscope system. The TA-PARS may provide unprecedented label-free contrast in any variety of biological specimens, providing otherwise inaccessible visualizations.