title: Spatial filter based bessel-like beam for improved penetration depth imaging in fluorescence microscopy.
abstract: Monitoring and visualizing specimens at a large penetration depth is a challenge. At depths of hundreds of microns, several physical effects (such as, scattering, PSF distortion and noise) deteriorate the image quality and prohibit a detailed study of key biological phenomena. In this study, we use a Bessel-like beam in-conjugation with an orthogonal detection system to achieve depth imaging. A Bessel-like penetrating diffractionless beam is generated by engineering the back-aperture of the excitation objective. The proposed excitation scheme allows continuous scanning by simply translating the detection PSF. This type of imaging system is beneficial for obtaining depth information from any desired specimen layer, including nano-particle tracking in thick tissue. As demonstrated by imaging the fluorescent polymer-tagged-CaCO₃ particles and yeast cells in a tissue-like gel-matrix, the system offers a penetration depth that extends up to 650 µm. This achievement will advance the field of fluorescence imaging and deep nano-particle tracking.
authors.affiliation: Nanobioimaging Laboratory, Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Subhajit B
authors.initials: SB
authors.lastname: Purnapatra
authors.forename: Sampa
authors.initials: S
authors.lastname: Bera
authors.forename: Partha Pratim
authors.initials: PP
authors.lastname: Mondal
mesh_terms: D008856:Microscopy, Fluorescence / Q000295:instrumentation* / Q000379:methods*; D053758:Nanoparticles; D012441:Saccharomyces cerevisiae / Q000166:cytology*