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Abnormal physiological processes and diseases can lead to content or activity fluctuations of biocomponents in organelles and whole blood. However, precise monitoring of these abnormalities remains extremely challenging due to the insufficient sensitivity and accuracy of available fluorescence probes, which can be attributed to the background fluorescence arising from two sources, 1) biocomponent autofluorescence (BCAF) and 2) probe intrinsic fluorescence (PIF). To overcome these obstacles, we have re‐engineered far‐red to NIR II rhodol derivatives that possess weak BCAF interference. And a series of "zero" PIF sensing‐platforms were created by systematically regulating the open‐loop/spirocyclic forms. Leveraging these advancements, we devised various ultra‐sensitive NIR indicators, achieving substantial fluorescence boosts (190 to 1300‐fold). Among these indicators, 8‐LAP demonstrated accurate tracking and quantifying of leucine aminopeptidase (LAP) in whole blood at various stages of tumor metastasis. Furthermore, coupling 8‐LAP with an endoplasmic reticulum‐targeting element enabled the detection of ERAP1 activity in HCT116 cells with p53 abnormalities. This delicate design of eliminating PIF provides insights into enhancing the sensitivity and accuracy of existing fluorescence probes toward the detection and imaging of biocomponents in abnormal physiological processes and diseases. [ABSTRACT FROM AUTHOR]

Fluorescence imaging utilizing traditional organic fluorophores is extensively applied in both cellular and in vivo studies. However, it faces significant obstacles, such as low signal‐to‐background ratio (SBR) and spurious positive/negative signals, primarily due to the facile diffusion of these fluorophores. To cope with this challenge, orderly self‐assembled functionalized organic fluorophores have gained significant attention in the past decades. These fluorophores can create nanoaggregates via a well‐ordered self‐assembly process, thus prolonging their residency time within cells and in vivo settings. The development of self‐assembled‐based fluorophores is an emerging field, and as such, in this review, we present a summary of the progress and challenges of self‐assembly fluorophores, focusing on their development history, self‐assembly mechanisms, and biomedical applications. We hope that the insights provided herein will assist scientists in further developing functionalized organic fluorophores for in situ imaging, sensing, and therapy. [ABSTRACT FROM AUTHOR]

Comprehensive Summary: Fluorophores play a significant role in achieving high‐contrast imaging in complex physiological environment. Herein, we present the results of our studies on the development of various fluorophores, such as anti‐solvatochromic fluorophores, long wavelength emission two‐photon fluorophores, large Stokes shift fluorophores and solid‐state fluorophores, to achieve high‐contrast bioimaging in physiological environments. In this study, we have primarily focused on the design, optical performance, imaging mechanisms, and the applications of these fluorophores with respect to complex physiological conditions. What is the most favorite and original chemistry developed in your research group? Orderly assembly of small‐molecule dyes and their applications for long‐term in situ bioimaging. How do you get into this specific field? Could you please share some experiences with our readers? We have long been engaged in the field of fluorescence imaging for biomedical applications, and realized that obtaining the target's in situ information is urgent and challenging. Our exploration revealed that the orderly assembly of specific molecular dyes is an effective strategy to overcome this problem. Keeping curiosity and thinking is the key to solving problems in scientific research. What is the most important personality for scientific research? Clear mind and perseverance. What's your hobby? Communicating with Youngs. How do you keep balance between research and family? To make a proper allotment of time. How do you supervise your students? Let them know what they want and fight for it. [ABSTRACT FROM AUTHOR]