This article reviews the state of the art of ultrafast transient absorption microscopy, discusses current experimental concepts and highlights future challenges. The advantages of transient absorption microscopy over other micro-spectroscopic techniques are its high optical resolution combined with high temporal resolution as well as its ability to study non-fluorescent and weakly fluorescent molecular species and to probe excited-state processes. In conventional transient absorption spectroscopy the spectroscopic information usually presents a spatial average over the focal spot of the typically weakly focused probe beam. Transient absorption microscopy, however, enables investigations of the excited state dynamics in individual microscopic areas of a sample. Hence, the technique does not only yield detailed morphological information based on a label-free molecular contrast, but also gives insight into the ultrafast morphology-dependent photoinduced processes in heterogeneous samples. Different variations of transient absorption microscopy have found a number of applications ranging from material sciences to biology, which are discussed in this review together with different setup modifications and approaches towards transient absorption spectroscopy with spatial resolution below the diffraction limit.

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An in cellulo study of the ultrafast excited state processes in the paradigm molecular light switch [Ru(bpy)2dppz]2+ by localized pump-probe spectroscopy is reported for the first time. The localization of [Ru(bpy)2dppz]2+ in HepG2 cells is verified by emission microscopy and the characteristic photoinduced picosecond dynamics of the molecular light switch is observed in cellulo. The observation of the typical phosphorescence stemming from a 3MLCT state suggests that the [Ru(bpy)2dppz]2+ complex intercalates with the DNA in the nucleus. The results presented for this benchmark coordination compound reveal the necessity to study the photoinduced processes in coordination compounds for intracellular use, e.g. as sensors or as photodrugs, in the actual biological target environment in order to derive a detailed molecular mechanistic understanding of the excited-state properties of the systems in the actual biological target environment.

Two‐photon laser writing is used here to fabricate 3D proteinaceous microstructures with photothermal functionality in the near‐infrared spectral region and tunable elasticity. The photo‐cross‐linking is initiated in bovine serum albumin (BSA) by rose bengal or methylene blue and the photo‐thermal effect arises from gold non‐spherically symmetric nanoparticles dispersed in the ink. Massive energy transfer of the plasmonic resonances of the gold nanoparticles to methylene blue prevents effective photo‐crosslinking of BSA. However, stable microstructures with photo‐thermal functionality can be fabricated in the rose bengal proteinaceous inks. On these microstructures, with a gold atom concentration as low as 1% w/w, a highly localized temperature increase can be quickly (≅1 s) reached and maintained under continuous wave laser irradiation at 800 nm. The photothermal efficiency under continuous wave laser irradiation depends on the thickness of the microstructure and can reach 12.2 ± 0.4 °C W−1 These proteinaceous microstructures represent therefore a promising platform for future applications in the fields like physical stimulation of cells for regenerative nanomedicine.

Spontaneous Raman microscopy reveals the chemical composition of a sample in a label-free and non-invasive fashion by directly measuring the vibrational spectra of molecules. However, its extremely low cross section prevents its application to fast imaging. Stimulated Raman scattering (SRS) amplifies the signal by several orders of magnitude thanks to the coherent nature of the nonlinear process, thus unlocking high-speed microscopy applications that provide analytical information to elucidate biochemical mechanisms with subcellular resolution. Nevertheless, in its standard implementation, narrowband SRS provides images at only one frequency at a time, which is not sufficient to distinguish constituents with overlapping Raman bands. Here, we report a broadband SRS microscope equipped with a home-built multichannel lock-in amplifier simultaneously measuring the SRS signal at 32 frequencies with integration time down to 44 µs, allowing for detailed, high spatial resolution mapping of spectrally congested samples. We demonstrate the capability of our microscope to differentiate the chemical constituents of heterogeneous samples by measuring the relative concentrations of different fatty acids in cultured hepatocytes at the single lipid droplet level and by differentiating tumor from peritumoral tissue in a preclinical mouse model of fibrosarcoma.

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We introduce a broadband coherent anti-Stokes Raman scattering (CARS) microscope based on a 2-MHz repetition rate ytterbium laser generating 1035-nm high-energy (≈µJ level) femtosecond pulses. These features of the driving laser allow producing broadband red-shifted Stokes pulses, covering the whole fingerprint region (400–1800 cm−1), employing supercontinuum generation in a bulk crystal. Our system reaches state-of-the-art acquisition speed (<1 ms/pixel) and unprecedented sensitivity of ≈14.1 mmol/L when detecting dimethyl sulfoxide in water. To further improve the performance of the system and to enhance the signal-to-noise ratio of the CARS spectra, we designed a convolutional neural network for spectral denoising, coupled with a post-processing pipeline to distinguish different chemical species of biological tissues.

The first key step in the detection and classification of most cancers is the microscopic assessment of thin tissue slices, the so-called "histopathology". This procedure is still nowadays, similarly to 150 years ago, performed by staining the tissue with two or more dyes able to bind to specific biological structures, followed by visual inspection by the histopathologist under the bright-field optical microscope. This approach involves long manual procedures which can be accompanied by human errors, subjectivity, and lack of reproducibility. Vibrational microscopies are capable of directly providing chemical and biomolecular information on tissues, identifying them through their fingerprint vibrational spectra without the need of staining and, thus, constitute powerful tools for label-free and objective tumour identification. The two most established techniques, spontaneous Raman microscopy and infrared absorption microscopy, suffer, respectively, from long acquisition times and low spatial resolution. These limitations can be overcome by novel and more technically demanding approaches such coherent Raman scattering and photothermal infrared microscopy. Here we present an extended overview of the major advances in the field of vibrational imaging for cancer diagnosis. We start from a detailed description of the different technologies and then present examples of their applications to tissue imaging for cancer assessment. We critically compare the presented approaches, discussing the steps required to bring these powerful technologies from bench to bedside.