biostudies-literatureNedosekin DANIBIB NIH HHSNCI NIH HHS672-81https://www.ebi.ac.uk/biostudies/studies/S-EPMC3274827biostudies-literatureUnknown102(3)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 PBiophysical journalPhotothermal confocal spectromicroscopy of multiple cellular chromophores and fluorophores.PMC3274827R01 CA131164R01EB009230R01CA131164R01 EB009230R21CA139373R01 EB000873R01EB000873R21 CA139373Ayyadevara SZharov VPNedosekin DAGalanzha EIShmookler Reis RJfalsePhotothermal confocal spectromicroscopy of multiple cellular chromophores and fluorophores.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 P2012-01-01T00:00:00Z2012 Feb2025-06-01T02:33:19.481Z2025-06-01T02:33:19.481ZS-EPMC32748272232529110.1016/j.bpj.2011.12.035