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.

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.

Multimodal microscopy for the simultaneous visualization of five different imaging modalities using a single light source.

Optical microscopy has been widely used in biomedical research as it provides photophysical and photochemical information of the target in subcellular spatial resolution without requiring physical contact with the specimen. To obtain a deeper understanding of biological phenomena, several efforts have been expended to combine such optical imaging modalities into a single microscope system. However, the use of multiple light sources and detectors through separated beam paths renders previous systems extremely complicated or slow for in vivo imaging. Herein, we propose a novel high-speed multimodal optical microscope system that simultaneously visualizes five different microscopic contrasts, i.e., two-photon excitation, second-harmonic generation, backscattered light, near-infrared fluorescence, and fluorescence lifetime, using a single femtosecond pulsed laser. Our proposed system can visualize five modal images with a frame rate of 3.7 fps in real-time, thereby providing complementary optical information that enhances both structural and functional contrasts. This highly photon-efficient multimodal microscope system enables various properties of biological tissues to be assessed.

Erratum: Multimodal microscopy for the simultaneous visualization of five different imaging modalities using a single light source: publisher's note.

[This corrects the article on p. 5452 in vol. 12, PMID: 34692194.].

Real-time visualization of two-photon fluorescence lifetime imaging microscopy using a wavelength-tunable femtosecond pulsed laser.

A fluorescence lifetime imaging microscopy (FLIM) integrated with two-photon excitation technique was developed. A wavelength-tunable femtosecond pulsed laser with nominal pulse repetition rate of 76-MHz was used to acquire FLIM images with a high pixel rate of 3.91 MHz by processing the pulsed two-photon fluorescence signal. Analog mean-delay (AMD) method was adopted to accelerate the lifetime measurement process and to visualize lifetime map in real-time. As a result, rapid tomographic visualization of both structural and chemical properties of the tissues was possible with longer depth penetration and lower photo-damage compared to the conventional single-photon FLIM techniques.