Significance  In recent years, numerous researchers have dedicated themselves to developing the label-free microscopic imaging techniques, which rely on the inherent characteristics of biological tissues to provide image contrast and yield image quality comparable to traditional H&E staining. Label-free microscopic imaging techniques offer unique advantages in laboratory and clinical medical research by allowing for the acquisition of detailed tissue and cell structure information without damaging the sample. This technology has shown significant potential in intraoperative cancer diagnosis, enabling surgeons to obtain high-resolution tissue images in real-time and more accurately delineate the boundaries of cancerous lesions, thereby enhancing surgical success rates and patient prognosis. The advancement of this technology not only provides new tools for basic research but also opens new avenues for clinical applications and medical imaging.

Progress  This review introduces the fundamental principles of several label-free cancer diagnostic techniques and summarizes recent advancements in label-free pathological diagnosis across various tumors.

Photoacoustic microscopy represents a non-invasive imaging modality that harnesses the photoacoustic effect, integrating the benefits of both optical and acoustic imaging. This technique mitigates the depth limitations inherent in optical imaging while addressing the resolution and contrast constraints associated with acoustic imaging, thereby delivering high-resolution and high-contrast images of deep biological tissues, which is advantageous for cancer diagnosis and evaluation.

Optical coherence tomography (OCT) is a high-resolution, non-invasive optical imaging technique extensively utilized in ophthalmology and other medical domains. OCT employs a broadband light source and a Michelson interferometer to capture tomographic images of biological tissues via low-coherence light interference. OCT provides real-time, non-destructive imaging capabilities, with subdivisions including time-domain OCT (TD-OCT) and Fourier-domain OCT (FD-OCT). FD-OCT facilitates faster imaging speeds through the elimination of mechanical scanning.

Coherent Raman scattering (CRS) involves inelastic photon scattering interactions with materials. Due to the low incidence of spontaneous Raman scattering, CRS techniques enhance imaging signals. CRS includes coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS). CARS relies on the frequency difference between pump and Stokes light for sample excitation but is susceptible to non-resonant background interference. SRS, which eliminates such background noise, offers clearer spectral information and is particularly suitable for label-free imaging by detecting pathological tissues based on chemical bond vibrational signatures.

Multiphoton microscopy (MPM) has extended from fundamental research to clinical applications, demonstrating significant potential in label-free cancer pathology analysis. MPM utilizes short-pulse focused lasers to induce nonlinear optical effects in samples, including two-photon and three-photon excited fluorescence microscopy, as well as second and third harmonic generation microscopy. In contrast to single-photon excitation, MPM employs longer-wavelength lasers, allowing for deeper tissue penetration up to several hundred micrometers. MPM’s label-free properties and deep penetration capabilities are promising for intraoperative tumor diagnosis, with future advancements potentially focusing on equipment miniaturization and the integration of deep learning to enrich tissue pathology information.

Multimodal microscopy synthesizes various label-free imaging techniques to offer a comprehensive assessment of cancer pathology features. Multimodal nonlinear optical microscopy synchronously acquires multidimensional information from biological tissues by combining techniques such as two-photon excited fluorescence (2PEF), three-photon excited fluorescence (3PEF), second harmonic generation (SHG), third harmonic generation (THG), and CARS. Progress in equipment miniaturization and the application of deep learning to enhance imaging speed and imaging quality are necessary for achieving rapid and accurate intraoperative diagnosis.

Fourier ptychographic microscopy (FPM) and lensless coded ptychographic microscopy (CPM) address the trade-off between field-of-view and resolution in traditional microscopy. These techniques present distinct advantages in high-throughput, label-free quantitative phase imaging of biological samples, facilitating non-invasive imaging of transparent specimens and advancing rapid pathological evaluation and digital pathology.

Conclusions and Prospects  Label-free microscopic imaging techniques facilitate rapid, high-resolution imaging while circumventing the limitations associated with traditional fluorescence methods. These techniques include multiphoton fluorescence microscopy, coherent Raman scattering, optical coherence tomography, harmonic microscopy, photoacoustic microscopy, and ptychographic microscopy. This review discusses their fundamental principles and examines their applications in intraoperative tumor diagnosis. Additionally, it evaluates their optical performance in terms of imaging speed, imaging depth, and spatial resolution. Future directions include the application of virtual staining techniques to convert label-free images into standard H&E-stained images. Moreover, the miniaturization of imaging devices and the development of novel imaging technologies, such as DRUM, are essential for improving diagnostic accuracy and treatment outcomes.