SNR enhanced high-speed two-photon microscopy using a pulse picker and time gating detection.

Two-photon microscopy (TPM) is an attractive biomedical imaging method due to its large penetration depth and optical sectioning capability. In particular, label-free autofluorescence imaging offers various advantages for imaging biological samples. However, relatively low intensity of autofluorescence leads to low signal-to-noise ratio (SNR), causing practical challenges for imaging biological samples. In this study, we present TPM using a pulse picker to utilize low pulse repetition rate of femtosecond pulsed laser to increase the pulse peak power of the excitation source leading to higher emission of two-photon fluorescence with the same average illumination power. Stronger autofluorescence emission allowed us to obtain higher SNR images of arterial and liver tissues. In addition, by applying the time gating detection method to the pulse signals obtained by TPM, we were able to significantly reduce the background noise of two-photon images. As a result, our TPM system using the pulsed light source with a 19 times lower repetition rate allowed us to obtain the same SNR image more than 19 times faster with the same average power. Although high pulse energy can increase the photobleaching, we also observed that high-speed imaging with low total illumination energy can mitigate the photobleaching effect to a level similar to that of conventional illumination with a high repetition rate. We anticipate that this simple approach will provide guidance for SNR enhancement with high-speed imaging in TPM as well as other nonlinear microscopy.

Label-free multimodal microscopy using a single light source and detector for biological imaging.

Multimodal nonlinear microscopy has been widely applied in biology and medicine due to its relatively deep penetration into tissue and its label-free manner. However, current multimodal systems require the use of multiple sources and detectors, leading to bulky, complex, and expensive systems. In this Letter, we present a novel method of using a single light source and detector for nonlinear multimodal imaging of biological samples. Using a photonic crystal fiber, a pulse picker, and multimode fibers, our developed system successfully acquired multimodal images of swine coronary arteries, including two-photon excitation fluorescence, second-harmonic generation, coherent anti-Stokes Raman scattering, and backreflection. The developed system could be a valuable tool for various biomedical applications.

Rapid histologic diagnosis using quick fluorescence staining and tissue confocal microscopy.

With the development of advanced and minimally invasive surgical techniques, and in view of the functional and cosmetic aspects, the need for rapid and accurate diagnosis during surgery is increasing. This study was conducted to develop a tissue diagnosis method using confocal microscopy after simple tissue staining that does not require freezing and slicing. At present, fluorescence staining with confocal microscopy is not generalized for real-time diagnosis during surgery. In this paper, we propose a fluorescence staining method using Hoechst 33342 and Eosin that does not require tissue freezing and slicing. The proposed method can be used as part of a rapid tissue diagnosis method that is suitable for use in the operating room, although further research is required before it can be applied in clinical practice.

Rapid tissue histology using multichannel confocal fluorescence microscopy with focus tracking.

BACKGROUND: Simplified hematoxylin and eosin (H&E) staining followed by cryo-sectioning enables rapid identification of cancerous tissue within the procedure room during Mohs micrographic surgery. Yet, a faster evaluation method is desirable as the staining protocol requires physically sectioning of the tissue after freezing, which leads to prolonged sectioning time along with the frozen artifacts that may occur in frozen sectioning. METHODS: We present a multichannel confocal microscopy system to rapidly evaluate cancerous tissue. Using the optical sectioning capability of the confocal microscope, optically sectioned images of the freshly excised mouse tissue were acquired and converted into images resembling H&E histology. To show details of the nuclei and structure of the tissue, we applied a newly developed rapid tissue staining method using Hoechst 33342 and Eosin-Y. Line scanning and stitching was performed to overcome the limited field of view of the confocal microscope. Unlike previous confocal systems requiring an additional mechanical device to tilt the sample and match the focus of the objective lens, we developed a focus tracking method to rapidly scan large sample area. The focus tracking provides an effective means of keeping the image of the thick tissue in focus without additional devices. We then evaluated the performance of the confocal microscope to obtain optically sectioned images in thick tissue by comparing fluorescence stained slide images. We also obtained the corresponding H&E histology image to assess the potential of the system as a diagnostic tool. RESULTS: We successfully imaged freshly excised mouse organs including stomach, tumor, and heart within a few minutes using the developed multichannel confocal microscopy and the tissue staining method. Using the pseudocolor method, colors of the acquired confocal grayscale images are converted to furthermore resemble Hematoxylin and Eosin histology. Due to the focus tracking and the line scanning, optically sectioned images were obtained over the large field of view. Comparisons with H&E histology have shown that the confocal images can acquire large details such as the ventricle as well as small details such as muscle fibers and nuclei. CONCLUSIONS: This study confirms the use of confocal fluorescence microscopy technique to acquire rapid pathology results using optical sectioning, line scanning and focus tracking. We anticipate that the presented method will enable intraoperative histology and significantly reduce stress on patients undergoing surgery requiring repeated histology examinations.