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<p>Fluorescence microscopy is indispensable for visualizing biological structures and dynamics, yet its efficiency is limited—over half of emitted photons fall outside the objective’s numerical aperture and go undetected. Here, we introduce Paired-Objectives Photon Enhancement (POPE) microscopy, which increases photon collection efficiency by up to two-fold using a single excitation source, single detector, and dual objectives. By integrating a 4f optical system with a reflective mirror positioned opposite the objective in an inverted microscope, POPE redirects a substantial portion of otherwise lost photons into the detection pathway. Compatible with super-resolution, confocal, epifluorescence, and autofluorescence modalities, POPE improves spatial resolution, acquisition speed, and signal-to-noise ratio, particularly under photon-limited conditions. It has been validated across fluorophore solutions, subcellular structures, live cells, and thick tissues, consistently enhancing imaging performance. As a modular and cost-effective upgrade for standard inverted microscopes, POPE extends access to high-sensitivity fluorescence imaging and enables new applications in cell biology, biophysics, and biomedical research.</p>

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Paired-objectives photon enhancement (POPE) microscopy: enhanced photon collection for fluorescence imaging

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月27日

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  • Mark Tingey,
  • Andrew Ruba,
  • Samuel L. Junod,
  • Coby Rush,
  • Jason Saredy,
  • William E. Brew,
  • Weidong Yang

摘要

Fluorescence microscopy is indispensable for visualizing biological structures and dynamics, yet its efficiency is limited—over half of emitted photons fall outside the objective’s numerical aperture and go undetected. Here, we introduce Paired-Objectives Photon Enhancement (POPE) microscopy, which increases photon collection efficiency by up to two-fold using a single excitation source, single detector, and dual objectives. By integrating a 4f optical system with a reflective mirror positioned opposite the objective in an inverted microscope, POPE redirects a substantial portion of otherwise lost photons into the detection pathway. Compatible with super-resolution, confocal, epifluorescence, and autofluorescence modalities, POPE improves spatial resolution, acquisition speed, and signal-to-noise ratio, particularly under photon-limited conditions. It has been validated across fluorophore solutions, subcellular structures, live cells, and thick tissues, consistently enhancing imaging performance. As a modular and cost-effective upgrade for standard inverted microscopes, POPE extends access to high-sensitivity fluorescence imaging and enables new applications in cell biology, biophysics, and biomedical research.